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Chromatography in Food Science and Technology
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Tibor Cserháti
Esther Forgács
Institute of Chemistry, Chemical Research Center
Hungarian Academy of Sciences
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aTECHNOMIC ®publication
Technomic Publishing Company, Inc.

851 New Holland Avenue, Box 3535
Lancaster, Pennsylvania 17604 U.S.A.
Copyright © 1999 by Technomic Publishing Company, Inc.

All rights reserved
No part of this publication may be reproduced, stored in a

retrieval system, or transmitted, in any form or by any means,
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A Technomic Publishing Company book

Bibliography: p.
Includes index p. 547
Library of Congress Catalog Card No. 99-62434

ISBN No. 1-56676-749-0
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Page vii
Table of Contents
Preface ix
List of Abbreviations xi
Chapter 1. Theory and Practice of Chromatography 1
1.1 Gas Chromatography 1
1.2 Liquid Chromatography 5
1.3 Capillary Electrophoresis 8
Chapter 2. Macrocomponents in Foods 11
2.1 Separation of Carbohydrates 11
2.2 Separation of Lipids 51
2.3 Separation of Amino Acids, Amines, Peptides and Proteins 158
Chapter 3. Microcomponents in Foods 299
3.1 Vitamins and Provitamins 299
3.2 Miscellaneous Bioactive Microcomponents 324
3.3 Separation of Food Additives 470
References 507
Index 547
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Page ix
Preface
Chromatography has been developed as a powerful and rapid technique for separation compounds with
highly similar molecular characteristics even from complicated matrices. Because of excellent
separation characteristics and versatility, chromatographic methods have found growing acceptance and
application in food science and technology for the separation and quantitative determination of a wide
variety of compounds such as food ingredients, additives, vitamins, environmental pollutants, and more.
The objectives of this monograph are compilation and concise evaluation of the newest results in this
rapidly developing domain of chromatography, brief enumeration of the methods applied, and critical
discussion of the results. The book is meant to be self-sufficient in terms of the needs of the average
professional who intends to work in this interesting field. We are confident that the book will be found
useful as a valuable reference book for researchers and serious students interested in the topics covered.
We are grateful to Viola Sándor-Orosz and Zsuzsa Bárányos for their valuable technical assistance.
TIBOR CSERHáTI
ESTHER FORGáCS
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List of Abbreviations
AA
ascorbic acid
ACN
acetonitrile
AE
anion exchange
AG
agmatine
A-LAGI
geometrical isomer of α-linolenic acid
AMF
anhydrous milk fat
ANN
artificial neural network
AOAC
Association of Official Analytical Chemists
APCI
atmospheric pressure chemical ionization
AQC
6-aminoquinolyl-N-hydroxy zuccinimidyl carbamate
ASTED
automated sequential trace enrichment of dialysate
BITC
butylisothiocyanate
BMIS
beet medium invert syrup
BO
borage oil
BSA
bovine serum albumin
BSTFA/TMS
bis (trimethylsilyl) trifluoracetamide trimethylchlorosilane
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BTC
butylthiocarbamyl
CA
cadaverine
β-CDS
β-cyclodextrin-bonded silica
CE
capillary electrophoresis
CGA
chlorogenic acid
CGE
capillary gel electrophoresis
CIEF
capillary isoelectric focusing
CITP
capillary isotachophoresis
CLND
chemiluminescent nitrogen detection
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CMP
caseinomacropeptide
CN
carbon number
CPP
caseinophosphopeptide
CV
coefficient of variation
CZE
capillary zone electrophoresis
d.b.
dry basis
DDT
dithiotreitol
DECTP
diethylchlorothiophosphate
DG
diacyl glycerol
DH
degree of hydrolysis
DHAA
dehydroascorbic acid
DHIAA
dehydroisoascorbic acid
DI
direct injection
DM
dry material
DMA
dimethyl amine
DMF
dimethylformamide
DMS
dimethyl sulphide
DNPH
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2,4-dinitrophenylhydrazine
DNPU
dinitrophenylurethane
Dns-AA
dansylamino acid
DTNB
5,5'-dithio-bis (2-nitrobenzoic acid)
ECN
equivalent carbon number
ED
electrochemical detection
ECD
electrochemical detector
EDTA
ethylenediametetraacetic
EI
electron impact
ELSD
evaporative light scattering detector
EOF
electroosmotic flow
EPO
Evening Primrose oil
EPT
endpoint temperature
ES
electrical stimulation
ESI-MS
electron spray ionization mass spectrometry
ETA
ethanol amine
F
furaldehyde
FAB-MS
fast atom bombardment mass spectrometry
FAME
methylated fatty acid
FBD
fermented bread dough
FD
flavor dilution
FDAA
(1-fluoro-2, 4-dinitrophenyl)-5-L-alanine amide
FFAP
free fatty acid column
Page xiii
FID
flame ionization detector
FMOC
fluoromethyl chloroformate
FPD
flame photometric detection
FPLC
fast protein liquid chromatography
FSD
fermented sourdough
FT-IR
Fourier transform infrared spectroscopy
GC
gas chromatography
GC-MS
gas chromatography–mass spectrometry
GCO
gas chromatography combined with olfactometry
GCO-H
gas chromatography–olfactometry of headspace
GL
galactolipid
GLC
gas-liquid chromatography
GPC
gel permeation chromatography
GSC
gas-solid chromatography
HBPF
hydrophobic bitter peptide fraction
HDM
hand-deboned meat
HFCS
high-fructose corn syrup
HI
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histamine
HMF
5-hydroxymethyl-2-furaldehyde
HP-FFAP
Hewlett-Packard free fatty acid column
HPGFC
high-performance gel filtration chromatography
HPLC
high-performance liquid chromatography
HPSEC
high-performance size exclusion chromatographic
HPTLC
high-performance thin-layer chromatography
HTCGC
high-temperature capillary gas chromatography
IAA
isoascorbic acid
IBLC
N-isobutyryl-L-cysteine
ICP-MS
inductive-coupled plasma-mass spectrometry
ID
isotope dilution
I.D.
inner diameter
IEC
ion-exchange chromatography
IGC
inverse gas chromatography
IMAC
immobilized metal ion affinity chromatography
IR
infrared
IRMS
isotope ratio mass spectrometry
IS
internal standard
isoBOC
isobutyloxycarbonylated
ISSPH
isoelectric soluble soybean protein hydrolysate
IV
iodine value
Page xiv
LC
liquid chromatography
LC-MS
liquid chromatography-mass spectrometry
LD
limit of detection
LLE
liquid–liquid extraction
LLE-EC
liquid–liquid extraction with external concentration
LLE-IC
liquid–liquid extraction with internal concentration
LQ
limit of quantification
MDGC
multidimensional gas chromatography
ME
mercaptoethanol
MECC
micellar electrokinetic capillary chromatography
MEEKC
microemulsion electrokinetic chromatography
MF
milk fat
MG
monoacylglycerol
MRM
mechanically recovered meat
MS
mass spectrometry
MSG
monosodium glutamate
na
not analyzed
NANA
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N-acetyl neuraminic acid
NBD
N-chloro-7-nitrobenzofurazan
NC
not calculable
nd
not determined
ND
not detected
NDMA
N-nitrosodimethylamine
NL
neutral lipid
NMR
nuclear magnetic resonance
NQ
not quantified
ODS
octadecylsilica
OPA
o-phthalaldehyde
OW
optothermal window
PAAGE
polyacrylamide agarose gel electrophoresis
PAD
pulsed amperometric detector
PAGE
polyacrylamide gel electrophoresis
PCR
principal component regression
PGA
pteroylmonoglutamic acid
PHE
phenyl ethylamine
PHVO
partially hydrogenated vegetable oil
PITC
phenyl isothiocyanate
PL
phospholipid
PP
1,3-dipalmitoyl-glycerol
Page xv
PrCN
propionitrile
PTC
phenylthiocarbamyl
PTV
programmed temperature vaporizer
PU
putrescine
PUFA-TG
triglyceride fraction containing polyunsaturated fatty acids
Py-GC-MS
pyrolysis–gas chromatography–mass spectrometry
PYR
pyrrolidine
RI
refractive index
RMT
relative migration times
RP
reversed phase
RPBA
radioprotein-binding assay
RP-HPLC
reversed-phase high-performance liquid chromatography
RP-TLC
reversed-phase thin-layer chromatography
RSD
relative standard deviation
RT
retention time
SD
standard deviation
SDEC
simultaneous distillation–extraction with concentration
SDENC
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simultaneous distillation–extraction without concentration
SDS
sodium dodecylsulfate
SE
serotonin
SEC
size exclusion chromatography
SE-HPLC
size exclusion high-performance liquid chromatography
SFC
supercritical fluid chromatography
SFE
supercritical fluid extraction
SI
silica
SIM
selected-ion monitoring
SLL
Syrian Local Large
SM
spermine
SMM
S-methylmethionine
SP
spermidine
SPE
solid-phase extraction
SPME
solid-phase microextraction
STP
sodium tripolyphosphate
TAA
total amino acid
TAG
triacylglycerol
TBDMS
tert-butylmethylsilylate
TCA
trichloroacetic acid
TEA
triethylamine
TEMED
N, N, N', N'-tetramethyl-ethyleme-diamine
TFA
trifluoroacetic acid
Page xvi
TG
triglyceride
THF
tetrahydrofolate
TIC
total ion chromatogram
TLC
thin-layer chromatography
tr
trace
TR
tryptamine
TY
tyramine
UBD
unfermented bread dough
UHT
ultra heat treatment
UV-VIS
ultraviolet-visible
VC
variation coefficient
VOME
vegetable oil methyl ester
WP
whey powder
WPC
whey protein concentrate
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Page 1
Chapter 1—
Theory and Practice of Chromatography
1.1—
Gas Chromatography
The term gas chromatography (GC) summarizes chromatographic methods when the mobile phase is
gas and the stationary phase is solid or liquid (gas-solid chromatography = GSC or gas-liquid
chromatography = GLC). GC has proven to be a powerful technique for the qualitative and quantitative
analysis of a wide range of materials in such diverse areas as environmental, clinical and food analysis,
and petroleum and chemical production. The analysis of flavor and aroma components, the types and
amounts of fatty acids, and the possible presence of adulterants are some applications of gas
chromatography in the food industry. There are serious limitations to the types of compounds suitable
for GC analysis. The most decisive physical property of a compound is its volatility. The compound
needs to have appreciable vapor pressure at a temperature below 350-400° C. Another important
characteristic is a compound's ability to vaporize rapidly without decomposing or reacting. Instability at
high temperatures usually renders a compound unsuitable for GC analysis.
Common gas chromatographic equipment consists of a carrier gas system, injector, gas
chromatographic column, detector, and data processing unit. Carrier gas is generally a permanent gas
with low or negligible adsorption capacity, such as hydrogen, helium, or nitrogen. The nature of carrier
gas may influence the separation characteristics of the GLC systems and can modify the sensitivity of
the detection. Use of hydrogen as a carrier gas is hampered by its high flammability and by the strict
safety regulations concerning its application. Helium is an excellent choice, but the elevated price of
helium considerably reduces its use. Nitrogen is the most frequently used carrier gas in laboratory
practice; it is not expensive, and many separations can be successfully
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carried out with nitrogen carrier gas. Some other gases have also been used for the solution of special
separation problems or for the elucidation of theoretical principles.
The stability and reproducibility of the carrier gas flow rate is a prerequisite of a successful gas
chromatographic analysis. Many injector types have been developed with the objective to deliver the
vaporized sample to the head of the GC column with a minimum initial bandwidth. Sample inlets,
referred to as injectors or injection ports, can be classified into two major groups: vaporization and on-
column injectors. Vaporization injectors utilize high temperatures to vaporize a liquid sample rapidly.
Usually a syringe is used to introduce the sample into the heated injector. In this case the sample rapidly
vaporizes, mixes with the carrier gas, and is transported into the column. Any portion of the sample that
does not rapidly vaporize either remains in the injector or at the front of the column. In either case,
these residues will eventually contaminate the GC apparatus and make cleaning necessary. On-column
injectors deposit the sample directly into the column without relying upon vaporization of the sample
and its subsequent transport into the column.
Headspace sampling involves placing a liquid or solid sample in a vial sealed with a septum cap. The
sample is thermostatted, and portions of the volatile components move into the space above the sample.
The amount of each compound in the headspace is dependent on the volatility of the compound and its
concentration in the sample. The procedure requires sufficient time for the sample components to
equilibrate between the headspace and the sample at the setpoint temperature before a measured portion
of the headspace is removed using a needle inserted into the vial headspace. The sampled portion is
transported through a heated transfer line to the column using carrier gas. Headspace sampling is well
suited for analysis of the volatile components in a liquid or solid sample.
The gas chromatographic column has to be thermostatted at constant temperature (isocratic separation
mode) or according to a predetermined temperature program (temperature gradient). Since the column
temperature is one of the most decisive parameters in GC analysis, its exact regulation is of paramount
importance. The stationary liquid phase of GC columns has to comply with the following requirements:
low vapor pressure, high chemical stability, and relatively low viscosity at the temperature of analysis;
selectivity for the sample components under investigation; and good wetting capacity both for the
surface of the inert support or for the possibly inert wall of the column. The length of a packed column
is limited to about 3 m because of the high pressures that are required to maintain the carrier gas flow
rates or velocities necessary for optimal performance. Solid supports wetted by the stationary liquid
phase are characterized by low adsorption capacity and by large surface area. Inert supports improve
peak symmetry and do not interfere with the partition
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of analytes between the gas and the liquid stationary phases. Both the absence of adsorption centers on
the support surface and the large surface area contribute to the even distribution of the wetting agent,
which is a prerequisite of high separation performance. Higher column efficiency is obtained with small
support diameters that vary over a narrow range. Common solid support diameters are 80-120 mesh
(125-177 µm). The amount of the stationary phase coated on a support is generally between 1% and
10%, with 30% being the highest normally encountered. In addition to column packings that consist of
supports coated with a stationary phase, porous polymer packings have also found application. These
packing materials are frequently used for the separation of highly volatile compounds.
Owing to their higher separation, capacity capillary (also called open tubular) columns have also been
extensively used in food analysis. The inner surface of a capillary column is coated with a thin layer of
the stationary phase. Most of the stationary phases are cross-linked and covalently bonded to the fused-
silica surface. The amount of stationary phase in a capillary column is denoted by the film thickness,
which is typically 0.1-5 µm. Compound retention is proportional to film thickness in capillary columns;
retention increases as the film thickness increases, and it decreases as the film thickness decreases.
Detectors interact with the solute molecules as they exit the column. This interaction is converted into
an electrical signal that is sent to a recording or data storage device. A chromatogram is then created,
which is a plot of the intensity of the signal versus elapsed time. The primary differences between
detectors are in the lowest amount of a compound that is detectable (sensitivity) and in which
compounds produce the strongest detector response (selectivity). Many different detectors (flame
ionization, nitrogen-phosphorus, flame photometric, electron capture, thermal conductivity, atomic
emission, electrolytic conductivity, chemiluminescence, etc.) and data handling devices have been
developed for the sensitive and selective detection and quantitation of sample components. The newest
developments are presented in the prospects of the firms producing gas chromatographs.
1.1.1—
Fundamentals
The distribution of solute molecules between the stationary and mobile phases (carrier gas) is defined
by the distribution constant (KD), which is the ratio of the concentration of the solute molecules in the
stationary phase to that in the mobile phase:
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The relationship between retention, column temperature, and solute properties is described by
where KD is the distribution constant, ∆G0 is the change in Gibbs free energy for the evaporation of
compound from the stationary phase, T is the column temperature (Kelvin degree), and R is the ideal
gas constant. Equation (2) shows that the differences in the Gibbs free energy for the evaporation of the
solutes from the stationary phase result in different distribution of the solutes. The amount of the time
that the compound spent in the column is called the retention time (tR). The partition ratio, also known
as the capacity factor (k′), is the amount of time that a compound spends in the stationary phase relative
to the mobile phase.
The retention index (I) method introduced by Kováts [ 1] highly increased the reproducibility and
reliability of the determination of the retention characteristics. The basis of the method is the
comparison of the retention time of a given analyte, with the retention times of normal hydrocarbons
eluting before and after the analyte. The retention index I can be calculated by the following equation:
where x refers to the analyte, c refers to the number of carbon atoms of the n-hydrocarbon eluting
before the analyte, and c + 1 refers to the number of carbon atoms on the n-hydrocarbon eluting after
the analyte. VN is the net retention volume. In practice the net retention time is usually obtained by
measuring the distance on the recorder chart between the peaks of nonsorbed and sorbed solutes.
The separation factor (alpha) characterizing the separation of two molecules can be calculated by the
following equation:
where k1 is the partition ratio of the earlier eluting peak and k2 is the partition ratio of the later eluting
peak.
Resolution (R) is a measure of peak separation that takes peak widths into account:
where tR1 and tR2 are the retention times of peaks 1 and 2, wh1 and wh2 are the peak widths at half height
of peaks 1 and 2, wb1 and wb2 are the peak widths at the base of peaks 1 and 2, respectively.
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Theoretical plate number (N) characterizes the separation capacity of a chromatographic column. There
are no plates in a GC column; however, this concept, borrowed from the distillation theory, was
successfully used to characterize column performance. The number of theoretical plates is calculated by
The theoretical aspects and practical applications of various gas chromatographic methods are discussed
in more detail in References [ 2,3,4,5].
1.2—
Liquid Chromatography
The term liquid chromatography (LC) summarizes chromatographic methods in which the mobile
phase is liquid and the stationary phase is an inorganic or organic solid. According to the shape of the
solid-phase matrix, liquid chromatographic methods can be roughly divided into planar and column
chromatography. According to the relative polarity of the stationary and mobile phases, LC methods are
classified as normal (adsorption) and reversed-phase separation mode. When the LC system consists of
a nonpolar mobile phase and a polar stationary phase, it is called normal-phase chromatography. In
contrast to the normal-phase separation mode, the reversed-phase mode requires the combination of a
polar mobile phase and a nonpolar stationary phase.
1.2.1—
Thin-Layer Chromatography
Thin-layer chromatography (TLC) is a planar chromatographic technique extensively used as a rapid

and easy to carry out analytical tool. TLC has proved to be suitable for the semiquantitative
determination of various organic and inorganic molecules in complicated mixtures. The principle of the
traditional TLC is very simple: the mixture of solutes to be separated is spotted on the solid surface and
moved with an adequate mobile phase system.
The different strength of interaction of solute molecules with both the stationary and mobile phase
results in different mobility and separation. A wide variety of inorganic and organic sorbents have been
tried as a stationary phase for TLC; however, mainly silica and silica with covalently bonded ligands on
the surface are the most frequently used supports. The polarity order of the various silica and bonded
silica plates is approximately the following: silica > amino silica > cyano silica > octadecyl (C18) silica.
Other sorbents such as alumina, diatomaceous earth (Kieselguhr), celluloses, and polyamides have
found only limited application. The performance of a TLC layer depends on the physicochemical
characteristics of the sorbent, such as specific surface area and pore volume, mean pore diameter and
pore size distribution, and particle distribution and size. The average particle size of TLC sorbents is
between 10 µm and 50 µm. A smaller particle size and narrower particle size distribution
Page 6
improve the resolution, decrease analysis time, and enhance the sensitivity of detection (high-
performance TLC = HPTLC). The solvent strength (elution capacity) of a solvent is determined by its
capacity to move a solute. Higher solute mobility indicates higher elution strength. The order of elution
strength on commonly used silica is: n-heptane > n-hexane > n-pentane > cyclohexane > carbon
tetrachloride > toluene > dichloromethane > diethylether > acetonitrile > 1-butanol > 2-propanol >
acetone > ethanol > dioxane > tetrahydrofuran > methanol > pyridine > water. The elution order is
nearly opposite in reversedphase TLC (RP-TLC).
1.2.1.1—
Fundamentals
The retention of a solute in TLC and in RP-TLC is characterized by the Rf value: the distance from the
origin to the center of the separated zone divided by the distance from the origin to the solvent front.
The capacity factor k can be defined by the ratio of retention times of a solute in the stationary (ts) and
mobile phase (tm), respectively:
The relationship between capacity factor and Rf is
Because the relationship between the Rf value of the solutes and the concentration of the stronger
component in the eluent (C) is logarithmic, the RM value was introduced to describe the correlation more
easily:
where RM0 is the theoretical RM value of a solute extrapolated to zero concentration of the stronger
component in the mobile phase and b is the change of the RM value caused by unit change of the
concentration of the stronger component in the mobile phase (C).
The general theory, fundamentals, and practical application of TLC have been described in other
excellent books [ 6,7,8] and reviews [9,10].
1.2.2—
High-Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) is the most frequently used column chromatographic
technique. Its popularity is caused by the high separation power, excellent selectivity, and the high
diversity of solutes that
Page 7
can be separated with this method. Similar to TLC, the majority of measurements are carried out either
in normal or in reversed-phase separation mode using silica and modified silica supports. However, in
contrast to TLC, reversed-phase HPLC (RP-HPLC) is a method of preference for the analysis of the
majority of solutes. RP-HPLC uses less toxic solvents (water and an organic modifier miscible with
water), causing less environmental pollution; the equilibrium time of RP-HPLC supports is
considerably lower than the normal-phase ones; and the retention of solutes can be more or less
predicted as the retention is mainly governed by the hydrophobicity of solutes, which can be calculated
with reasonable accuracy. In addition to normal- and reversed-phase separation modes, HPLC offers a
unique possibility to employ supports with different separation mechanisms, such as ion-exchange, size
exclusion, and gel permeation chromatography.
Separation in ion-exchange HPLC is based on the interaction of the charged solute with the oppositely
charged surface of the stationary phase. The retention of the electrostatically bonded solutes can be
easily influenced by adding salts to the mobile phase or by modifying its pH value. Size exclusion
chromatography (SEC) is a technique for separating molecules according to their effective shape and
size in the aqueous mobile phase. Gel permeation chromatography (GPC) is similar to SEC, but it
generally uses organic solvents for the separation of water-insoluble macromolecules. The stationary
phases used in these separation modes are porous particles with closely controlled pore size. The
molecules of solute can enter the pores of the support according to their size and shape. The choice of a
mobile phase for this separation mode is simpler than other HPLC modes since only one solvent is
required.
A common HPLC system consists of an injection device, a column, the mobile phase delivery system, a
detector, and a signal output device. Injector is an important part of the HPLC system because it has to
inject a precise volume of solute solution into the mobile phase under high pressure. The dimensions of
HPLC columns and the character of supports vary considerably according to the retention behavior of
the analytes. Information about the use of various columns and HPLC supports in food analysis is
included in the corresponding chapters of the book. The mobile-phase delivery system includes a pump
with filter, a degasser, and transfer tubing. It transfers the mobile-phase components into the separation
system with a constant, precise, and reproducible flow rate. When the composition of the mobile phase
is held constant during the separation process, the method is called isocratic elution. In order to increase
the efficiency of the separation or eluting solutes with highly different physicochemical characteristics
in one run, the composition of the mobile phase can be continuously changed in a predetermined
manner (gradient elution). A wide variety of detectors are used in HPLC, applying different
physicochemical principles or exploiting different molecular characteristics of solutes. The most
commonly used detectors are ultraviolet-visible (UV-VIS), fluorescence,
Page 8
refractive index (RI) detector), and various electrochemical detectors (ECD). Recently, hyphenated
techniques (a combination of HPLC with a detector suitable for the identification of the solutes) have
gained growing acceptance and application in HPLC. Thus, HPLC was successfully combined with
other analytical spectroscopic methods such as mass spectrometry (MS), infrared spectrometry (IR),
and Fourier transform infrared spectrometry.
1.2.2.1—
Fundamentals
The capacity ratio (k′) can be defined in both adsorption and reversed-phase chromatography by
where the tR is the retention time (the time passed from the injection to the appearance of the solute peak
maximum) and t0 is the dead time. The relationship between the logarithm of the capacity ratio (log k′)
and the volume fraction of the stronger component in a mobile phase composed of two solvents can be
described by
where the log k′0 value is the log k′ value of the solute extrapolated to zero concentration of the stronger
component in the mobile phase (C); the b (slope) value shows the sensitivity of the change in the
retention of a given solute caused by unit change in the concentration of the stronger component in the
mobile phase.
The characteristic chromatographic parameters (separation factor, resolution, theoretical plate number)
in HPLC can be calculated similarly like in gas chromatography [see equations (5)-(8)]. The theoretical
aspects [ 11] and practical applications of HPLC [12,13], the support most frequently used for the
various HPLC separations [14], and the application of HPLC for sample preparation [15] have been
described earlier and discussed in more detail.
1.3—
Capillary Electrophoresis (CE)
Separation by electrophoresis is obtained by differential migration of solutes in an electric field. In CE,

separation is performed in narrow-bore capillaries, typically 25-75 µm inner diameter (I.D.), usually
filled with a buffer. Use of the capillary has numerous advantages, particularly with respect to the
detrimental effects of Joule heating. The high electrical resistance of the capillary enables the
application of very high electrical fields (100-500 V/cm) with minimal heat generation. Moreover, the
large surface area to volume ratio
Page 9
of capillary efficiently dissipates the generated heat. The use of the high electrical fields results in short
analysis time, high efficiency, and resolution. Peak efficiency, often in excess of 105 theoretical plates
number, is caused by the plug profile of the electroosmotic flow, an electrophoretic phenomenon that
generates the bulk flow of solution within the capillary. This flow also enables the simultaneous
analysis of all solutes, regardless of charge. Minimal sample volume requirements (1-10 nL), on-
capillary detection, and the potential for quantitative analysis and automation make CE a rapidly
developing separation technique. CE is characterized by its simple experimental setup. Both ends of a
filled capillary are dipped into two buffer reservoirs maintained at the same level. A high voltage is then
applied to this capillary. Generally, the sample is injected at the anodic end, and the detector is placed
near the cathodic end.
Various modes of capillary electrophoresis can be performed using the same instrument, including
capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic
capillary chromatography (MECC), capillary isoelectric focusing (CIEF), and capillary
isotachophoresis (CITP). In MECC, surfactant above its critical micellar concentration is added to the
background electrolyte. Solutes with varying degrees of hydrophobicities are partitioned between the
hydrophobic core of the micelles and aqueous buffer [ 16].
1.3.1—
Fundamentals
Principal processes governing separation in CE are the electroosmotic flow (µEOF) and electrophoretic
flow (µep). Electrophoretic flow is the bulk flow of solvent in the capillary under an applied potential.
Electrophoretic flow is the flow of ions owing to charge. Electroosmotic flow can be expressed by
where ∈ = dielectric constant, ζ = zeta potential, and η = bulk viscosity.
Equation (16) indicates that the mobility is independent on the applied electric field. The zeta potential
is essentially determined by surface charge on the capillary wall. Since this charge is strongly pH
dependent, the magnitude of the electroosmotic flow (EOF) varies with pH. Depending on the specific
conditions, the EOF can vary by more than an order of magnitude between pH 2 and 12. The zeta
potential is also dependent on the ionic strength of the buffer, as described by double-layer theory.
Increased ionic strength results in double-layer compression, decreased zeta potential, and reduced
EOF. The analytical parameters for capillary electrophoresis can be described in similar terms to those
for column chromatography. The time required for a solute to migrate to the point of detection is called
the migration time and is given by the quotient
Page 10
of migration and velocity. The migration time and other experimental parameters can be used to
calculate the apparent solute mobility:
and
where l is the effective capillary length, L is the total length, t is the migration time, E is the electric
field, and V is the applied voltage. In the presence of EOF, the measured mobility is called the apparent
mobility (µa). The effective mobility (µe) can be determined by extracting from the apparent mobility by
independently measuring the EOF using a neutral marker that moves at a velocity equal to the EOF.
The separation of neutral solutes in MEKC can be described using by modified chromatographic
relationships. The capacity factor k′ is given by
where tr is the retention time of the solute, t0 is retention time of unretained solute moving at the EOF
rate, tm is the micelle retention time, K is the partition coefficient, Vs is the volume of the micellar phase,
Vm is the volume of the mobile phase.
Various capillary electrophoretic methods are discussed more profoundly in references

[ 16,17,18,19,20,21].
Page 11
Chapter 2—
Macrocomponents in Foods
The nutrition value of various foods and food products mainly depends on the quantity and quality of
the three most important classes of compounds:
1. Carbohydrates (in mono-, oligo-, and polymeric forms)
2. Triglycerids and other compounds of lipoidic character
3. Nitrogen-containing compounds such as amino acids, peptides, and proteins
Since each class of compounds consists of a considerable number of highly similar molecules, their
analysis presents a special challenge for the chromatographers. Virtually each chromatographic
technique can be used in the separation and quantitation of these compounds; however, the application
field of each chromatographic method is limited because of the different physico-chemical
characteristics of the compounds to be separated.
2.1—
Separation of Carbohydrates
Many chromatographic methods (TLC, GC, electrophoresis on supporting media, HPLC, and CE) have
been used for the analysis of carbohydrates. TLC is a rapid and relatively simple method used
extensively over the last few decades; however, the separation capacity and reproducibility of TLC are
sometimes not satisfactory. GC methods are generally sensitive, but the derivatization needed for the
nonvolatile carbohydrates is often time-consuming and results in more than one derivative, which
makes the evaluation of the chromatograms complicated. The accuracy of the quantitative
determination of the carbohydrate fractions separated by electrophoresis on supporting media is often
low. Because of the high reproducibility, separation power, and rapidity,
Page 12
HPLC is the method of preference for the separation of various carbohydrates. Since CE has similar—
sometimes better—separation characteristics than HPLC, the more frequent application of CE in the
carbohydrate analysis can be expected in the near future. The chromatographic analysis of
carbohydrates may promote the determination of the authenticity and quality of various foods and food
products.
2.1.1—
Mono- and Disaccharides
The majority of chromatographic analyses in the field of carbohydrates were dealing with the separation
of well-defined mono-, di-, and trisaccharides. This preference may be because of the fact that smaller
molecules can be more easily separated and pure standards of these saccharides can be purchased for
the quantitative analysis. Earlier results in the chromatographic separation of mono- and disaccharides
have been reviewed in reference [ 22].
HPLC and other analytical methods were used for the determination of sugars, nonvolatile acids, 13 C/12
C ratios, and minerals in 46 samples of red raspberry (Rubus idaeus) [23]. The influence of cultivar,
maturity, geographic origin, mold contamination, and harvesting procedures on the parameters
mentioned above was evaluated. Sugar samples were prepared by mixing 5 mL juice with 5 mL
mannitol internal standard solution. The mixture was passed through a C18 cartridge activated with 5 mL
methanol and 5 mL water. The first 2-3 mL was discarded, the remaining eluate was passed through a
10-mL column containing 1.5 mL hydrated Bio-Rex 5 anion exchange resin. The first 2-3 mL were
discarded, and the remaining eluate was used for HPLC analysis. The separation of sugars was carried
out on an Bio-Rad Aminex HPX-87C column (300 × 7.8 mm I.D.) using an aqueous solution of 200 mg
Ca(NO3)2 /L as eluent. The minimum and maximum concentrations and the mean of the parameters are
compiled in Table 2.1. The data in Table 2.1 clearly show that the composition of the juice of red
raspberry is highly variable; therefore, their use as quality markers is questionable.
HPLC using anion-exchange support [24] and pulsed amperometric detector (PAD) has also been used
for the analysis of fruit juices [25]. The efficacy of HPLC-PAD has recently been compared with that of
CZE in the analysis of the sugar composition of fruit juices [26]. Juices were filtered, diluted with
deionized water (50- to 100-fold for CE and 2000- to 10,000-fold for HPLC), and used for the analyses
without any other pretreatment. The CZE background electrolyte was 6 mM potassium sorbate (pH
adjusted to 11.9-12.4 with 1 M NaOH). Fused-silica capillaries of 42 cm and 90 cm were used in the
experiments, the effective separation distances being 35 cm and 83 cm, respectively. Detection was
carried out at 256 nm, and the capillaries were always thermostatted. Samples were injected by applying
105 mbar for 1 or 2 s. The HPLC column was of 250 × 4 mm I.D. (Carbopac PA 1), and the flow
Page 13
TABLE 2.1. The Minimum and Maximum Concentration and the Mean of Sorbitol, Sucrose, Glucose/Fructose
Ratio, Citric Acid, Malic Acid, Isocitric Acid, Potassium, Sodium, Magnesium, and Calcium in Juices of Red
Raspberry.
Component Minimum Maximum Mean
Sorbitol (percentage of total sugars) 0 0.5 0.15
Sucrose 0 24.1 1.4
Glucose/fructose ratio 0.76 1.03 0.93
Citric acid (percentage of total acids) 89.8 98.9 95.6
Malic acid 0.3 9.6 3.3
Isocitric acid 0.2 2.6 1.1
Potassium (mg/mL) 108 400 227.8
Sodium 0.2 4.0 2.1
Magnesium 11.3 29.4 18.6
Calcium 8.0 16.3 12.5
Reprinted from Reference [ 23] with permission, copyright © 1995, by AOAC International.
rate was 1 mL/min. The detector consisted of a gold working electrode and an Ag/AgCl reference
electrode. The pulse sequence was different for sampling (50 mV, 480 ms), cleaning (600 mV, 120 ms),
and sugar reduction (-600 mV, 60 ms).
A typical electropherogram and chromatogram are shown in Figures 2.1 and 2.2. It is interesting to note
that no matrix peaks interfere with the separation of sugars in CZE and HPLC. Sugars are well
separated in both systems; however, their retention order is strikingly different. This discrepancy was
tentatively explained by the influence of the strong electroosmotic flow in CZE. The results of the
comparison of the various chromatographic parameters of CZE and HPLC are compiled in Tables 2.2
and 2.3. The data prove that the separation efficiency of CZE is superior to that of HPLC, and—taking
into consideration the injection volume—CZE is more sensitive in terms of mass detection. Both
methods showed good linearity, the correlation coefficients always being over 0.993. The repeatability
of the retention (migration) times was similar: <0.3%. The day-to-day reproducibility of the CZE
method was fairly high (10-18%). This result was explained by the uncontrollable modification of the
inner capillary wall at high pH values. The sugar contents determined by CZE using external calibration
deviated considerably from those obtained by HPLC, whereas the results of CZE using internal
calibration showed acceptable agreement with the results of HPLC. It was stated that this discrepancy
may be caused by the poor reproducibility of the very low injection volumes.
An interesting application of the HPLC determination of carbohydrates is the measurement of the

activity of β-galactosidase in yoghurt [27]. Since the disaccharide lactose present in yoghurt can be
hydrolyzed by the microbial
Page 14
Figure 2.1.
Capillary zone electrophoresis of apple juice (diluted 1:50). Running conditions:
buffer 6 mM sorbate adjusted to pH 12.2; 230 V/cm; 15°C; indirect UV
detection at 256 nm; 1-s injection: 1 = sucrose; 2 = glucose;
3 = fructose; 4 = glucuronic acid.
(Reprinted from Reference [ 26], copyright © 1994. With kind permission
from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055
KV Amsterdam, The Netherlands.)
enzymes to the monosaccharides glucose and galactose, the separation and quantitative determination of
these carbohydrates may contain information on the enzymatic activity of the microorganisms in
yoghurt. Samples for HPLC analysis were prepared by diluting yoghurt with bidistilled water (1:10)
and incubating overnight at 55°C with 0.1 M potassium phosphate buffer solution (pH 7) containing 2%
w/v lactose and 5 mM dithioerythrol. After incubation, the samples were filtered and injected without
any pretreatment. Yoghurt without incubation and buffer served as blanks. An ion-exchange column
(300 × 7.6 I.D.; Aminex HPX-87C, Ca2+ form; column temperature 85° C) was used for the separation
of carbohydrates. The eluent was bidistilled water; flow rate was set to 0.6 mL/min. Sugars were
detected with a refraction index detector. A typical chromatogram is shown in Figure 2.3. Sugars are
well separated in this HPLC system in a relatively short analysis time. The day-to-day repeatability was
2.4%, and the run-to-run repeatability was 3.1%, showing the good reproducibility of the method. The
method was proposed for the simple and rapid control of the microbiological quality and enzymatic
activity of yoghurt. HPLC was also applied for the determination of the lactose content of milk
Page 15
Figure 2.2.
HPLC analysis of an orange juice (diluted 1:2000) on a Carbopac PA1
column with 200 mM NaOH as eluent at a flow rate
of 1 mL/min: 1 = sucrose; 2 = glucose; 3 = fructose; 4 = glucuronic acid.
from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055
KV Amsterdam, The Netherlands.)
[28]. Because of the high separation capacity of HPLC, the simultaneous determination of sugars and
other food components in one run has been demonstrated many times [29]. The combination of an
HPLC separation method with on-line dialysis facilitates the automation of both the sample preparation
and analytical steps, resulting in higher laboratory output [30]. This elegant technique has been
successfully used for the simultaneous separation and quantitative determination of sugars and organic
acids in juice and wines [31].
The working scheme of automated sequential trace enrichment of dialysates (ASTED XL) is shown in
Figure 2.4. The sample was loaded into the donor channel and the solutes diffused across the
membrane. The dialysate from the recipient channel was directly transferred into the injection loop of
the HPLC column because the enrichment step was not needed. The dialysis was optimized (pulsed
dialysis for donor channel, static mode for the recipient channel, flow rate 0.18 mL/min). The ASTED-
HPLC method was used for the determination of sugars in orange syrup, cola drinks, reconstituted baby
milk,
Page 16
TABLE 2.2. Comparison of the Separation Efficiency and Limits of Detection in CZE and
HPLC.
Separation Efficiency (N) Limit of Detection (LOD)
Sugar CZE HPLC CZE (mM) HPLC (µM)
Sucrose 49 7000 4 350 0.29 0.58
Glucose 29 300 3 400 0.23 1.11
Fructose 69 600 3 100 0.24 1.11
Reprinted from Reference [ 26], copyright © 1994. With kind permission from Elsevier
Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.
and liquid chocolate yoghurt (method a) and for the simultaneous determination of sugars, organic
acids, glycerol, and ethanol in grape and apple juices, red and white wines, and cider (method b).
The analytical conditions are compiled in Figure 2.5. To compensate the various recovery ratios of the
sample pretreatment, internal standards were
TABLE 2.3. Sucrose, Glucose, and Fructose Contents in Fruit Juices.

Concentration
Sample Sugar Method Calibration Found (g/L) RSD (%)
Apple juice Sucrose CZE External 16.6 ± 0.42 2.5
Internal 17.1 ± 0.83 4.9
HPLC External 19.2 ± 0.50 2.6
Glucose CZE External 23.3 ± 0.50 2.2
Internal 22.7 ± 0.74 3.3
Fructose CZE External 56.5 ± 0.35 0.6
Internal 62.8 ± 2.04 3.3
Orange juice Sucrose CZE External 31.9 ± 1.03 3.2
Internal 34.6 ± 0.70 2.0
Glucose CZE External 21.5 ± 0.50 2.3
Internal 22.0 ± 0.10 0.5
Internal 25.6 ± 0.90 3.5
Grape juice Glucose CZE External 87.1 ± 0.76 0.9
Internal 73.5 ± 1.01 1.4

Internal 81.2 ± 1.57 1.9
Reprinted from Reference [26], copyright © 1994. With kind permission from Elsevier Science—NL, Sara
Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.
Page 17
Figure 2.3.
Typical chromatogram of lactase activity from a sample incubated
at 55°C for 17 h: lactose (1; 1.610 g/100 mL), glucose (2),
and galactose (3; 0.232 g/100 mL).
Reprinted from Reference [ 27] with permission from Chromatographia.
used for the analysis of each food and food product. The sugar content of four foodstuffs is shown in
Table 2.4. The analysis time was relatively short (15 min when lactose was present). The
chromatograms did not show additional peaks originating from the accompanying matrix. This fact
indicates the efficiency of the sample clean-up procedure. The interfering compounds were effectively
removed from the samples of fruit juices and fermented beverages as demonstrated in Figure 2.6. The
components determined in five beverages are listed in Table 2.5. In the case of grape juice, the
concentration of malic acid cannot be determined because of the large neighboring peaks of glucose and
fructose. Under these conditions malic acid can be determined by lowering the column temperature. It
was found that on-line dialysis, combined with HPLC, is a suitable method for the removal of various
polymer matrix components such as proteins, polysaccharides, and condensed phenolic derivatives.
Because of the low cost of consumables and the environmental friend character of the eluents, the
technique has been proposed as a routine analysis for the food industry.
Various ion-exchange HPLC methods have been previously used for the simultaneous determination of
sugars, organic acids and alcohols in juice [32], must, and wine [33,34,35,36,37]. The determination of
sugars and organic acids can be carried out by first separating the neutral and acidic components of
wine on an ion-exchange cartridge and then separating them using the same column and
Page 18
Figure 2.4.
ASTED XL sample preparation (dialysis and injection process): L = load position;
P = HPLC pump; C = chromatography column; W = waste.
(Reprinted from Reference [ 31], copyright © 1995. With kind permission from Elsevier
Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)
Page 19
Figure 2.5.
ASTED XL and HPLC conditions.
(Reprinted from Reference [ 31],
copyright © 1995. With kind permission from Elsevier Science—NL, Sara
Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)
Page 20
TABLE 2.4. Carbohydrate Content of Four Foodstuffs, with Corresponding RSD (IS = internal standard; n = 10).
Baby Milk Chocolate Yoghurt Orange Syrup Cola Drink
g/L RSD(%) g/L RSD g/L RSD(%) g/L RSD(%)
Sample (%)
Fructose — — — — 46.4 3.4 30.1 2.5
Glucose — — IS — 60.2 3.8 31.5 4.4
Sucrose IS — 80.0 3.4 7.8 7.6 63 2.3
Maltose — — — — 7.0 7.0 IS —
Lactos 20.9 3.5 42.6 4.7 IS — — —

Reprinted from Reference [31],copyright © 1995. With kind permission from Elsevier Science—NL, Sara
Burgerhartstraat 25, 1055 KV Amdsterdam, The Netherlands.
eluent system [38]. The separation of sugars and carboxylic acids was carried out on an HPX-87 H
column, using 0.01 M aqueous sulfuric acid as eluent. The column was thermostatted at 65° C; the flow
rate was 0.6 mL/min. The wine components were detected by RI and UV detectors. The recovery of the
sample purification step varied between 83.75% and 100.98%, and the correlation between detector
response and solute concentration was linear in a wide
Figure 2.6.
Chromatogram of a dry white wine after clean-up using ASTED XL. Peaks:
1 = citric acid, 2 = tartaric acid, 3 = glucose, 4 = malic acid, 5 = fructose,
6 = succinic acid, 8 = glycerol, 9 = acetic acid, I.S. = propionic acid, 10 = ethanol.
(Reprinted from Reference [31], copyright © 1995. With kind
permission from Elsevier Science—NL, Sara Burgerhartstraat 25,
1055 KV Amsterdam, The Netherlands.)
TABLE 2.5. Organic Acid, Sugar, Glycerol, and Ethanol Content of Five Beverages, with Corresponding RSD.
Grape Juice Red Wine White Wine Apple Juice
Sample g/L RSD(%) g/L RSD(%) g/L RSD(%) g/L RSD(%)
Citric acid 0.44 2.9 0.45 4.0 0.73 7.2 0.76 3.9
Tartaric acid 2.22 2.9 1.99 3.4 2.64 2.5 — —
Malic acid ND * — 0.46 8.0 2.82 2.4 8.99 0.9
Succinic acid — — 0.70 1.6 0.71 2.0 — —
Lactic acid — — 1.91 1.4 0.34 1.0 — —
Acetic acid — — 0.51 3.2 0.22 8.0 — —
Glucose 44.1 1.4 0.15 4.6 0.7 7.0 30.7 1.3
Fructose 39.1 1.2 <LOD 3.0 1.0 1.1 39.0 1.6
Glycerol 0.9 3.0 6.6 1.0 7.4 1.4 — —
Ethanol 2.2 7.8 122.0 2.2 123.0 2.0 — —
* ND = not detected.
Reprinted from Reference [ 31], copyright © 1995. With kind permission from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV A
Netherlands.
Page 22
concentration range. Both sugars (Figure 2.7) and carboxylic acids (Figure 2.8) were successfully
separated in this HPLC system. This HPLC method has been proposed for the quantitative
determination of sugars and carboxylic acids in wine and the detection of wine adulteration. A slightly
modified automated HPLC method was developed for the determination of glucose, fructose, glycerin,
and ethanol in wine, and the validation parameters were calculated [ 39 ]. Wine samples were diluted
with bidistilled water to 1:40 at 20°C. The neutral and acidic fractions were separated on an anion-
exchange column (90 × 4 mm I.D., particle size 7-10 µm, PS-DVB with -NR3 + in the Cl-form).
Separation was carried out on a cation-exchange column (300 × 4 mm I.D., particle size 5-8 µm, PS-
DVB with -SO3- in the Ca-form). The column was thermostatted at 80°C, the eluent was distilled water,
and the flow rate was set to 0.4 mL/min. The collaborative study was simultaneously carried out by 19
official and private laboratories. The validation parameters are found in Tables 2.6 and 2.7. No
significance differences were found between the repeatability and reproducibility of the results of the
collaborative study and the conven-
Figure 2.7.
Chromatogram of a sweet wine: A = glucose, B = fructose, C = glycerin,
E = ethanol. Reprinted from Reference [ 38] with permission.
Page 23
tional methods in the European Economic Community (EEC) Commission Regulation No. 2676/90.
This finding indicates that this HPLC method can be used for the determination of glucose, fructose,
glycerol, and ethanol in wine.
The separation and quantitation of sugars, carboxylic acids, glycerol, and ethanol were achieved by
using two columns (Amines HPX-87H; 300 × 7.8 mm I.D.) and two detectors (UV detector at 214 nm
and RI detector thermostatted at 50°C) connected in series [ 40 ]. Eluent was 0.65 mM aqueous sulfuric
acid at a flow rate of 0.7 mL/min. Samples were injected after filtering without any other pretreatment.
A typical chromatogram detected with both detectors is shown in Figure 2.9. Since the peak of malic
acid was not separated from those of glucose and fructose in RI detection, malic acid and fructose were
quantitated with a UV detector. The validity parameters of the method (between-day repeatability and
reproducibility, recovery) are compiled in Tables 2.8 and 2.9. The correlation coefficient of the linear
relationship between the peak areas and the concentration of solutes was always higher than 0.9990, and
the limit of detection varied between 1.84. 10-3 (g/L
Figure 2.8.
HPLC separation of carboxylic acids of wine: F = citric acid, G = tartaric acid,
H = malic acid, I = succinic acid, J = lactic acid, K = acetic acid.
(Reprinted from Reference [ 38] with permission.)
Page 24
TABLE 2.6. Reproducibility of the Measurements and the Upper Confidence Limit (95%) with Five
Replicates.
Limit (%) (N1 = 34;
Mean Upper Confidence n 2
(n = 34) Standard Deviation RSD (%) = 5; p = 95%)
Glucose (g/L) 18.65 0.0385 0.19 0.6
Fructose (g/L) 20.28 0.0368 0.18 0.5
Glycerin (g/L) 5.29 0.0244 0.46 1.3
Ethanol (g/L) 66.76 0.2398 0.36 1.1
Reprinted from Reference [ 39] with permission.
acetic acid) and 0.162 (g/L glycerol). This method has also been proposed for quality control in the
wine industry.
HPLC methods were used for the measurement of the carbohydrate content of various foodstuffs of
plant origin such as raw and baked sweet potatoes [41 ], presweetened cereals [ 42 ], and breads and
biscuits [ 43 ]. HPLC has been successfully used for the elucidation of the change of mono- and
disaccharides in dough samples during processing [ 44 ]. The mono- and disaccharide content of various
doughs and baked products was also measured by HPLC [ 45 ]. Dried samples of 5-10 g weight were
ground, boiled for 20 min in 100 mL of 60% ethanol, cooled, and filtered. The filtrate was further
purified on a C18 cartridge. Separation was carried out on an aminopropyl-silica column (particle size 4
µm) thermostatted at 30°C. The eluent was acetonitrile:water (75:25) mixture containing 0.125% w/v
sodium chloride. The addition of sodium chloride to the mobile phase effectively decreased the
interference from the salt content of the samples. Sugars were detected with a refractive index detector.
The retention time of sugars was fructose = 5.0 min; glucose = 5.8 min; sucrose = 8.2 min; maltose =
9.8 min; and lactose = 11.4 min. The composition of sugars (%) in the various products during
processing is compiled in
TABLE 2.7. Repeatability (r) and Compatibility (R) for the HPLC Determination of Glucose, Fructose,
Glycerol, and Ethanol.
Glucose (g/L) Fructose (g/L) Glycerin (g/L) Ethanol (g/L)
Sr 0.11796 0.14506 0.12157 0.43660

SR 0.14178 0.16551 0.13910 0.48814
r 0.3338 0.4105 0.3440 1.2355
0.029 xi 0.024 xi 0.16 %vol

R 0.4012 0.46839 0.39365 1.3814
0.036 xi 0.027 xi 0.18 %vol

xi = mean; Sr and SR = standard deviations of repeatability (r) and compatibility (R), respectively; r = 2.83
Sr; R = 2.83 SR. Reprinted from Reference [ 39] with permission.
Page 25
Figure 2.9.
Chromatograms of Treixadura wine according to the described procedure by
(A) refractive index and (B) UV (at 214 nm). Conditions: mobile phase, 0.65 mM
sulfuric acid; flow rate, 0.7 mL/min; column temperature, 75°C. Peaks: 1 = citric
acid; 2 = tartaric acid; 3 = malic acid; 4 = fructose; 5 = lactic acid; 6 = succinic
acid; 7 = glycerol; 8 = acetic acid; and 9 = ethanol.
(Reprinted from Reference [ 40] with permission from Preston Publications,
A Division of Preston Industries, Inc., and the corresponding author.)
Page 26
TABLE 2.8. Study of Between-Day Repeatability and Reproducibility for a White Wine.
Repeatability Reproducibility
(n = 10) (n = 10)
Compound Detector Mean (g/L) RSD (%) Mean (g/L) RSD (%)
Citric acid RI 0.301 2.00 0.301 2.20
Tartaric acid RI 1.50 1.40 1.50 1.42
Glucose RI ND — ND —
Malic acid UV 2.45 1.60 2.45 1.43
Fructose UV 0.199 1.95 0.199 1.94
Succinic acid RI 0.360 2.04 0.360 1.52
Lactic acid RI 0.180 2.40 0.180 1.98
Glycerol RI 6.45 0.90 6.45 0.92
Acetic acid RI 0.01 2.80 0.01 2.97
Ethanol * RI 9.16 0.95 9.16 0.73

* Expressed in %v/v
Abbreviations: RI, refractive index; ND, not detected.
Reprinted from Reference [ 40] with permission from Preston Publications, A Division of Preston Industries, Inc.
Table 2.10. Sugar composition was unchanged in baking powder biscuits but changed in the fermented
products. Saccharose was completely hydrolyzed before the first sampling step, and then glucose was
preferentially used by the yeasts. The decomposition rate of sugars depended on both the type of
product and the chemical structure of the saccharides.
TABLE 2.9. Recovery Efficiency of the High -Performance Liquid Chromatographic Method for Treixadura Wine.
Concentration (g/L)
Compound In Wine Added Calculated Found Recovery (%)
Citric acid 0.301 0.5 0.801 0.798 99.6
Tartaric acid 1.50 0.5 2.00 2.04 102.0
Glucose ND 0.5 0.50 0.53 106.0
Malic acid 2.45 0.5 2.95 2.93 99.3
Fructose 0.199 0.5 0.699 0.690 98.7
Succinic acid 0.360 0.5 0.860 0.842 97.9
Lactic acid 0.180 0.5 0.680 0.670 98.5
Glycerol 6.45 0.5 6.95 6.94 99.8
Acetic acid 0.013 0.010 0.023 0.025 108.7
Ethanol * 9.16 1 10.16 10.18 100.2
* Expressed in %v/v
Reprinted from Reference [ 40] with permission from Preston Publications, A Division of Preston Industries, Inc.
Page 27
TABLE 2.10. Composition of Sugars (%) in the Various Products during Processing, Reported on an As-Consumed
Basis.
Fructose Glucose Sucrose Maltose Lactose
Baking Powder Biscuit

End of mix ND ND 2.61a* ND 0.76a
Crumb ND ND 2.55a ND 0.67a
No-Time Bread
End of mix 1.34a 1.50a ND 0.72a ND
End of proof 1.32a 0.92b ND 1.48b ND
Crumb 1.31a 0.62b ND 2.01c ND
Straight-Dough Bread
End of mix 1.31a 1.28a ND 0.52a 0.66a
End of fermentation 1.15a 0.67b ND 1.72b 0.59a
End of proof 0.52b 0.10b, c ND 1.88b 0.49a
Crumb 0.44b 0.06c ND 2.04b 0.52a
Sponge-and-Dough Bread
End of sponge mix 0.09a 0.09a ND 0.86d ND
End of sponge
fermentation 0.00a 0.00a ND 0.00a ND
End of dough mix 1.11b 1.53d ND 0.25c ND
End of dough mix
fermentation 1.35c 1.62d ND 0.23b,c ND
End of proof 1.42c 1.33c ND 0.08a, b ND
Crumb 1.06b 1.06b ND 0.11a, b ND

* Means followed by the same letter within columns of the same product are not significantly different at p = 0.05.
The gas-liquid chromatographic analysis of nonvolatile sugars can be carried out only after increasing
the volatility of sugars with derivatization [46 ]. Because the derivatization step is sometimes time-
consuming and of poor reproducibility, GC has not been used frequently in the analysis of various food
products such as wine [ 47 ], must [48 ], and starch [ 49 ]. The GC analysis of carbohydrates was applied for
the determination of the authenticity of honey [ 50 ]. For the development of a reliable method to detect
added sweeteners in honey, 68 authentic honey samples and five commercial invert sugar samples were
analyzed by capillary gas chromatography. The samples were freeze-dried and derivatized by adding
0.5 mL silylation agent (Tri-Sil Z) and heating the mixture at 80°C for 1 h. The GC capillary column
was 30 m × 0.25 mm I.D. DB-5 (film thickness 0.25 µm). The injector temperature was 250°C, and the
injected volume was 1 µL (spitless injection mode). The carrier gas was helium at a flow rate of 0.75
mL/min. The temperature program was as follows: 210°C for 15 min, 1°C/min to 290°C, 290°C
maintained for 20 min. A flame ionization detector was used at 300°C. The mean and ranges for the
macrocomponents in the honey samples are compiled in Table 2.11.
Page 28
TABLE 2.11. Means and Ranges for Moisture, Glucose (G), Fructose (F), Sucrose, and
F/G Ratio.
Analyte Mean Range
Moisture, % 17.2 14.1–21.9
Glucose, % 32.3 24.9–40.0
Fructose, % 38. 534.0–46.4
Sucrose, % 1.9 0.1–4.9
F/G ratio 1.20 0.91–1.61
The chromatograms of a pure honey and a commercial beet invert syrup are shown in Figures 2.10 and
2.11. Under these GC conditions the derivatized oligosaccharides elute after 30 min (disaccharides 40-
64 min; tri- and tetrasaccharides >80 min). Disaccharides eluting at 43, 46 and 47 min were identified
as sucrose, turanose, and maltose, respectively. It was established that the oligosaccharide composition
of the honey samples depended considerably on the pH, mineral content, storage time, temperature, and
so on. Two characteteristic oligosaccharide peaks were found on the chromatograms of sweeteners
(57.6 and 57.9 min elution time), which were absent in 46 honey samples and were present at very low
concentrations in the other honey samples. It was stated that the adulteration of honey with 5%
sweetener can be significantly detected by measuring the peak area of these two oligosaccharides.
2.1.2—
Oligo- and Polysaccharides, Decomposition Products
The number of studies dealing with the chromatographic separation and quantitative determination of
oligo- and polysaccharides in various foodstuffs is relatively low. Although the number of
monosaccharides is fairly limited in foodstuffs, a huge amount of structurally different oligomers and
polymers can exist together in a sample. Owing to their similarity, the separation of oligo-and
polysaccharides is very complicated and up until now not entirely solved. Because of the high
molecular mass, gas chromatographic methods cannot be used for such a type of analysis.
Various HPLC methods have been preferentially used for the separation of oligo- and polysaccharides.
Thus, HPLC using an anion-exchange column and pulsed amperometric detector was used for the
determination of the oligosaccharide profile of honey [ 51 ] and beet medium invert syrup [ 52 ], and for
the detection of an inexpensive sweetener addition for grapefruit juice [ 53 ]. The mono- and
oligosaccharide content of beer was also measured with HPLC [ 54 ]. Samples were prepared by
removing the carbon dioxide under gentle shaking and then injecting 0.5 mL beer into a C18 cartridge
and washing the cartridge with water to a final volume of 10 mL.
Page 29
Figure 2.10.
Capillary gas chromatogram of a pure honey sample (Geographical origin = North
Dakota, Botanical origin = alfalfa/clover).
(Reprinted from Reference [ 50] with permission, copyright
© 1995, by AOAC International.
Mono- and oligosaccharides were separated on a polymeric amino column (250 × 4.6 I.D.)
thermostatted at 40°C. The gradient elution was 70% acetonitrile in water for 5 min, 50% acetonitrile in
20 min, and 50% acetonitrile for 5 min. Nitrogen was used as the evaporation gas (pressure 1.5 bar) for
the evaporative light scattering detector (detector voltage and temperature being
Figure 2.11.
Capillary gas chromatogram of a commercial beet invert syrup.
© 1995, by AOAC International.
Page 30
600 V and 90°C, respectively). Mono- and oligosaccharides in beer were well separated in this HPLC
system (Figure 2.12). The chromatogram indicates that oligomers with a higher number of monomeric
units are also present in beer; however, they were not identified because of the lack of appropriate
standards. The method showed only limited precision, and the coefficient of variation of the linearity of
the detector response varied between 6.2 and 11.1 for the different solutes. The recoveries were high,
but its relative standard deviation (SD) was 5.5-23.6%. The range of carbohydrate contents in 48
Finnish and 48 foreign beers is compiled in Table 2.12. The data in Table 2.12 indicate that the
carbohydrate content of beers shows a high variation according to the type of the beer. Maltotetraose
was found to be the most abundant carbohydrate in beers. The detector response of mono- and
disaccharides is given in Table 2.13. The data clearly show that the detector response depends
considerably on
Figure 2.12.
Chromatogram of a beer sample obtained with evaporative light
scattering detection: 1 = fructose, 2 = glucose, 3 = maltose,
4 = maltotriose, 5 = maltotetraose, 6 = maltopentaose,
7 = maltohexaose, 8 = maltoheptaose.
(Reprinted from Reference [ 54] with permission from
the Institute of Brewing, London.)
Page 31
TABLE 2.12. Carbohydrate Contents in 48 Finnish and 48 Foreign Beers (g/L).

Sugar Finnish Foreign
Fructose 0–tr * tr –5.4
Glucose 0–1.2 tr –5.3
Maltose tr–0.5 0.1–5.2
Maltotriose 0.4–3.6 0.5–7.0
Maltotetraose 1.9–3.5 0.7–8.4
Maltopentaose 0.3–2.0 tr –4.7
Maltohexaose 0–1.5 tr –6.4
Maltoheptaose 0–0.3 tr –2.3

* tr = trace amount.
Reprinted from Reference [ 54] with permission from the Institute of Brewing, London.
the character of the carbohydrate molecule influencing the detection limit. Because of its higher
sensitivity and good separation power, the method was proposed for the separation and quantitative
determination of mono- and oligosaccharides in beer.
Not only gas chromatography, but also HPLC-PAD, was used for the verification of the authenticity of
honey [55 ]. Honey samples (0.8 ± 0.15 g) were dissolved in water; 400 µL of a 20 mg/mL aqueous
maltotetraose internal standard was added and the mixture filled up to 20 mL with distilled water. Prior
to HPLC analysis, samples were purified on anion-exchange resin.
TABLE 2.13. Relative Response (glucose = 1.0) to Mono- and Oligosaccharides Obtained with
Evaporative Light Scattering Detector. *
Sugar Retention Time (min) Relative Response
Fructose 5.2 1.15
Galactose 5.8 0.34
Glucose 6.0 1.0
Lactose 7.8 0.64
Maltose 8.0 0.60
Mannose 5.8 0.25
Sucrose 7.2 1.01
Sorbitol 5.4 1.26
Xylitol 5.0 1.36
Xylose 5.1 0.43
Xylulose 5.0 0.57

*Each sugar was chromatographed separately by isocratic elution adjusting the amount of acetonitrile in
water so that the retention time of each sugar was between 5 min and 8 min.
Page 32
Oligosaccharides were separated on two Dionex PA1 guard columns (50 × 4.6 mm I.D.) and a Dionex
PA100 column (250 × 4.6 mm I.D). The chromatographic conditions are listed in Table 2.14. Eluents
were prepared quickly to decrease the possibility of the absorption of atmospheric carbon dioxide. Ater
injection, the eluent passes through the guard columns, and it is directed to the waste for 3 min. During
this time, the majority of monosaccharides elute from the guard column and do not interfere with the
separation of the oligosaccharide fraction.
The HPLC profile of oligosaccharides in honey was characteristic for the type of honey, the peak of
maltotetraose being well separated from the peaks of other oligosaccharides (Figure 2.13). Forty
different oligosaccharide peaks were observed in the 91 authentic honey samples. Since standard
compounds were not available for many of the oligosaccharides, peaks were quantified relative to the
peak height of maltotetraose. Canonical discriminant analysis was used for the classification of honey
samples according to the quality and quantity of oligosaccharides. It was established that the
determination of the oligosaccharide profiles may help the identification of honey types. Similar
methods have been used previously for the determination of honey authenticity, too [ 56 ]. Slightly
different methods were used for the detection of orange juice adulteration with beet medium sugar [ 57 ]
and for the determination of the addition of high-fructose corn syrup and beet medium invert syrup to
maple syrup [ 58 ].
TABLE 2.14. HPLC-PAD Gradient System for the Separation of Honey Oligosaccharides.
0.1 M Sodium
Time after %A 0.1 M Sodium Hydroxide and 0.1 M %C 0.3 M Sodium
Line Injection Flow (mL/min) Hydroxide Sodium Acetate Hydroxide
1 0.0 0.75 100 0 0
2 10.0 0.75 100 0 0
3 45.0 0.75 0 100 0
4 46.0 0.75 0 100 0
5 47.5 1.50 0 0 100
6 55.5 1.50 0 0 100
7 57.0 1.50 100 0 0
8 65.0 1.50 100 0 0
9 70.0 0.75 100 0 0
10 75.0 0.75 100 0 0

Mobile phase composition and flow rate changes between adjacent lines were linear with respect to time.
Reprinted from Reference [ 55], copyright © 1995. With kind permission from Elsevier Science—NL, Sara
Page 33
Figure 2.13.
Oligosaccharide profiles of three typical unifloral honeys: (A) bramble, (B) ling,
and (C) oil seed rape.
(Reprinted from Reference [ 55], copyright © 1995. With
kind permission from Elsevier Science—NL, Sara Burgerhartstraat 25,
Page 34
Diluted samples of 80 pure maple syrups, high-fructose corn syrup (HFCS), beet medium invert syrup
(BMIS), and maple syrups intentionally adulterated with 5%, 10%, and 20% HFCS and BMIS were
purified by passing through an AG 50W-X8 cation-exchange, an AG1 -X8 anion-exchange column, and
a C18 cartridge. Separation was carried out on a CarboPac PA1 column (250 × 4 mm I.D.) coupled with
a CarboPac PA1 guard column 50 × 4 mm I.D.). The gradient program is shown in Table 2.15, and the
flow rate was 1 mL/min. Like the oligosaccharide profiles of pure maple syrup, HFCS and BMIS
differed considerably; this difference was succesfully used for the detection of HFCS and BMIS in
maple syrup (Figures 2.14 and 2.15). The reproducibility of retention times and peak areas of six maple
syrup samples intentionally adulterated with HFCS or BMIS are given in Table 2.16. The relative
standard deviations of the HPLC characteristics of the marker peaks were very low, indicating the good
reproducibility of the method. It was stated that the method is suitable for the detection of the
adulteration of the maple syrup with 5% of HFCS and BMIS.
Various HPLC methods have been frequently used for the determination of the carbohydrate content of
legume seeds [ 59 ]. Thus, the effect of germination on the oligosaccharide content of lupin species [ 60 ]
and lentils [ 61 ], the influence of soaking and cooking on the carbohydrate composition of legume [ 62 ],
and the impact of processing on the carbohydrate content of lentils have been elucidated [ 63 ]. The
performance of a reversed-phase (RP) column + refractive index detector and that of an anion-exchange
column + pulsed amperometric detector was compared in the analysis of oligosaccharides in lentils [ 64 ].
Single embryos of the Syrian Local Large (SLL) genotype of lentil (Lens culinaris) were ground, and
the flour was extracted with 5 mL of boiling 80% ethanol under reflux for 15 min and then cooled and
centrifuged. The procedure was repeated twice, and the extract was collected and concentrated in an
TABLE 2.15. HPLC-PAD Elution Program for Oligosaccharide Separation.

Composition*
Time (min) %A %B %C
0.00 100 0 0
8.00 100 0 0
40.00 0 100 0
41.00 0 0 100
70.00 0 0 100
71.00 100 0 0
116.00 100 0 0
*%A, 100 mM NaOH (sodium hydroxide); %V, 100 mM NaOH/250 mM NaOAc (sodium acetate; %C, 300 mM
NaOH. All gradients were linear.
Reprinted from Reference [ 58] with permission, copyright © 1995, American Chemical Society.
Page 35
evaporator. The RP -HPLC system consisted of a C18 precolumn (40 × 3.2 mm I.D.), a
µBondapack/carbohydrate column (300 × 3.9 mm I.D.) thermostatted at 35°C and a differential
refractometer detector. The isocratic eluent was acetonitrile:water, 75:25. The other HPLC system used
a CarboPac PA-100 guard column (25 × 3 mm I.D.), a CarboPac PA-100 anion-exchange column (250
× 4.0 mm I.D.) at room temperature. The eluent was 145 mM aqueous sodium hydroxide solution, and
the flow rate was set to 1 mL/min. The PAD conditions were: E1 = 0.05 V (t1 = 300 msec); E2 = 0.60 V
(t 2 = 120 msec); E3 = -0.60 V (t3 = 300 msec). Typical chromatograms showing the separation of
standard mixtures and lentil samples on RP and anion-exchange columns are presented in Figures 2.16
and 2.17. The chromatograms unambigously prove that both HPLC systems are suitable for the
separation of mono- and oligosaccharides of cotyledon lentil samples. Because of the different
mechanism of retention, the retention order of carbohydrates is different on the columns. The
chromatographic parameters of the HPLC systems are shown in
Figure 2.14.
HPLC-PAD chromatogram of a maple syrup (64; 5.5° Brix) and the same sample
intentionally adulterated with HFCS 55 at levels of (A) 5%, (B) 10% and
(C) 20% ( * indicates the fingerprint oligosaccharide used as a marker).
(Reprinted with permission from Reference [ 58], copyright
© 1995, American Chemical Society.)
Page 36
Figure 2.15.
HPLC-PAD chromatogram of a maple syrup (17; 18° Brix) and the same
sample intentionally adulterated with BMIS at levels of (A) 5%, (B) 10%
and (C) 20% ( * indicates the fingerprint oligosaccharide used as a marker).
(Reprinted with permission from Reference [ 58], copyright
Page 37
TABLE 2.16. Reproducibility of Retention Times and Peak Areas of Six Maple Syrup Samples Intentionally
Adulterated with 10% HFCS and 10% BMIS.
HFCS Fingerprint Peak BMIS Fingerprint Peak
Sample tr* Area ** tr Area
3 39.04 0.58 39.66 0.60
3 39.18 0.58 39.51 0.51
31 38.91 0.57 40.01 0.60
31 38.79 0.58 40.12 0.59
45 38.77 0.57 40.06 0.59
45 38.60 0.57 40.31 0.59
59 38.92 0.58 40.03 0.60
59 38.87 0.57 40.17 0.62
71 39.00 0.58 40.09 0.60
71 38.61 0.57 40.02 0.61
79 38.86 0.58 40.37 0.60
79 38.43 0.59 40.07 0.60

* Retention time (min).
** × 10 6 µV × s.
Reprinted from Reference [ 58], copyright © 1995, American Chemical Society.
Table 2.17. Although both methods were suitable for the separation of the carbohydrates in cotyledon
lentil, because of its higher sensitivity, the use of the anion-exchange column connected with PAD was
proposed for the analysis of various parts of seeds, embryos, and testas of lentils.
Since the number of the individual carbohydrate polymers in a foodstuff is extremely high, the analysis
of soluble carbohydrate polymers is extremely difficult. Because of the elevated molecular mass of
solutes, the analysis requires special chromatographic supports and techniques. The high similarity of
the polymers generally results in poor resolution. Sometimes the analyst is interested in the type and
relative concentration of the mono- and oligosaccharides forming the polymer and not in the separation
of the polymers themselves. In this instance the polymer can be hydrolyzed and the carbohydrates
determined by any of the methods discussed above. Thus, the free and total carbohydrate profile of
soluble coffee was determined on an anion-exchange column and PAD [ 65 ].
For the determination of the free carbohydrates in soluble coffee, an aliquot was dissolved in water,
passed through a C18 cartridge, and used for HPLC analysis. The total carbohydrate content was
determined by hydrolyzing the sample with 1.00 N HC1 in boiling water for 150 min. After hydrolysis,
the samples were filtered and passed through an anion-exchange (AE) cartridge in the silver form to
eliminate chlorides and to neutralize the hydrolysate. Coffee husks were ground and extracted with
water at 70°C for 30 min to measure the
Page 38
Figure 2.16.
Chromatograms obtained using an RP-HPLC system:
µBondapack/carbohydrate column (300 × 3.9 mm I.D.)
(Waters Associates) at 35 °C; eluent acetonitrile:water
(75:25): (a) standard solution; (b) SLL cotyledon lentil sample,
where 1 = fructose; 2 = sucrose; 3 = raffinose; 4 = stachyose;
5 = ciceritol; 6 = verbascose.
(Reprinted from Reference [ 64], by courtesy
of Marcel Dekker, Inc.)
free carbohydrates. The hydrolysis time for the determination of the total carbohydrate content of coffee
husks was increased to 240 min. Separation of carbohydrates was carried out on a CarboPac PA1
(Dionex) column (250 × 4 mm I.D.) using distilled water as eluent. The HPLC parameters are compiled
in Table 2.18. Each carbohydrate was well separated from the others, indicating the good separation
power of the method. The free and total carbohydrate profiles of a pure soluble coffee sample are
shown in Figure 2.18. The free and total carbohydrate composition of a pure soluble coffee differs
considerably. Pure soluble coffee contains a low concentration of free sugars and high quantities of total
galactose and mannose. When roasted coffee beans are
Page 39
Figure 2.17.
Chromatograms obtained using a CarboPac PA 100 column
(250 × 4.0 mm I.D.) (Dionex); eluent, 145 mM NaOH: (a) standard
solution; (b) SLL cotyledon lentil sample, where
1 = fructose; 2 = sucrose; 3 = raffinose;
4 = stachyose; 5 = ciceritol; 6 = verbascose.
(Reprinted from Reference [ 64], by courtesy of Marcel Dekker, Inc.)
coextracted with coffee husks and/or parchments, the concentration of free mannitol, free fructose, free
glucose, total glucose, and total xylose increases considerably, as demonstrated in Figure 2.19. The
adulteration of pure soluble coffee with coextracted cereals, malt, or caramelized sugar results in an
increased quantity of free fructose, free glucose, free sucrose, and total glucose. The chromatogram of a
free and total carbohydrate profile of soluble coffee containing added cereals or caramelized sugar is
shown in Figure 2.20.
The free and total carbohydrate content of 63 soluble coffee samples and 11 parchments/husks can be
found in the original publication [65 ]. The results obtained with this method were compared with those
of an HPLC method using an amino-bonded silica column and those of enzymatic hydrolysis.
Significant differences were found only in the case of free arabinose (0.10%) and
TABLE 2.17. Chromatographic Parameters in the Detection of Low Molecular Mass Sugars by RP-HPLC and HPLC -PAD.
Parameters
RP -HPLC HPLC-PAD
Detection Detection
Sugars tR k'** N*** Limit **** tR k' N Limit
Fructose 2.90 1.38 1098 1300 5.40 1.97 5184
Sucrose 5.00 3.10 1600 450 7.90 3.34 3994
Raffinose 9.00 6.38 2304 170 15.10 7.30 8635
Stachyose 16.50 12.52 3951 1380 17.00 8.34 6400

*RP-HPLC: µBondapack/carbohydrate column (300 × 3.9 mm I.D.) (Waters Associates) at 35°C; eluent acetonitrile:water (75:25);
HPLC-PAD: CarboPac PA100 (250 × 4.0 mm I.D.) (Dionex); eluent, 145 mM NaOH.
** k' = (tR - tM )/TM , where tR = retention time of solute and tM = retention time of solvent front.
*** N = number of theoretical plates = 16 [tR/W]2, where tR represents retention time and W represents peak width.
**** µg/mL and ng/mL for RP-HPLC and HPLC-PAD, respectively.
Reprinted from Reference [ 64], by courtesy of Marcel Dekker, Inc.
TABLE 2.18. Retention Time, Relative Response Factor, Linearity Range, and Detection Limit
of Carbohydrates.
Relative
Retention Response Linearity range Detection Limit
Carbohydrate Time (min) Factor* (µg/mL) ** (ng) ***
Mannitol 4.2 1.14 0.5–2000 10
Fucose 6.6 1.00 1.0–2000 20
Rhamnose 15.2 0.77 1.0–2000 20
Arabinose 16.1 1.06 1.0–2000 20
Galactose 21.2 1.01 1.0–2000 20
Glucose 24.3 1.00 1.0–2000 20
Sucrose 26.7 0.64 4.0–1000 80
Xylose 29.3 0.98 1.0–2000 20
Mannose 2.1 0.98 2.0–2000 40
Fructose 41.4 0.36 4.0–2000 80
Ribose 45.6 0.87 4.0–2000 80

* Response factor is relative to glucose.
** r = 0.9999 for all listed carbohydrates.
*** Detection limit was measured on a standard solution and defined arbitrarily as the amount of
carbohydrate on the column that produced a signal-to -noise ratio of 5.
Page 41
Figure 2.18.
Chromatogram of typical (A) free and (B) total carbohydrate profile of a pure
soluble coffee.
(Reprinted from Reference [ 65 ] with permission, copyright © 1995, by AOAC
International.)
Page 42
Figure 2.19.
Chromatogram of typical (A) free and (B) total carbohydrate profile of a
soluble coffee containing coffee husks or parchments.
© 1995, by AOAC International.)
free mannose (0.05%); however, the precision of the present method was clearly superior. The mean
repeatabilities were 0.03% (CV = 2.23%) and 0.11% (CV = 1.70%) for free and total carbohydrates,
respectively. It was concluded from the data that this HPLC-PAD method is suitable for the routine
analysis of the free and total carbohydrate content of soluble coffee and for
Page 43
Figure 2.20.
Chromatogram of typical (A) free and (B) total carbohydrate profile
of a soluble coffee containing cereals or caramelized sugar.
purity control. The repeatability of the method was evaluated in an interlaboratory study including 11
laboratories in four countries [ 66 ]. The reproducibility relative standard deviations were 9.9-59.5% for
mannitol, 35.6-72.6% for fucose, 4.9-21.1% for arabinose, 4.1-13.0% for galactose, 6.1-24.3% for
glucose, 10.0-41.6% for sucrose, 20.2-37.7% for xylose, 10.6-40.0% for mannose, 15.5-71.7% for
fructose, and 17.8-97.9% for ribose. It was concluded
Page 44
from the results of the interlaboratory study that the precision of the method is good. It allows the
reproducible separation of all major sugars present in soluble coffee; therefore, it is suitable for routine
analysis.
Dietary fibers (sometimes referred to as nonstarch polysaccharides) exert a beneficial effect on human
health. Because of their insolubility in common solvents, they cannot be analyzed as polymers by any
traditional chromatographic methods. HPLC has been used for the determination of the neutral
saccharides in neutral detergent fiber [ 67 ]. The composition of fibers of wheat bran and apple was
measured with anion-exchange HPLC connected with PAD [ 68 ]. The effect of processing conditions on
the composition of the dietary fiber of carrots was studied in detail [ 69 ].
Fiber samples were prepared from freeze-dried raw and autoclaved (121°C, 15 min) carrots. Samples
were dispersed in 2 mL of dimethylsulfoxide and hydrolysed with α-amylase and pullulanase. After
hydrolysis, the starch-free samples were washed with ethanol and acetone. The dry residue was
hydrolysed with 0.3 mL of 12 M H2SO 4 at 40°C for 2 h under continuous stirring (primary hydrolysis);
then 8.4 mL of deionized water was added to the mixture and stirred for 3 h in a boiling water bath
(secondary hydrolysis). The hydrolysate was purified by passing 5 mL of sample through a Bio-Rad
resin (40 × 9 mm I.D). After elution, the column was washed with 3 × 5 mL of water. The combined
eluates were dried, redissolved in 2 mL of water and passed through a C18 cartridge. Separation was
carried out on an HPX-87P column (300 × 7.8 mm I.D., particle size 9 µm) thermostatted at 85°C.
Eluent was deionized water, and the flow rate was 0.5 mL/min. Carbohydrates were detected with a
refractive index detector. Galacturonic acid was extracted with hot oxalic acid—ammonium oxalate (pH
4)—and determined with spectrophotometry. Except rhamnose and galactose, carbohydrates were well
separated under these HPLC conditions. The recoveries were 89.21% for cellobiose, 91.24% for
glucose, 88.39% for xylose, 89.34% for galactose/rhamnose, 95.28% for arabinose, and 98.12% for
mannose.
The composition of dietary fiber of carrot before and after processing is given in Tables 2.19 and 2.20.
The data show that glucose is the most important monosaccharide in both raw and processed carrot; the
amount of galacturonic acid is the highest in the samples. The quantity of the individual carbohydrate
increases under heat treatment; however, this phenomena may be caused by the loss of carbohydrates
during processing. It has been stated that the HPLC-PAD method developed for the determination of
dietary fiber is rapid and easy to carry out.
Because of the inherent difficulties of polymer separation, the number of papers dealing with the
chromatographic separation of carbohydrate polymers in their original polymeric form is surprisingly
low. A two -column, size exclusion HPLC method was used for the separation of amylopectin from
amylose [ 70 ]. The HPLC separation of debranched starches was carried out by size
Page 45
TABLE 2.19. Monomeric Composition of Polysaccharides That Constitute Dietary Fiber in Raw
Carrot (grams per 100 g dry matter).
Constituent Mean* RSD**
Cellobiose 0.5567 0.5064
Glucose 3.9375 0.1844
Xylose 0.4180 0.0947
Galactose/rhamnose 2.2252 0.1587
Arabinose 1.6330 0.2718
Mannose 0.3546 0.1153
Galacturonic acid 5.1833 0.0868
Total 14.3083 0.0936

* n = 18.
Reprinted from Reference [ 69], copyright © 1994. With Kind permission from Elsevier
Science—NL, Sara Burgerhartstraat 25, 1055 KY Amsterdam, The Netherlands.
exclusion chromatography, too [ 71 ]. Starch samples were defatted by overnight extraction with ethanol
in a Soxhlet apparatus and then dried under vacuum at room temperature. Debranching of starches was
carried out by suspending 50 mg of starch in 1 mL of 0.25 M sodium hydroxide and by heating the
mixture at 100°C for 5 min under continuous stirring. After cooling, 32 µL of glacial acetic acid and 4
mL of 0.05 M sodium acetate (pH 4.0) were added. The mixture was incubated with 25 µL isoamylase
at 37°C for 2 h. After completing the hydrolysis, the sample was heated at 100°C for 20 min in order to
denaturate the enzyme. The reaction mixture was centrifuged (12,000 g; 10 min), passed through a
mixed bed, ion-exchange resin, and filtered.
The HPLC system used for the analysis of debranched starches consisted of Waters Ultahydrogel
columns (UHG-120, UHG-250, and UHG-500) and a
TABLE 2.20. Monomeric Composition of Polysaccharides That Constitute Dietary Fiber in

Processed Carrot (grams per 100 g dry matter).
Constituent Mean* RSD*
Cellobiose 0.8123 0.3882
Glucose 5.2812 0.1158
Xylose 0.5399 0.5051
Galactose/rhamnose 3.9278 0.2910
Arabinose 3.3835 0.2773
Mannose 0.4130 0.1770
Galacturonic acid 5.8344 0.1121
Total 20.1921 0.0990

* n = 18.
Reprinted from Reference [ 69], copyright © 1994. With kind permission from Elsevier Science—
NL, Sara Burgerhartstraat 25, 1055 KY Amsterdam, The Netherlands.
Page 46
refractive index detector. Support of the columns was a hydrophilic crosslinked methacrylate matrix
with molecular mass cut-offs of 5.80 kDA and 400 kDa. An aqueous solution of 0.05 M ammonium
acetate was the eluent, and the flow rate was 0.5 mL/min. The hydrolysis of debranched starches by β-
amylase was studied by adding β-amylase (500 units/mL reaction solution) to an aliquot of starches
after denaturation of the isoamylase and before the treatment on ion-exchange resin. After 1 h
incubation at 37°C, an aliquot of the reaction mixture was used for HPLC analysis. The same amount of
β-amylase was added to the rest and the hydrolysis was continued for another hour.
The denaturation of the enzyme and the desalting of the sample were carried out as described above.
Two Bio-Rad HPX-42A columns connected in series and the same refractive index detector were used
for the analysis of the debranched starches treated with β-amylase. Columns were thermostatted at 80°
C, and the eluent was deionized water at a 0.6 mL/min flow rate. Debranched starches were separated
into two fractions on UHG columns, as demonstrated in Figure 2.21. It was established that peak I
corresponds to amylose with a high degree (500+) of polymerization, whereas peak II contains chains
with less than 50 glucose units from the amylopectin. The best separation was achieved on the UHG-
250 column. Interestingly, the first peak was absent in debranched waxy starches isolated from maize
and barley (Figure 2.22). The ratio on the higher molecular weight fraction (peak I) was higher in wheat
starch, barley starch, and normal maize starch, and the results of HPLC analysis correlated well with
those obtained by the traditional colorimetric iodine procedure. The chromatographic profile of starches
depended highly on the origin and the mode of preparation, as demonstrated in Figure 2.23. The
separation of the hydrolysis products of starch treated with β-amylase revealed that the end product
contains mainly maltose, a small amount of maltotriose, and 4-10% of high molecular weight fraction
(Figure 2.24). It was found that the repeatability of the method is better than that of the
spectrophotometric iodine binding, and the results were not affected by the lipid content of the starch.
The reactions occurring during cooking and baking of foods are of great importance for the production
of aroma, taste, and color; however, the same reactions may cause a reduction in nutritive value and the
formation of toxic substances. Carbohydrates decompose under heat treatment and can form various
volatile [ 72 ] and nonvolatile products [ 73 ] which considerably influence the organoleptic characteristics
of food products [ 74 ,75 ]. Gas chromatography has been frequently used for the study of volatile
decomposition products [ 76 ,77 ]. Solid-phase microextraction (SPME) combined with gas
chromatography-mass selective detection was applied for the study of the products of the Maillard
reaction using model compounds [78 ]. It was established that, not only the chemical structure of the
compounds, but also the composition of the accompanying matrix, have a marked effect on the
recovery. Gas chromatography-mass Spectrometry (GC-MS) was also used for the separation and
identification of
Page 47
Figure 2.21.
Separation of wheat starch debranched with isoamylase
on (a) UHG-120, (b) UHG -250, and (c) UHG-500
columns. An aliquot of 20 µL containing 10 mg/mL
debranched starch was applied to each column. For all
columns, the mobile phase was 0.05 M ammonium
acetate at a flow rate of 0.5 mL/min.
Wiley—VCH and the corresponding author.)
Page 48
Figure 2.22.
Separation of debranched waxy starches from (a) maize and (b) barley on a
UHG-250 column. An aliquot of 20 µL containing 10 mg/mL debranched starch was
applied to each column. For all columns, the mobile phase was
0.05 M ammonium acetate at a flow rate of 0.5 mL/min.
Page 49
Figure 2.23.
Separation of debranched starches from (A) pea (45%
amylose), (B) amylomaize (50% amylose), and (C)
amylomaize (80% amylose) on a UHG-250 column.
An aliquot of 20 µL containing 10 mg/mL debranched
starch was applied to each column. For all columns, the
mobile phase was 0.05 M ammonium
acetate at a flow rate of 0.5 mL/min.
Page 50
Figure 2.24.
Separation on a Bio-Rad HPX-42A column of (a)
wheat starch, (b) wheat amylose, and (c) wheat
amylopectin digested with iso-amylase followed
by β-amylase. An aliquot of 20 µL containing 10 mg/mL
debranched polysaccharide, which had been digested
with β-amylase, was applied to the column and
eluted with water at 0.6 mL/min.
Page 51
the degradation products of starch-based polymers [ 79 ]. Methylglyoxal formed during the heat
treatment of carbohydrates can be determined by both GC [ 80 ] and HPLC methods [ 81 ]. Fructose
solutions were heated at 130, 140, and 150° C for 5, 10, 20, 40, and 80 min. Samples (1 mL, containing
1-100 µg methylglyoxal) were reacted with 60 µL of O-phenylenediamine solution for 5 min under
buffered conditions (pH 6 adjusted with phosphate buffer). The stable methylchinoxalin derivative was
separated by HPLC using a Partisil ODS2 column (250 × 4.6 mm I.D.) and acetonitrile:water (1:1)
isocratic eluent. The flow rate was 2 mL/min, and the detection wavelength was set to 313 nm. It was
established that the formation of methylglyoxal during heat treatment of an aqueous fructose solution
follows a reaction order of 1.5, and the energy of activation is relatively low (0.5 kJ/mol).
2.2—
Separation of Lipids
Oils and fats are primary sources of lipids, which provide a considerable portion of the energy supply in
human diets.
2.2.1—
Fatty Acids
The composition of fatty acids in various oils and fats has a marked impact on nutritional value. Thus,
monounsaturated fatty acids generally present in cis configurations reduce the intake of saturated fat,
decreasing in this manner the serum level of the atherogenic low-density lipoprotein cholesterol.
However, during processing, the cis fatty acids can be converted into trans isomers, which exert an
unfavorable effect on the serum lipoprotein composition. The composition of fatty acids influences not
only the nutritional value of the foodstuffs, but also the application parameters.
The beneficial or adverse biological activities of chemically different fatty acids make the analysis of
fatty acids in many areas of food science and technology important. Gas chromatography is the method
of preference for the separation and quantitation of fatty acids. To increase the volatility and to improve
separation efficiency, fatty acids are generally derivatized before GC analysis. Because of its simplicity
and low cost, methylation is frequently used for the derivatization of fatty acids. Because of the higher
separation capacity, capillary column is preferentially used in the analysis of fatty acids [ 82 ]. Because of
the considerable nutritional importance, many efforts have been devoted to the separation and
quantitative determination of trans isomers of fatty acids [ 83 ]. The geometrical isomers of α-linolenic
acid (A -LAGIs) in heat-treated and deodorized rapeseed and soybean oils were separated by capillary
GC [ 84 ]. Methylation was carried out with 10% BF 3 in methanol. A fused-silica capillary column (CP
Sil 88, 50 m × 0.25 mm I.D., 0.20 µm film thickness) was used for isothermal separation. The column
temperature varied
Page 52
between 155° C and 185° C; the temperature of the split injector port and the flame ionization detector
(FID) was held at 250° C. Helium was the carrier gas (inlet pressure: 120 kPa). It has been established
that the retention order of fatty acid methyl ester markedly depends on the column temperature (Figure
2.25). The chromatograms clearly show that the separation of fatty acid methyl esters is incomplete at
higher temperatures, resulting in erroneous quantification of the individual fatty acid fractions. The use
of a column temperature under 160° C is proposed for such a type of fatty acid analysis. A similar
method was used for the identification of minor C18 triene and conjugated diene isomers in
hydrogenated soybean and margarine [ 85 ].
A combined GC-IR method was developed for the determination of trans unsaturation in partially
hydrogenated vegetable oils (PHVOs) [ 86 ]. The methyl esters of fatty acids were separated on a fused-
silica capillary GC column (100 m × 0.25 mm I.D., coated with SP -2560) using an FID. The
chromatographic conditions were split ratio, 1:100; injector, 225° C; detector, 250° C; column
temperature gradient initial, 150° C; program rate 1.0° C/min;
Figure 2.25.
Influence of temperature on the elution order of cis-11 20:1 acid and linolenic
acid geometrical isomers. Selected chromatograms obtained with a CP Sil 88
capillary column (50 m × 025 mm I.D., 0.20 µm film, Chromapack, Middelburg,
The Netherlands) of fatty acid methyl esters prepared with a sample of
commercial deodorized rapeseed oil from St. Hyacinthe, Canada. Inlet pressure
of the carrier gas (helium): 120 kPa. Temperature of the column as indicated on
chromatograms. All injections at the same load. Identification of peaks: (a)
trans-9, cis -12, trans-15 18:3 acid; (b) cis-9, cis-12, trans-15 18:3 acid;
(c) cis-9, trans-12, cis-15 18:3 acid; (d) trans-9, cis-12, cis-15 18:3
acid; (e) cis-9, cis-12, cis-15 18:3 acid. 20:1 acid is the cis-11 isomer.
(Reprinted from Reference [ 84] with permission from AOCS Press.)
Page 53
final, 200°C; final hold, 20 min. A characteristic chromatogram is shown in Figure 2.26. The method
separates well the majority of cis and trans fatty acids, and it is suitable for the determination of the
trans fatty acid content of partially hydrogenated vegetable oils and terrestrial animal fats.
The performance of GLC, thin-layer chromatography followed by gas liquid chromatography (GLC +
TLC), Fourier transform infrared spectroscopy (FT-IR), and optothermal window (OW) (open
photoacoustic cell) was
Figure 2.26.
C18 region of the gas chromatogram of the fatty acid methyl esters from
partially hydrogenated soybean oil, using 100 m × 0.25 mm I.D. fused-silica
capillary column coated with SP2560. Peak identification: 1 = 18:0; 2 = 18:1
6-8t; 3 = 18:1 9t; 4 = 18:1 10t; 5 = 18:1 11t; 6 = 18:1 12t; 7 = 18:1 6-8c + 18:1 9c
(major component) + 18:1 13-14t; 8 = 18:1 10c + 18:1 15t; 9 = 18:1 11c;
10 = 18:1 12c; 11 = 18:1 13c; 12 = 18:2n; 13 = 18:1 14c + 18:1 16c; 14 = 18:2n;
15 = 18:1 15c; 16,17 = 18:2n; 18 = 18:2 9t,12t; 19,20 = 18:2 9c,13t + 18:2 8t,12c;
21 = 18:2 ct/tc; 22 = 18:2 9c,12t; 23 = 18:2 8c,13c; 24 = 18:2 9t,12c;
25 = 18:2 9t,15c + 18:2 10t,15c + 18:2 9c,13c; 26 = 18:2 9c,12c (linoleate);
27 = 18:2 9c,15c; 28 = not identified; 29 = 20:0; 30 = 18:3; 31 = 18:3 9c,12c,15t;
32 = 18:3 9c,12t,15c; 33 = 18:3 9t,12c,15c; 34 = 18:3 9c,12c,15c
(linoleate); 35 = 20:1 c; 36-40 = 18:2 conjugates.
Page 54
compared for the detection of the total trans fatty acid content in margarine [ 87 ]. The GC separation of
methylated fatty acids was carried out on an SIL88 column (100 m × 0.25 mm I.D.). The injector and
detector temperatures were 275° C and 250° C, respectively. The carrier gas was hydrogen (inlet
pressure = 167 kPa). The temperature program begins at 150° C, and it is raised to 155°C at a heating
rate of 2°C/min; this temperature was held constant for 60 min. Then, a temperature of 200°C was
achieved at 40°C/min. The total time of an analysis was 89 min. The total trans fatty acid content of
four samples of margarine determined by the four methods is compiled in Table 2.21. The data showed
reasonable agreement, and the relative error of each method was about 2%. The results of GLC analysis
are generally lower than those of the other methods.
This phenomenon was explained by the supposition that the peak of some trans fatty acids is marked by
the peak of the corresponding cis fatty acids. The use of FT-IR and OW techniques were proposed
when the total content of trans fatty acid is a priority. When the distribution of trans fatty acid is of
interest, the more time- and labor-consuming GLC + TLC technique is preferable. The GC analysis of
fatty acid composition combined with isotope ratio mass spectrometry (GC/IRMS) has been
successfully used for the detection of the adulteration of maize oil with less expensive oils [ 88 ]. Because
of the different photosynthetic fixation route of CO 2 in maize and in other commercial oil-producing
plants, the ratio of12 C and 13C ( δ13C value) can be used for the detection of the adulteration of maize oil.
The combined GC-isotope ratio mass spectrometry allows the simultaneous separation of fatty acids
and the determination of the δ13C value of the individual fractions. For the saponification and
methylation of the oils, about 100 µL oil was mixed with 2 mL of 0.5 M sodium hydroxide in methanol
and refluxed at 70°C for 45 min. The hydrolysates were cooled, the pH was adjusted to 3 with 1 M
HC1, and the free fatty acid was extracted three times with 5 mL of n-hexane. One milliliter of the
combined extracted was dried under nitrogen stream; 100 µL of 14% boron trifluoride was added and
heated at 70°C for 45 min. After cooling, 2 mL of water and 1 mL of diethyl ether were added to the
reaction mixture, and the fatty acid methyl esters were extracted into the organic phase. The diethyl
ether was removed with a nitrogen stream, and the rest was dissolved in
TABLE 2.21. Total Trans Fatty Acid Content (%) in Different Samples of
Margarine by Various Methods (for symbols, see text).
Margarine Sample (code) FT-IR OW GLC GLC + TLC
6337 59.3 62.2 50.5 58.3
6338 64.9 63.6 53.6 62.7
6339 42.9 42.0 35.9 41.8
6340 41.8 42.4 39.8 41.0
Reprinted from Reference [ 87] with permission, copyright © 1996, American
Chemical Society.
Page 55
n-hexane for GC analysis. A highly polar capillary column (SGE BPX-70; 25 m × 0.32 mm I.D., 0.25
µm film thickness of 70% cyanopropyl [equivalent poly(silphenylene)siloxane] was employed for the
separation. The temperature program was 80° C (1 min) to 200° C (10 min) at 4° C/min. The
combustion interface contained a ceramic furnace with a copper oxide and platinum catalyst at 850° C.
Typical traces of GC/IRMS measurements are compiled in Figure 2.27. The profiles for maize and
rapeseed oils are markedly different,
Figure 2.27.
GCC/IRMS profiles for (A) pure maize and (B) pure rapeseed oils. The
upper trace represents the instantaneous ratio of ion currents m/z45 to
m/z44 (i.e., 13CO 2 to 12CO 2 ), while the lower trace
corresponds to the m/z44 ion current.
from Elsevier Science—NL, Sara Burgerhartstraat 25,
Page 56
allowing the identification of the addition of rapeseed oil to maize oil samples. The fatty acid
composition and calculated iodine values for selected vegetable oils are listed in Table 2.22. The
following mathematical model of mixing was used for the construction of calibration curves:
where A is the added oil and M is maize oil; δ13C(A+M ) is the predicted isotope ratio of the fatty acid of
the mixed oil; δ13C(A) is the isotope ratio of the fatty acid in the added oil; δ13 C(M) is the isotope ratio of
the fatty acid in the maize oil; A is the concentration of the added oil present (%, by weight); M is the
concentration of the maize oil present (%, by weight); percent in A is the amount of the fatty acid in the
added oil; and percent in M is the amount of the fatty acid in the maize oil. It has been concluded from
the data that the GC/IRMS technique is a powerful method for the detection of adulteration of vegetable
oils. The adulteration of maize oil can be easily detected at a 5% (w/w) level.
The determination of the fatty acid composition by capillary GC has also been used for the
determination of the individual free fatty acids in cheese [ 89 ] and for the detection of milk fat
adulteration [ 90 ]. The transesterification of the samples was done by shaking a 10% solution of fat in n-
hexane with 200 µL of 2 M KOH in dry methanol for 1 min. After a further reaction time of 5 min, 0.5
g KHSO4 was added to prevent saponification. Esters were extracted with 1 mL of redistilled diethyl
ether. Separation of fatty acid methyl esters was carried out on a fused-silica capillary column (CP -Wax
58 CB, 25 m × 0.32 mm I.D., film thickness 0.2 µm). The temperature program was 35°C for 3 min and
then rising to 110°C at 10°C/min, 1 min at 110°C, rising to 230°C at 6°C/min, and finally 10 min at
230°C. A flame ionization detector and hydrogen carrier gas (35 kPa) were used. The average
concentration values of fatty acids are listed in Table 2.23. The low standard deviation of the results
indicates that the method is reliable and can be successfully used for the elucidation of the fatty acid
composition of milk fat. Linear discriminant analysis suggested that the saturated and unsaturated fatty
acids with 18 carbon atoms are the best discriminating variables for the detection of the adulteration of
milk fat. This method allowed the detection of the presence of >3% tallow, lard, olive oil, or palm oil in
milk fat samples.
In order to improve the application parameters of anhydrous milk fat (AMF), a continuous supercritical
fluid processing method was developed, and the fatty acid composition and the cholesterol content of
the fractions were determined by GC [ 91 ]. The schematic diagram of continuous supercritical fluid
fractionation system is shown in Figure 2.28. Fatty acid methyl esters were separated on a capillary
column (15 m × 0.25 mm I.D., Durabond-225).
TABLE 2.22. Fatty Acid Composition and Calculated Iodine Values for Selected Vegetable Oils.
Sample Origin C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0
Maize** nd *** 10.4 Nd nd nd 1.8 28.2 57.6 1.3 nd
Groundnut** nd 10.3 Nd nd nd 3.6 62.3 18.0 2.6 nd
Rapeseed 214 Europe <0.1 4.7 0.2 <0.1 0.1 1.4 58.7 21.3 10.2 0.6
Maize E Argentina <0.1 13.1 0.1 0.1 <0.1 2.0 34.7 48.0 0.7 0.4
Maize 15 Italy 0.1 10.7 0.2 0.1 <0.1 2.0 25.5 58.6 1.0 0.4
Maize 2 France <0.1 11.5 0.1 0.1 <0.1 1.8 25.0 58.9 0.9 0.4
* Iodine value, as calculated by AOCS Recommended Practice Cd 1c -85.

** As used in the mixing experiments.
*** nd, not determined.
Reprinted from Reference [ 88], copyright © 1995. With Kind permission from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KY
Page 58
TABLE 2.23. Fatty Acid Concentration Values (grams individual fatty acid/100 g of total fatty
acid) of 352 Austrian Milk Fat Samples.
Fatty Acid Mean Std. CV *(%) Maximum Minimum
C4:0 3.71 0.132 3.56 4.02 3.17
C6:0 2.20 0.099 4.52 2.38 1.88
C8:0 1.22 0.077 6.27 1.36 0.99
C10:0 2.63 0.235 8.95 3.07 2.03
C12:0 2.98 0.315 10.58 3.62 2.25
C14:0 10.51 0.761 7.23 11.73 8.66
C14:1 0.88 0.097 10.94 1.13 0.67
C15:0 ISO** 0.35 0.023 6.70 0.42 0.29
C15:0 AISO *** 0.55 0.037 6.74 0.69 0.48
C15:0 1.34 0.065 4.82 1.52 1.18
C16:0 30.00 2.931 9.77 34.74 25.05
C16:1 1.54 0.085 5.56 1.77 1.27
C17:0 ISO** 0.59 0.041 6.95 0.72 0.44
C17:0 AISO *** 0.50 0.039 7.74 0.84 0.43
C17:0 0.81 0.108 13.29 1.04 0.05
C18:0 9.13 0.994 10.88 11.42 7.45
C18:1 (omega)9 19.60 1.724 8.80 23.62 16.79
C18:1 (omega)7 **** 2.75 1.195 43.45 5.85 1.21
C18:2 1.21 0.115 9.54 1.93 0.88
C18:3 0.84 0.186 22.17 1.32 0.31
C18:2 CONJ§ 0.95 0.411 43.14 2.15 0.38

* CV = coefficieint of variation.
** Iso -branched fatty acid.
*** Antiiso -branched fatty acid.
**** Mixture of cis and trans isomers.
§ 9 =cis, 11 = trans-octadecadienoic acid.
The initial column temperature was 60°C for 2 min and then was raised to 220°C at 4°C/min and held
for 10 min. The injector and detector temperatures were 200°C and 240°C, respectively. The effect of
extraction conditions on the composition of AMF is demonstrated in Table 2.24. The fatty acid
composition did not show marked changes between the fractions, suggesting that this experimental
approximation cannot be used for the separation of fractions with a given fatty acid composition. AMF
was separated by extraction and four-step fractionation with SC -CO 2 at 40°C and 80°C and 241 to 69
bar. The results are compiled in Table 2.25. The data indicate that SC-CO2 fractionation of AMF can
successfully modify the fatty acid composition of the fractionated products. The short- and medium-
length fatty acids are concentrated in the extracts, their ratio depending on the extraction conditions. It
has been further proposed that the use of this method can produce potentially new dairy products.
Page 59
Figure 2.28.
Schematic diagram of continuous supercritical fluid fractionation system
(AC: adsorption column; B: back pressure regulator; D: dry test meter; EC:
extraction column; EV: extraction vessel; H: heat exchanger; MV: metering
valve; P: pressure reading; PP: high pressure pump; PR: pressure regulator;
R: rotameter; RV: AMF reservoir; T: temperature reading; TK:
carbon dioxide cylinder; V: on/off valve; VC: view cell).
Various other multivariate mathematical-statistical methods such as principal component analysis,

cluster analysis and discriminant analysis have also been used for the evaluation of the authenticity of
eight different types of edible plant oils [ 92 ]. The oils were transesterified with sodium methylate, and
the methyl esters of fatty acids were separated by GC-FID using a capillary column (DB 225, 30 m ×
0.25 mm I.D., film thickness 0.25 µm). Carrier gas was helium. The concentration of the most
important fatty acids in the different oils is given in Table 2.26. Each multivariate mathematical-
statistical method differentiated exactly the various edible plant oils, indicating that these calculation
methods can help the elucidation of the authenticity of these products.
The determination of the fatty acid composition of various foodstuffs by GC has been used for the
quantitation of total fat and saturated fat content [ 93 ]. Acid hydrolysis was carried out on 2 g or 4 g of
food samples by adding 10 mL or 20 mL of 6 N HCl and 2 mL or 4 mL of ethanol, depending on the
weight of the sample. The mixture was hydrolyzed at 70-80° C for 30 min with frequent shaking. After
cooling, it was extracted with 100 mL of diethyl
Page 60
TABLE 2.24. Effects of Processing Parameters on Extraction of Anhydrous Milk Fat at 40°
C/241 Bar.
Temp
Original No Gradient Recycle Reflux
Operation AMF Treatment (40-60 °C) (R = 12.3) (R = 1.3)
S/F g/g — 48 48 46 49
AMF feed (g/hr) — 53 54 55 52
CO 2 feed (g/hr) — 2580 2630 2540 2570
Extracts
Triglycerides — 66 56 59 43
Yield (wt%)
Chol. yield (wt%) — 71 61 60 47
Chol. changes (%) — +8.3 +7.6 +1.8 +7.7
Fatty acids (mol%)
C4-C8 17.6 20.8 23.4 19.6 24.8
C10-C12 6.1 7.2 7.2 7.0 7.5
C14-C18 76.2 71.8 69.2 73.2 67.5
Unsaturated 26.3 23.6 21.4 24.0 21.0
Saturated 49.8 48.1 47.8 49.1 46.4
Unsaturated/saturated 0.52 0.49 0.44 0.49 0.45

Raffinates
Triglycerides — 34 38 42 56
Yield (wt%)
Chol. yield (wt%) — 25 38 39 53
Chol. changes (%) — –26.9 –11.6 –1.8 –5.6
Fatty acids (mol%)
C4-C8 — 3.3 5.0 9.2 8.3
C10-C12 — 3.9 4.6 4.9 4.8
C14-C18 — 92.6 90.3 85.8 86.7
Unsaturated — 37.2 36.1 32.7 34.2
Saturated — 55.3 54.2 53.1 52.5
Unsaturated/saturated — 0.67 0.66 0.61 0.6

ether:petroleum ether (boiling range 30-60° C), 1:1. The aquous phase was extracted again with 2 × 60
mL diethyl ether:petroluem ether 1:1. The combined extract was dried with anhydrous sodium sulfate,
and the solvents were removed with a stream of nitrogen in a steam bath, and the rest of the solids were
dried in vacuum. After methylation, the samples were dissolved in n-hexane for GC analysis.
Separation was carried out on a packed glass column (180 cm × 2.6 mm I.D.; 100/120 mesh GCQ
support with 10% Silar 10C). The column temperature was 200° C, and the injector and detector
temperatures were 250° C. Carrier gas was nitrogen. For control purposes, crude fat was determined
gravimetrically according to the Association of Official Analytical Chemists (AOAC) method. Typical
chromatograms are shown in Figure 2.29. Under these chromatographic conditions, the geometric
configu-
Page 61
TABLE 2.25. Fatty Acid Compositions (mol%) of AMF and Its SC -CO 2 Fractions.
SF (g/g) 61
Extract loading (wt%) 1.3
Triglycerides recovery (%) 101
AMF R S1 S2 S3 S4
Temperature (°C) — 40 60 80 80 60
Pressure (bar) — 241 241 207 172 69
PCO2(kg/m3) — 873 779 608 503 153
Triglycerides yield (wt%) — 19.5 11.8 36.1 17.8 15.4
C4:0 6.8 1.0 3.9 7.5 10.9 10.6
C6:0 3.5 0.7 2.4 5.2 5.2 5.5
C8:0 1.5 0.4 1.1 1.7 2.0 2.2
C10:0 3.2 1.2 2.5 3.4 3.5 4.0
C12:0 3.6 1.9 3.1 3.7 3.7 4.4
C14:0 11.4 8.4 10.8 11.8 11.7 12.7
C14:1 0.8 0.6 0.9 0.9 0.8 0.8
C15:0 1.0 0.9 1.0 1.0 1.0 1.1
C16:0 28.9 28.5 29.5 29.3 28.9 29.0
C16:1 1.3 1.3 1.4 1.3 1.2 1.2
C18:0 12.0 18.4 13.4 10.8 9.6 8.9
C18:1 22.5 32.2 25.8 20.8 18.2 16.9
C18:2 2.7 3.4 3.1 2.5 2.2 1.8
C18:3 0.3 0.4 0.4 0.3 0.3 0.3
SCFA(C 4-C8) 11.8 2.2 7.5 13.6 18.2 18.4
MCFA(C10-C12) 6.8 3.1 5.6 7.2 7.3 8.4
LCFA(C 14-C18) 81.2 94.6 86.7 79.1 74.3 73.0
Unsaturated 27.8 38.2 31.7 26.1 23.0 21.2
Saturated 53.4 56.4 54.9 53.0 51.3 51.8
Unsaturated/Saturated 0.52 0.68 0.58 0.49 0.45 0.41

Abbreviations: SCFA: short -chain fatty acids; MCFA; medium-chain fatty acids; LCFA: big -
chain fatty acids.
ration of the double bonds do not influence the retention. The results of the AOAC gravimetric method
and the acid hydrolysis-GC method are given in Table 2.27. The fat contents determined by the acid
hydrolysis-GC method are generally lower than those determined by the traditional gravimetric method;
however, it was established that the absolute differences were relatively low, and the results of both
methods showed significant linear correlation. The high variations of the fat content of some foodstuffs
were tentatively explained by the marked inhomogeneity of the raw material. The fatty acid
composition of some products is shown in Table 2.28. The coefficient of variation showed high
diversity between the individual fatty acid and samples; however, no relationship was found between
the sample characteristics and the value of the
Page 62
TABLE 2.26. Results of Gas Chromatographic Determination of Palmitic, Stearic, Oleic

and Linoleic Acid in Edible Plant Oils.
Oil Type Palmitic Acid Stearic Acid Oleic Acid Linoleic Acid
(%) (%) (%) (%)
Olive Oil (n = 43)
Mean 11.34 2.90 72.47 8.68
Min.-Max. 8.9-15.5 2.9-3.8 63.1-77.9 5.1-13.8
Standard deviation 1.210 0.321 3.112 1.981

Thistle Oil (n = 41)
Mean 6.64 2.47 10.82 78.58
Min.-Max. 5.7-7.9 2.1-2.8 9.0-14.1 74.2-81.7

Sunflower Oil (n = 33)
Mean 6.44 4.62 20.81 65.96
Min.-Max. 5.4-7.7 3.8-5.5 15.4-29.0 58.4-71.3

Rapeseed Oil (n = 20)
Mean 4.87 1.60 54.41 23.57
Min.-Max. 3.9-5.8 1.3-1.9 49.2-58.2 19.5-31.0

Maize Germ Oil (n = 18)
Mean 11.41 1.87 26.66 58.14
Min.-Max. 10.2-12.1 1.5-2.0 25.4-29.1 56.1-59.6

Wheat Germ Oil (n = 10)
Mean 17.14 0.85 13.8 58.35
Min.-Max. 14.9-19.4 0.7-1.1 10.4-18.3 54.5-61.5

Pumpkin Seed Oil (n = 9)
Mean 11.68 5.68 28.8 51.44
Min.-Max. 10.9-12.2 5.3-6.1 24.9-33.4 47.9-55.6

Soybean Oil (n = 4)
Mean 11.03 3.82 19.4 55.28
Min.-Max. 10.6-11.3 3.7-4.1 18.2-20.5 54.1-56.2

Reprinted from Reference [ 92] with permission, copyright © 1994, Wissenschaftliche

Verlagsgesellschaft mbH, Stuttgart, Germany.
coefficient of variation. It was concluded from the data that acid hydrolysis-GC is a suitable technique
to be used as a quality control standard.
The fatty acid composition of virgin and refined olive oils was also determined by GC using the methyl
esters of fatty acids. The aim of the study was the elucidation of the effect of natural antioxidants on the
oxidative stability of various olive oils [ 94 ]. The results of GC analysis are shown in Table 2.29. The
experiments showed that the oxidation rate of linoleic acid is higher than that
Page 63
Figure 2.29.
Gas chromatograms of fatty acid methyl esters prepared from test portions
of ravioli containing meat sauce (upper panel) and grilled snack chips (lower
panel) after acid hydrolysis of sample composites. Mean fat contents of the
products determined by the acid hydrolysis -packed column GLC
methodology were 3% (ravioli) and 28% (grilled snack chips).
Page 64
TABLE 2.27. Determination of Total Fat (%) by AOAC Gravimetric Methodology (A) and by Acid
Hydrolysis-Packed Column-GLC Methodology (B).
Product A B CV(%) D E
Tapioca pudding, fat-free 0.84 0.16 ± 0.03 21.2 0.68 –81
Honey mustard chicken, low-fat 1.50 0.88 ± 0.19 21.7 0.62 –41
Waffles, fat-free 1.73 0.56 ± 0.05 8.2 1.17 –68
Chicken chow mein, low-fat 1.91 1.57 ± 0.31 19.6 0.34 –18
Crackers, fat-free 2.55 0.94 ± 0.19 20.5 1.61 –63
Hard pretzels, no-fat 2.88 0.92 ± 0.25 27.1 1.96 –68
Rice cakes, fat-free 3.14 1.58 ± 0.24 15.2 1.56 –50
Cookies, fruit-center, fat-free 3.48 1.31 ± 0.16 12.3 2.17 –62
Instant breakfast powder, low-fat 3.57 1.44 ± 0.19 12.9 2.13 –60
Turkey gravy dressing meal 3.94 2.93 ± 0.19 6.5 1.01 –26
Beef ravioli in meat sauce 4.06 3.14 ± 0.29 9.0 0.92 –22
Muesli cereal, low-fat 6.61 3.54 ± 0.70 19.6 3.04 –46
Cereal with raisins, low-fat 8.77 5.28 ± 0.71 13.5 3.49 –40
Breaded fish filets 9.84 8.22 ± 0.32 3.9 1.62 –16
Taco dinner without meat 11.7 10.7 ± 0.4 3.7 1.0 –9
Infant formula powder 20.0 17.4 ± 0.7 4.1 2.6 –13
Chocolate chip cookies 26.2 20.8 ± 0.2 1.2 5.4 –20
Chicken-flavored snack crackers 26.8 24.8 ± 0.6 2.4 2.0 –8
SRM1846 powdered infant 26.8 23.8 ± 0.6 2.6 3.0 –11
formula
Grilled snack chips 29.3 27.8 ± 1.0 3.6 1.5 –3
Cheese cracker sandwiches 30.0 27.4 ± 0.8 3.0 2.6 –9
Corn chips 36.5 34.4 ± 1.4 4.0 2.1 –6
Chocolate bar with almonds 36.9 29.7 ± 1.3 4.5 7.2 –20
Mayonnaise 72.6 75.9 ± 0.9 1.1 –3.3 +5
Values for percent fat by acid hydrolysis -packed column -GLC methodology are means ± SD out of
four or five independent replications; coefficient of variation (CV,%) = [(SD/Mean) × (100)]; D =
difference (AOAC -GLC) fat (%); E = relative difference; % = [(GLC) - (AOAC direct)]/[AOAC
direct) × (100)].
Reprinted from Reference [ 93], copyright © 1995 with kind permission of Elsevier Science —NL,
Sara Burgerhartstraat 25, 1055 KY Amsterdam, The Netherlands.
of oleic acid, and the antioxidant effect of phenolic compounds is lower in oils with a high peroxide
value.
Capillary GC was also used for the study of the thermal stability and frying performance of a
genetically modified sunflower seed (Helianthus annuus L.) oils [ 95 ].
Although the overwhelming majority of gas chromatographic studies dealing with the separation and
quantitative determination of fatty acids were varied after methylation, other derivatization methods
such as ethylation [ 96 ] and silylation have also found application. The free and combined fatty acids and
sterols were measured in grape must and yeasts as trimethylsilyl derivatives [ 97 ]. The separation of
silylated fatty acids and sterols was carried out on a fused-silica
Page 65
TABLE 2.28. Distribution of Fatty Acids in Several Food Products.

Turkey Gravy Beef Ravioli Breaded Fish Fillets
Dressing Meal in Meat Sauce
Fatty Acid mg/g CV (%) mg/g CV (%) mg/g CV (%)

10:0 0 — 0 — 0 —
12:0 0 — 0 — 0 —
14:0 0.19 14.2 0.82 10.1 0.12 22.9

14:1 0.06 43.0 0.32 12.7 0 —
16:0 5.59 9.5 7.68 9.3 9.53 3.6

16:1 0.88 12.6 1.53 9.4 0.22 15.1
18:0 2.48 5.4 4.28 9.8 3.48 2.8
18:1 12.78 9.2 13.42 9.6 37.81 3.3
18:2 6.71 5.3 2.89 11.7 28.81 4.8
20:0 0 — 0 — 0 —
20:1 0.08 22.9 0.10 25.0 0.52 10.12

18:3 0.38 18.9 0.41 21.5 2.22 9.9
22:0 0.10 53.6 0 — 0.49 4.1
22:1 0 — 0 — 0 —
Sum, mg/g 29.25 31.44 82.49
Fat, % 2.93 3.14 8.25
Taco Dinner Infant Formula Grilled Snack Chips

without Meat Powder
Fatty Acid mg/g CV (%) mg/g CV (%) mg/g CV (%)

10:0 0 — 1.01 13.7 0 —
12:0 0 — 14.19 5.8 0.06 11.3

14:0 0.09 3.2 7.03 4.5 2.26 4.0
14:1 0 — 0.05 5.4 0 —
16:0 12.20 3.2 42.34 3.9 67.03 3.5

16:1 0.22 3.9 0.51 5.2 1.93 3.3
18:0 7.06 3.9 6.45 4.0 7.14 3.2
18:1 69.93 3.4 58.68 3.9 45.69 3.5
18:2 16.23 4.3 39.24 3.9 151.66 3.6
20:0 0 — 0 — 0 —
20:1 0.30 14.5 1.14 5.8 0.41 4.8

18:3 0.41 13.5 3.10 3.1 1.57 1.8
22:0 0.54 24.0 0.40 2.9 0.61 8.6
22:1 0 — 0.02 — 0 —
Sum, mg/g 106.97 174.17 278.25
Fat, % 10.7 17.4 27.8

Values (mg/g) are means of four or five independent replicates. CV (%) =
[(SD/Mean) × (100)]. The value for C 20:1 in grilled snack chips (0.41 mg/g)
represents the mean of three independent determinations. A value of 0.00
mg/g was obtained for a fourth determination. Mean ± SD of all four
determinations = 0.31 ± 0.20 mg/g; CV = 66.9%.
Reprinted from Reference [ 93], copyright © 1995. With kind permission
from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam,
The Netherlands.
Page 66
TABLE 2.29. Analysis of Fatty Acid Composition (%) of the Olive Oils. *
No. of Olive Oil
Fatty Acid I II III IV V

Origin California Italy Italy Spain California
C16:0 9.0 (0.01) 14.8 (0.04) 14.5 (0.1) 8.9 (0.06) 14.9 (0.05)
C16:1n-7 0.5 (0.004) 1.8 (0.005) 1.7 (0.01) 0.5 (0.005) 1.3 (0.007)
C18:0 3.5 (0.02) 2.3 (0.003) 2.4 (0.03) 3.4 (0.002) 2.6 (7.10-4)
C18:1n-9 76.9 (0.04) 65.0 (0.01) 66.2 (0.04) 79.4 (0.03) 71.7 (0.03)
C18:2n-6 8.5 (0.02) 14.1 (8.10 -3) 13.3 (7.10 -3) 5.1 (2.10-3 ) 5.7 (3.10-3)
C18:3n-3 0.6 (0.01) 0.8 (3.10-3) 0.7 (8.10-3) 0.7 (1.10-4 ) 1.2 (3.10-3)
C20:0 0.5 (10-3) 0.5 (10-3) 0.5 (5.10-3) 0.5 (4.10-3 ) 0.6 (2.10-3)
C20:1n-9 0.3 (0.01) 0.3 (8.10-3) 0.3 (0.01) 0.3 (0.01) 0.4 (0.01)
C33:0 0.1 (7.10-3) 0.2 (6.10-3) 0.2 (0.01) 0.2 (7.10-3 ) 0.2 (1.10-3)
*Mean (standard deviation in parentheses), n = 2; I = refined, bleached, and deodorized; II = refined
extra light; 3 = refined pure; 4 = extra virgin; 5 = extra virgin.
Reprinted from Reference [ 94] with permission by AOCS Press.
DB1 column (30 m × 0.25 mm I.D.; film thickness 0.25 µm). The temperature program was initially
40°C, rising to 200°C at 6°C/min and held for 15 min and then rising to 260°C at 6°C/min and from
260 to 290°C at 2°C/min. The temperature of the flame ionization detector and injector were 300 and
280°C, respectively. Helium was used as carrier gas at a flow rate of 1.5 mL/min.
For the extraction of free fatty acids and sterols, the must samples (50 mL) were coextracted with the
internal standards (C 9 , C19 , and cholesterol) using 3 × 25 mL chloroform. The combined chloroform
extracts were concentrated at 30°C to about 1 mL and dried with anhydrous Na2SO 4. An aliquot of 200
µL was dried under nitrogen flux, then 100 µL pyridine, and 100 µL bis(trimethylsilyl)
trifluoracetamide, and 10 µL trimethylchlorosilane was added and the mixture incubated at 80°C for 1
h.
For the determination of the combined fatty acids and sterols in musts, 25 mL absolute ethanol were
added to the aqueous phase remaining after the chloroform extraction and then centrifuged (5000 rpm,
15 min). The supernatant was discarded, and the rest was suspended in 10 mL water for saponification.
For the determination of the combined fatty acids and sterols in yeasts, 0.02-1.0 g (wet weight) of yeast
was suspended in 10 mL water. Saponification was carried out on the suspensions after the addition of
the same internal standards. Suspensions and 10 mL of 2 N KOH in ethanol were refluxed for 1.5 h.
After cooling, sterols and nonsaponifiable lipids were extracted with 3 × 15 mL of chloroform, and the
combined organic phase was concentrated to about 2 mL. The aqueous phase was acidified with 10 N
H2SO4, extracted with 3 × 15 mL of chloroform, and evaporated to 2 mL. The chloroform concentrates
were unified and silylated as described above.
Page 67
Figure 2.30.
Example of a chromatogram of the free fatty acids and sterols in a sample must.
Peaks: 1 = ethyl caprylate; 2 = caprylic acid; 3 = nonanoic acid; 4 = ethyl decanoate;
5 = capric acid; 6 = undecanoic acid; 7 = lauric acid; 8 = tridecanoic acid;
9 = tetradecanoic acid; 10 = palmitic acid; 11 = heptadecanoic acid;
12 = linolenic acid; 13 = linoleic acid; 14 = oleic acid; 15 = stearic acid;
16 = non-adecanoic acid; 17 = 5-α -cholestane; 18 = cholesterol;
19 = desmosterol; 20 = ergosterol; 21 = campesterol;
22 = stigmasterol; 23 = β-sitosterol; 24 = stigmasterol.
from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)
The chromatograms of free fatty acids and sterols in must, the combined fatty acids and sterols in must,
and the combined fatty acids and sterols in yeasts are shown in Figures 2.30, 2.31, and 2.32,
respectively. The chromatograms clearly show that each component is well separated from the others,
and no interfering peak was observed in the samples of musts and yeasts. The correlation coefficient
was over 0.99 for each compound, indicating the reliability of the quantitative determination. The
average coefficient of variation was found to be 5.63%, showing the good reproducibility of the
method. It was further established that the trimethylsilyl derivatives are relatively stable up to 5 days.
The method has been proposed for the simultaneous determination of fatty acids and sterols in foods
and natural plant products.
High-performance liquid chromatographic methods have found only limited application in the analysis
of the fatty composition of foodstuffs. Thus, the separation of n-3 fatty acids in fish oils was carried out
on an ODS column. Fatty acids were not derivatized; the detection wavelength was set to 210 nm [ 98 ].
Size exclusion chromatography and adsorption chromatography successfully separated fatty acid
monomers, dimers and polymers [ 99 ]. However, HPLC has been used in combination with GC either
for prefractionation or for
Page 68
Figure 2.31.
Example of a chromatogram of the combined fatty acids and sterols in a sample
must. Peaks: 1 = ethyl caprylate; 2 = caprylic acid; 3 = nonanoic acid; 4 = ethyl
decanoate; 5 = lauric acid; 6 = tridecanoic acid; 7 = palmitic acid; 8 = heptadecanoic
acid; 9 = linolenic acid; 10 = linoleic acid; 11 = oleic acid; 12 = stearic acid;
13 = nonadecanoic acid; 14 = cholesterol; 15 = β-sitosterol.
(Reprinted from Reference [ 97], copyright © 1994. With kind permission from
Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)
the simultaneous determination of triglycerides in the same sample before transesterification. A

combined method was used for the study of the alterations in fats occurring during frying [ 100]. First,
the various fatty acid fractions were separated on a high-performance size-exclusion chromatographic
(HPSEC) column; and then the individual fatty acids were separated by GC as methyl esters using a
fused-silica capillary column (SP -2380; 30 m × 0.25 mm I.D.) at 180°C. The HPSEC chromatograms of
total fatty acids and the nonpolar and polar fractions obtained by silica column chromatography are
shown in Figure 2.33.
The compositions of some oils before and after the frying experiment are listed in Table 2.30. The
concentration of dimeric and polymeric compounds after frying was the highest in the most unsaturated
oil. The fact that neither the amount of free fatty acids nor that of diglycerides changed considerably
during the frying experiment indicates that the contribution of hydrolysis to the alteration of oils is
negligible. The study suggested that the separation and quantitative determination of altered fatty acids
provide useful information on the quality of frying oils and fats and can be used for the quality control
of these products.
Another combined method was used for the characterization and purification of fatty acid methyl esters
from the liver oil of the deep sea shark (Cen-
Page 69
Figure 2.32.
Additional example of combined fatty acid and sterol chromatographic analysis
in (S22b) yeast cells. Peaks: 1 = heptanoic acid; 2 = ethyl caprylate; 3 = caprylic
acid; 4 = nonanoic acid; 5 = ethyl decanoate; 6 = capric acid; 7 = undecanoic
acid; 8 = ethyl laurate; 9 = lauric acid; 10 = tridecanoic acid; 11 = tetradecanoic
acid; 12 = palmitic acid; 13 = heptadecanoic acid; 14 = linolenic acid;
15 = linoleic acid; 16 = oleic acid; 17 = stearic acid; 18 = nonadecanoic
acid; 19 = 5- α-cholestane; 20 = cholesterol; 21 = desmosterol; 22 = ergosterol;
23 = campesterol; 24 = stigmasterol; 25 = lanosterol;
26 = β-sitosterol; 27 = stigmasterol.
trophorus squamosus) [ 101]. Crude shark liver oil (20 g) was prefractionated on a silica column (300 ×
40 mm I.D., 230-400 mesh) using n-heptane-dichloromethane mixtures of increasing elution strength.
The first and second fractions contained the esters and mainly squalene, respectively (sample 1). The
purity of fractions was controlled with thin-layer chromatography (silica layer; eluent:
cyclohexane/diethyl ether/fromic acid 32/3/0.2, v/v/v; visualization: iodine vapor). The ester fraction
was saponified in 2 N KOH in methanol/n-heptane (1/1, v/v) and then esterified with BF 3 in methanol
(14%, w/w). After esterification, n-heptane and saturated aqueous NaCl were added to the mixture; the
organic phase was separated and dried with Na 2SO 4 (sample 2). Sample 3 was prepared by
hydrogenating an aliquot of sample 1 in n-heptane with Pd on charcoal as catalysator (room
temperature, hydrogen pressure 3 bars).
After hydrogenation, the saponification and the synthesis of methyl esters of fatty acids were carried out
as described above. Sample 1 was fractionated with RP-HPLC using a C18 column (150 × 3.9 mm I.D.).
The gradient elution was 0% to 100% acetonitrile in dichloromethane in 30 min. The flow rate was 1
mL/min and the peaks were detected with a light scattering detector.
Page 70
Figure 2.33.
Significant parts of high-performance, size-exclusion chromatograms corresponding
to total fatty acids and the nonpolar and polar fractions obtained by silica column
chromatography. (FAM: fatty acid monomers; NPFAM: nonpolar fatty acid
monomers: OxFAM, oxidized fatty acid monomers: FAD, fatty acid dimers:
NPFAD, nonpolar fatty acid dimers: OxFAD, oxidized fatty acid dimers:
and FAP, fatty acid polymers.
(Reprinted from Reference [ 100 ] with permission from AOCS Press.)
Fatty acid methyl esters in samples 2 and 3 were separated by GC on a fused-silica capillary column (50
m × 0.25 mm I.D.; coated with CP-Sil-88). Separation was carried out under both isothermal and
temperature programming mode using different mass selective detectors. Some fractions were also
analyzed with countercurrent chromatography in an n-heptane/acetonitrile biphasic system. Separations
were carried out in two steps, both n-heptane and acetonitrile being the mobile phase. HPLC separated
16 fractions of sample 1 (Figure 2.34). The result of the HPLC separation of sample 1 indicates that,
similar to other natural oils, shark liver oil also consists of a high variety of triglycerides. Typical gas
chromatograms of samples 3 and 2 are shown in Figures 2.35 and 2.36, respectively.
Only the methyl esters of saturated fatty acids were observed on the chromatogram of the hydrogenated
sample, indicating the efficacy of the hydro-
Page 71
TABLE 2.30. Total Polar Compounds (wt% on oil), Polar Compound Distribution (mg/g oil), and
Fatty Acid Composition (wt% on oil) of Oils before and after Frying Experiment.
Before Frying Experiment Polar Compounds Distribution*
Sample** Total TGP TGD OxTGM DG FFA
SO 2.8 — 4.0 8.3 10.6 5.1
HOSO 3.1 — 3.4 6.9 16.3 4.4
PO 7.7 — 4.6 6.1 65.2 1.1
Fatty Acid Composition
Sample C16:0 C18:0 C18:1 C18:2 Others
SO 6.3 4.6 20.0 66.8 2.3
HOSO 4.0 4.3 72.3 16.8 2.6
PO 31.4 4.4 48.6 12.2 3.4
After Frying Experiment Polar Compounds Distribution
Sample Total TGP TGD OxTGM DG FFA

SO 28.9 50.0 125.1 98.0 11.8 4.1
HOSO 23.0 33.4 82.3 91.3 19.3 3.7
PO 25.5 26.8 71.1 86.4 69.4 1.3
Altered Fatty Acid Distribution***

Sample Total FAP NPFAD OxFAD OxFAM
SO 11.6 10.7 42.1 32.2 31.0
HOSO 9.0 6.1 27.0 24.0 32.9
PO 7.4 5.0 15.5 26.1 27.4
* Abbreviations: TGP = triglyceride polymers; TGD = triglyceride dimers; OxTGM = oxidized

triglyceride monomers; DG = diglycerides; FFA = free fatty acids.
** SO = sunflower oil; HOSO = high -oleic sunflower oil; PO = palm olein.
***FAP = fatty acid polymers; NPFAD = nonpolar fatty acid dimers; OxFAD = oxidized fatty acid
dimers; OxFAM = oxidized fatty acid monomers.
Reprinted from Reference [ 100] with permission from AOCS Press.
genation process. Fatty acids with 14-24 carbon atoms were found. The chromatogram of sample 2
indicates that monounsaturated fatty acids also occur in the shark liver oil. The fatty acid composition
of the oil is shown in Table 2.31.
It was established that countercurrent chromatography can be used for the isolation of fatty acid methyl
esters on the preparative scale.
Page 72
Figure 2.34.
HPLC chromatogram of ester fraction (sample 1). Column: 150 × 3.9
mm I.D., Waters Noca-Pack C18 ; mobile phase, A: acetonitrile, B:
dichloromethane; from 0 to 100% in 30 min; flow rate: 1 mL/min;
sample 0.2 µL of ester fraction in 20 µL.
Chromatographia and the corresponding author.)
Figure 2.35.
GC-MS-electron impact (EI) chromatogram of hydrogenated fatty acid
methylesters (sample 3). Column: 50 m × 0.25 mm I.D., CP-SIL-88;
pressure: 0.15 MPa; column temperature: 190°C.
Page 73
Figure 2.36.
GC -MS-EI chromatogram of fatty acid methyl esters (sample 2). Column:
50 m × 0.25 mm I.D., CP-SIL-88; pressure: 0.15 MPa; column temperature: 190°C.
2.2.2—
Triglycerides
The separation and quantitation of fatty acids after saponification provide useful information on the
fatty acid content of oils and fats, but it cannot be used for the exact assessment of the triglyceride (TG)
composition in the original products. Owing to their lower volatility, triglycerides can be analyzed with
GC at higher temperatures than free fatty acids. Triglycerides are the major components of oils and fats,
which are important food products. The
TABLE 2.31. Nature and Percentage of the Major Fatty Acids in the Shark Liver Oil.
Fatty Acids Percent Fatty Acids Percent
C14:0 2 C18:1 (isomers) 44.9
C16:0 20.6 C20:1 (isomers) 12.1
C16:1 7.9 C22:1 (isomers) 8.5
C17:1 1.1 Unidentified compounds 0.7
C18:0 2.2

Page 74
analysis of oils and fats of natural origin is extremely difficult because of the large potential complexity
of triglycerides.
Triglycerides in milk fat were separated according to the carbon number on a short capillary column
and on a packed column, and the performance of the two separation processes was compared [ 102]. The
chromatographic conditions were as follows: packed column GC: glass column of 50 cm × 2 mm I.D.,
packed with 3% OV-1 on 100/120 mesh Gas Chrom Q. Detector and injector temperatures were 370°C.
The temperature gradient was 210°C, 1 min isothermal and then to 350°C at 6°C/min, 5 min isothermal.
Carrier gas was nitrogen at a flow rate of 40 mL/min. Capillary column GC: Metal tubing of 5 m × 0.5
mm I.D., coated with deactivated dimethylpolysiloxane (film thickness 0.15 µm). Nitrogen flow rate: 3
mL/min. Other conditions were the same as for the packed column. A flame ionization detector was
used in both cases. Triglycerides were well separated on both the packed and capillary columns, as
demonstrated in Figure 2.37. TGs were eluted according to the carbon num-
Figure 2.37.
Comparison of milk fat triglyceride chromatogram obtained with
packed and capillary columns; heating rate 6 °C/min. respectively.
Page 75
ber, saturated and unsaturated TGs forming one peak. The separation capacity of both columns was
similar, indicating that a short capillary column can be used for the analysis of TGs in milk fat. It was
further established that cold-on-column injection increases the reliability of the method.
A combined GC method was developed for the determination of milk fat in chocolates [ 103]. Methyl
esters of fatty acids were separated on a capillary column (25 m × 0.32 mm I.D.); the injector and
detector (FID) temperatures were 250°C, and the column temperature was 185°C. Helium was used as
the carrier gas. TG analysis was also carried out on a capillary column (5 m × 0.33 mm I.D.). The
working temperatures were: injector, 380°C; detector (FID), 360°C; column, 255-355°C at 10°C/min.
The preliminary results of the calculation of standard deviations indicated that the triglycerides
containing 40, 42, and 44 carbon numbers can be determined with the lowest standard deviation;
therefore, these fractions were used for further analysis. The analytical results of pure milk fat (MF),
MF stearins, and MF oleins are compiled in Table 2.32. It was concluded from the results of analyses
that the best way for the determination of the MF content in chocolates is the measurement of the
concentration of TGs with carbon numbers of C40 , C42 , and C44 , and the determination of the ratios
between lauric acid/minor fatty acids and C14/C 16 fatty acids.
Since the presence of glycerol and mono-, di-, and triglycerides considerably impair the performance of
methylated vegetable oils used as diesel fuel substitutes, their separation and quantitative determination
is of paramount
TABLE 2.32. Analytical Results of Pure Milk Fat (MF), MF Stearins and MF Oleins.*
40-44(%) ** C12(%) MINFA*** Ratio**** C14(%) C16(%) Ratio§
Pure milk fat (n = 15)

Average 21.9 3.9 3.65 1.06 12.15 31.55 0.39
SD 0.69 0.39 0.17 0.72 3.4
SD % of average 3.15 10.0 4.65 5.9 10.8

Stearins (n = 5)
Average 21.5 3.9 3.65 1.06 12.65 34.65 0.37
SD 1.46 0.39 0.43 0.80 3.35
SD % of average 6.8 10.0 11.7 6.3 9.65

Oleins (n = 2)
O1. 1 22.0 3.5 3.55 1.24 12.9 33.55 0.38
O1. 2 20.0 3.7 4.0 0.80 12.05 27.1 0.44

* All results given in percent, except ratios.
** Triglycerides with carbon number 40-44.
*** MINFA = minor fatty acids between C 14 and C 16, i.e., C 14:1 , iso C 15, antiiso C 15, C 15:1 , and iso C16.
**** Ratio = C 12 /MINFA.
§ Ratio = C14/C 16.

Page 76
importance. Glycerol has been previously determined by GC using N, O-bis (trimethylsilyl)

trifluoroacetamide as a silylating agent [ 104]. A method for the simultaneous capillary gas
chromatographic determination of glycerol and mono-, di-, and triglycerides in vegetable oil methyl
esters (VOME) was developed [ 105]. Samples were silylated with N-methyl-N-
trimethylsilyltrifluoroacetamide after adding the internal standards 1,2,4-butanetriol and tricaprin
(100.0-110.0 mg of VOME, 100µL silylating agent, 15 min at room temperature). An uncoated,
deactivated fused-silica precolumn (2 m × 0.53 mm I.D.) and a DB-5 fused-silica column (10 m × 0.32
mm I.D.; film thickness 0.1 µm) were used for the separation. The temperature program was 50°C for 1
min, raised to 180°C at 15°C/min, to 230°C at 7°C/min, and to 370°C ballistically and held for 10 min.
Detector temperature was 370°C, and hydrogen was the carrier gas. The gas chromatogram of silylated
rapeseed oil with 1,2,4-butanetriol and tricaprin internal standards is shown in Figure 2.38. The
validation parameters of the quantitative analysis were very good (coefficient of correlation of the linear
relationship between peak area and weight over 0.99; recovery between 95-109% except triolein;
relative standard deviation (RSD) of repeatability between 0.4-1.4%), indicating the reliability of the
method. It was established that this GC procedure is suitable for the analysis of sunflower, soybean, and
used frying oil methyl esters without any modification of the GC parameters; however, the method
cannot be used for the investigation of lauric oil methyl esters.
Gas chromatography can be used not only for the separation and quantitative determination of fatty
acids and triglycerides in foodstuffs, but also for the measurement of various physicochemical
parameters. Thus, the solubility parameters of soybean oil were determined by inverse gas
chromatography (IGC) [106]. An acid-washed and silylated Chromosorb G (354-250 µm, 45/60 mesh)
was coated with soybean oil. The coating was carried out by dissolving soybean oil in n-hexane (5-20%
of support weight) and evaporating on the support surface with a rotary evaporator. A copper column of
about 1 m × 6.4 mm I.D. was filled with the soybean-coated support and thermostatted overnight at
123°C. The specific retention volume of a wide variety of compounds such as n-alkanes, aromatic
hydrocarbons, chlorinated hydrocarbons, aliphatic acids, and alcohols was determined at different
column temperatures. The solute-solvent interaction parameter χ was calculated by
where R = 6.236 × 104 mL-mm Hg/mole-K; Vg = specific retention volume; M1 = molecular weight of
the solute; p01 = vapor pressure of solute at column temperature, T; B11 = second pure virial coefficient
of the solute at T; V1 = molar volume of the solute at T; T = column temperature; v1 = specific volume of
the solute at T; v2 = specific volume of the solvent (soybean oil) at T; and
Page 77
Figure 2.38.
(a) Gas chromatogram of silylated rapeseed oil methyl ester with
1,2,4-butanetriol and tricaprin as internal standards.
(b) Enlargements of the regions, where the signals of mono-, di-,
and triglycerides appear in the upper gas chromatogram. GC column:
10 m × 0.32 mm I.D. fused-silica capillary column coated with DB-5 (0.1 µm
film thickness), equipped with a 2 m × 0.53 mm I.D. uncoated, deactivated
pre-column. GC temperature program: 50°C (1 min), at 15°C/min to 180°C,
at 7°C/min to 230 °C; ballistically to 370°C (10 min). Peak assignment: 1 = glycerol;
2 = 1,2,4 -butanetriol, I.S.; 3 = monopalmitin; 4 = monoolein, monolinolein,
monolinolenin; 5 = monostearin; tricaprin, I.D.; C34 -C38 = diglycerides with
carbon numbers of 34, 36 and 38; C 52 -C58 = triglycerides
with carbon numbers of 52, 54, 56, and 58.
M2 = molecular weight of the solvent. The second virial coefficient was calculated by
where Tc = critical temperature of the solute; Vc = critical volume of the solute; n = hypothetical number
of carbon atoms for a given solute that yields a p01
Page 78
equivalent to that of a corresponding n-alkane solute. The interaction parameters determined by the IGC
method are given in Table 2.33. A lower value of the interaction parameter indicates a stronger
interaction or a better miscibility between the solutes and soybean oil. In most cases the miscibility
increases with increasing temperature as expected from the general rule. Solubility parameters of
soybean oil at different temperatures were calculated from the interaction parameters and are presented
in Table 2.34. It has been stated that the determination of the solubility parameters of oils may help for
a better understanding of the technological processes in the industry and the behavior of oils in
connection with other oils or solvents.
Because of the advantageous separation characteristics, supercritical fluid chromatography (SFC) has
also found application in the analysis of various triglycerides in vegetable and fish oils [ 107], in Baltic
herring [ 108], and in butter fat [109]. However, it has been recently demonstrated that SFC can be used
for the simultaneous separation and quantitation of triglycerides and
TABLE 2.33. Interaction Parameters for Solute/Soybean Oil Systems at Specified Temperatures.
Temperature (°C)
Solute 58.6 79.0 100.9 123.4
n-Hexane 0.561 0.540 0.492 0.474
n-Heptane 0.568 0.520 0.473 0.442
n-Octane 1.11 0.954 0.813 0.697
n-Decane 2.36 2.11 1.89 1.70
Cyclohexane 0.411 0.367 0.335 0.318
Benzene 0.232 0.201 0.212 0.165
Toluene 0.162 0.132 0.134 0.118
Ethylbenzene 0.178 0.142 0.140 0.120
Methylene chloride 0.300 0.288 0.299 0.327
Carbon tetrachloride 0.150 0.163 0.147 0.157
Trichloroethylene 0.095 0.101 0.105 0.128
Acetone 1.22 1.15 1.06 1.01
Methyl ethyl ketone 0.899 0.804 0.771 0.722
Methyl isobutyl ketone 0.629 0.582 0.537 0.496
Furfural 1.70 1.49 1.34 1.21
Methanol 2.76 2.58 2.35 2.14
Ethanol 2.41 2.11 1.87 1.66
Isopropanol 1.94 1.68 1.46 1.15
n-Butanol 1.77 1.51 1.28 1.07
Acetic acid 1.40 1.48 1.50 1.51
Propionic acid 1.38 1.40 1.36 1.33
Butyric acid 1.58 1.47 1.40 1.32
Reprinted from Reference [ 106] with permission from Academic Press Ltd., a subsidiary of
Harcourt Brace and Company Ltd.
Page 79
TABLE 2.34. Variation in the Solubility Parameters of Soybean Oil with Temperature.
Solubility Parameter
Temperature (°C) x Sx
58.7 7.9 0.2
79.0 7.6 0.2
100.9 7.3 0.2
123.4 6.9 0.2
In units of cal 1/2 /cm 3/2 . x = mean; Sx = standard deviation.
Reprinted from Reference [ 106] with permission from Academic Press Ltd., a subsidiary
of Harcourt Brace and Company Ltd.
other lipoid compounds in various foodstuffs. Thus, free fatty acids, retinol, ergocalciferol,
cholecalciferol, squalene, tocopherols, wax esters, diacylglycerols, cholesterol esters, and
triacylglycerols in marine oils were determined in one run by SFC [110].
Samples of standards and marine oils were dissolved and used without any prepurification step for SFC
analysis. Separation was carried out on a fused-silica capillary column (20 m × 0.1 mm × 0.4 µm, 5%-
phenyl-methylpolysiloxane DB-5). Detection: FID; isothermal column temperature: 170°C. The mobile
phase density programs are shown in Figure 2.39. Typical chromatograms of
Figure 2.39.
Course of the three mobile-phase density programs employed.
(Reprinted from Reference [ 110] with permission from Chromatographia.)
Page 80
fish oils are shown in Figure 2.40. These SFC programs do not separate α-t ocopherol, cholecalciferol,
and ergocalciferol well; further, the related compounds, with different structure and degree of
unsaturation but the same carbon numbers, are also coeluted. The results of analyses are compiled in
Table 2.35. The relative standard deviation depends considerably on the concentration of the
component; it varied between 1% and 4% for major components and between 2% and 10% for major
components. The composition of marine oils showed marked differences that can be used for their
identification.
It has been established that the separation of the SFC method is superior to those of the GC and HPLC
methods described in the literature; therefore, its future use for the examination of these oils is
recommended. A similar SFC method was used for the separation and quantitative determination of
triacyl-glycerols in various edible oils and fats [ 111]. The composition of sea buckthorn (Hippophae
rhamnoides) seed and pulp oil, cloudberry (Rubus chamaemorus) seed oil, bovine milk fat, and rainbow
trout (Salmo gairdneri) lipids were determined. Separations were carried out on SB-Octyl-50 (50%
octyl/50% methylpolysiloxane) and SB-Cyanopropyl-25 (25% cyanopropyl/25% phenyl/50%
methylpolysiloxane) columns (both: 10 m × 50 µm I.D., film thickness 0.25 µm). A linear density
gradient of CO2 was used from 0.140 g/mL with 0.010 g/mL at constant 140°C. The temperature of the
FID was 340°C. It was found that the nonpolar stationary phase (SB -Octyl-50) can be successfully used
for the separation of triglycerides according to the number of acyl carbon atoms; however, the
separation of triglyceride mixtures containing a considerable number and variety of unsaturated
triglycerides cannot be carried out on the nonpolar stationary phase. The polar stationary phase (SB-
Cyanopropyl-25) showed a better separation capacity as demonstrated in Figures 2.41 and 2.42. The
chromatograms clearly show the good separation power of SFC; however, some saturated and
unsaturated triacylglycerols are coeluted and cannot be separated by this SFC method. The oils and fats
show marked differences in their triacylglycerol composition, which can be used for identification
purposes. The triacylglycerol compositions of the various oils and fats are given in Tables 2.36 and
2.37. The proportions of triacylglycerols were similar, using SFC and negative-ion chemical ionization
mass spectrometry, showing the reliability of the SFC method. The good repeatability of retention times
and the acceptable repeatability of the relative amounts indicate the good application parameters of the
method. Tocopherols can also be determined by this method. Because their concentration is relatively
low, a higher amount of sample has to be injected, resulting in the loss of the separation of
triacylglycerols.
Owing to its simplicity, TLC has also found application in the separation of triacylglycerols [ 112]. The
effect of different diets on the composition of hydrogenated milk fat was also elucidated by using TLC
[113]. The diets included in the study were hay and concentrate with no added lipid (A); grass (B); grass
and supplemental concentrate containing soy oil (C); and grass
Page 81
Figure 2.40.
Supercritical fluid chromatogram of tuna oil employing density program
B (I) and of basking shark oil employing density program
C (II). Numbers refer to compounds in Table 2.35.
Page 82
TABLE 2.35. Compounds, Chromatogram Peak Numbers (no.), and Composition (wt%) of the Marine Oils.
Basking Cod Sand
Compound* No. Herring Mackerel Tuna Shark Liver Eel Seal
FFA 14 1 nd nd 0.5 nd 0.1 0.1 nd
FFA 16 2 0.1 nd 4.4 0.2 1.9 0.4 0.0
FFA 18 3 0.1 nd 5.0 0.2 2.6 0.5 0.0
FFA 20 4 0.1 nd 1.6 0.2 1.2 0.6 0.0
FFA 22 5 0.2 nd 3.3 0.2 0.5 0.9 0.0
FFA 24** + Vitamin A*** 6 nd nd 0.2** 0.04** 0.1*** nd nd
Squalene 7 0.004 0.002 0.1 16.7 0.1 0.02 0.00
δ-Tocopherol 8 nd nd 0.02 nd 0.1 0.03 nd
α-tocopherol**** + Vitamin 9 nd nd 0.03**** 0.005**** 0.1§ nd nd

D 3§
WE 32 10 nd nd nd nd nd 0.1 nd
Cholesterol 11 0.1 0.1 1.8 0.2 0.3 0.5 0.1
DAG28 12 nd nd 0.1 0.01 nd nd nd
WE 34 13 nd nd nd nd nd 0.2 nd
DAG30 14 nd nd 0.1 0.02 0.03 0.03 nd
WE 36 15 0.01 nd 0.1 nd nd 0.3 nd
DAG32 16 0.02 0.03 0.3 0.1 0.2 0.1 nd
WE 38 17 0.03 nd 0.1 0.03 nd 0.3 nd
DAG34 18 0.04 0.03 0.4 0.1 0.5 0.1 0.0
WE 40 19 0.02 nd 0.1 nd nd 0.4 nd
DAG36 20 0.02 0.1 0.4 0.1 0.6 0.2 0.0
WE 42 21 0.01 nd 0.03 nd nd 0.2 nd
DAG38 + TAG 42 22 0.04 0.1 0.5 0.2 0.9 0.2 0.0
WE 44 23 0.02 nd 0.04 nd nd 0.1 nd
DAG40 + TAG 44 24 0.1 0.3 0.5 0.3 0.8 0.3 0.0

DAG41 + TAG 45 25 0.05 0.1 0.04 nd nd 0.1 nd
(table continued on next page)

Page 83
(table continued from previous page)
Basking Cod Liver Sand Eel

Compound* No. Herring Mackerel Tuna Shark Seal
TAG 46 + DAG 42 26 0.5 0.9 0.8 0.6 0.6 0.9 0.2
CE-FA 14 + TAG 47 27 0.1 0.3 0.3 0.1 0.1 0.2 nd
TAG 48 + DAG 44 28 1.4 2.5 2.7 1.8 1.6 2.3 1.1
CE-FA 16 + TAG 49 29 0.3 0.6 0.9 0.3 0.5 0.5 0.2
TAG 50 30 3.4 6.0 5.7 4.5 5.4 5.3 4.1
CE-FA 18 + TAG 51 31 0.5 1.0 1.6 0.6 1.4 0.8 0.5
TAG 52 32 6.8 10.8 9.2 7.6 13.6 9.4 9.0
CE-FA 20 + TAG 53 33 0.7 1.1 1.6 0.9 nd 1.0 0.7
TAG 54 34 11.7 16.0 12.5 11.4 17.1 13.6 15.6
CE-FA 22 + TAG 55 35 nd nd 1.9 0.7 nd 0.9 nd
TAG 56 36 15.3 18.0 14.8 15.1 18.1 17.4 19.8
TAG 57 37 nd nd nd 1.0 nd nd nd
TAG 58 38 17.5 16.8 11.2 14.1 15.1 16.1 19.4
TAG 60 39 16.7 13.0 9.3 9.4 9.6 12.2 15.5
TAG 62 40 12.1 7.5 5.7 6.9 4.8 7.7 9.4
TAG 64 41 7.0 3.5 1.6 4.3 1.6 4.3 3.5
TAG 66 42 3.7 1.2 0.7 1.2 0.4 1.7 0.7
TAG 68 43 1.2 0.2 0.2 0.6 0.04 0.2 0.1
TAG 70 44 0.2 nd nd 0.3 nd nd nd
TAG 72 45 nd nd nd 0.1 nd nd nd
*Abbreviations: FFA = free fatty acids; WE = wax esters; DAG = diacylglycerols; TAG = triacylglycerols; CE-FA =
cholesterol esters. The subscripts of FFAs, DAGs, TAGs, and CE-FAs refer to the total number of acyl carbon atoms of the
components; that is, TAG72 includes, among others, TAGs with three tetracosanoic acids attached to the glycerol molecule.
For WEs, the subscripts refer to the total number of carbon atoms of the molecule.
** Assumed to be FFA 24 .
*** Assumed to be predominantly retinol.

**** Assumed to be α-tocopherol.
§ Assumed to be predominantly cholecalciferol.

Page 84
Figure 2.41.
The separation of triacylglycerols on an SB -Cyanopropyl -25 (Dionex, Salt Lake
City, UT) column: (A) sea buckthorn pulp oil, (B) sea buckthorn seed oil,
(C) a mixture of saturated triacylglycerols, and (D) a mixture of unsaturated
triacylglycerols. For peak identification, see Table 2.36.
and supplemental concentrate containing palm oil (D). Lipids were extracted from milk, hydrogenated,
and separated into three fractions with TLC. The composition of the fractions was determined by using
HPLC and GC. The composition of MF samples are shown in Tables 2.38 and 2.39. The statistical
analysis of the data proved that the nature of diet does not have a significant influence on the
triglyceride composition of hydrogenated milk fat. Theoretically, a large number of triglycerides can be
derived by the permutation of the component fatty acids. The results prove that only the formation of a
small number of triglycerides is preferred. It has been concluded from the results that this method may
help the food industry to produce, isolate, and select specific triglycerides required for the processing of
specific food products.
Page 85
Figure 2.42.
The separation of triacylglycerols on an
SB-Cyanopropyl-25 UT) column: (A) milk fat
and (B) rainbow trout oil. For peak
identification, see Table 2.37.
(Reprinted from Reference [ 111] withi
permission from AOCS Press.)
TLC has also been employed for the determination of cholesterol in egg yolk [ 114]. This method was
modified for the simultaneous determination of cholesterol esters, triacylglycerols, free fatty acids, and
cholesterol in hens' egg yolk [ 115]. Lipids were extracted from 100 mg samples with 2 mL of
chloroform:methanol (2:1). The extract was evaporated to dryness after passing through a column
containing glass wool. For the determination of cholesterol esters and free fatty acids, the residue was
redissolved in 3 mL chloroform:methanol (2:1). This solution was diluted 1:1 with the same solvent
mixture for the determination of cholesterol and 1:16 for the determination of triacylglycerols.
Separations were carried out on 10 × 20 cm high performance silica plates using petroleum ether (37.5-
52°C):diethyl ether:acetic acid (80:20:2) for cholesterol, triacylglycerols, and free fatty acids and n-
hexane: petroleum ether:ethyl ether:glacial acetic acid (50:20:5:1) for cholesterol
Page 86
TABLE 2.36. Retention Times and Proportions of Triacylglycerols in Sea Buckthorn Pulp and
Seed Oil and in Cloudberry Seed Oil.
Peak Compound * tR**
Number Major/Minor(s)
(min) SD Proportion (%) SD
Sea Buckthorn Pulp Oil
1 48:1 35.93 0.03 8.03 0.05
2 48:2/50:1*** 36.71 0.03 19.77 0.10
3 50:2/48:3,52:1 37.34 0.03 21.63 0.06
4 52:2/50:3 37.99 0.03 19.15 0.37
5 52:3/50:4,54:2 38.61 0.03 9.77 0.05
6 52:4/54:3 39.24 0.03 6.22 0.09
7 54:4/52:5 39.89 0.03 3.85 0.06
8 54:5/52:6 40.49 0.03 3.06 0.19
9 54:6 41.11 0.03 3.04 0.03
10 54:7 41.77 0.03 3.24 0.03
11 54:8 42.44 0.03 1.79 0.14
12 54:9 43.13 0.02 0.66 0.04
Sea Buckthorn Seed Oil

1 48:1 35.92 0.02 1.29 0.03
2 50:1/48:2 36.66 0.03 2.99 0.19
3 50:2/52:1,48:3 37.28 0.03 3.55 0.16
4 52:2/50:3 37.96 0.03 4.55 0.11
5 52:3/54:2,50:4,56:1 38.63 0.03 5.19 0.09
6 52:4/54:3,56:2 39.30 0.04 8.78 0.06
7 54:4/52:5,56:3 39.98 0.04 11.70 0.10
8 54:5/52:6,56:4 40.64 0.05 14.30 0.11
9 54:6/56:5 41.27 0.04 16.36 0.13
10 54:7 41.94 0.05 16.84 0.15
11 54:8 42.58 0.04 10.57 0.10
12 54:9 43.22 0.03 3.72 0.06
Cloudberry Seed Oil

4 52:2/50:3 37.71 0.12 1.37 0.12
5 52:3/54:2 38.45 0.11 2.05 0.40

6 52:4/54:3 39.07 0.09 4.59 0.23
7 54:4/52:5,56:3 39.77 0.08 8.56 0.12
8 54:5/52:6,56:4 40.42 0.06 14.79 0.25
9 54:6/56:5,58:4 41.11 0.06 22.52 0.24
10 54:7/56:6 41.80 0.05 24.88 0.20
11 54:8/56:7 42.46 0.04 15.74 0.15
12 54:9 43.08 0.05 5.50 0.13

* Based on the use of spiked reference compounds and mass spectrometry data.
** Five replicates.
*** Partial separation, integrated as one peak.

Page 87
TABLE 2.37. Retention Times and Proportions of Triacylglycerols in Milk Fat and Rainbow
Trout Oil.
Peak No. tR* (min) SD Proportion (%) SD
Milk Fat
1 23.89 0.03 0.17 0.05
2 25.24 0.01 0.49 0.04
3 26.24 0.02 0.18 0.05
4 26.60 0.01 0.80 0.01
5 27.30 0.02 0.26 0.02
6 27.53 0.02 0.27 0.03
7 27.84 0.02 1.72 0.10
8 28.45 0.02 0.62 0.03
9 28.71 0.02 0.74 0.06
10 29.06 0.03 4.60 0.28
11 29.61 0.02 1.32 0.05
12 29.86 0.03 2.04 0.12
13 30.25 0.04 8.27 0.53
14 30.74 0.04 4.30 0.13
15 30.97 0.04 3.39 0.15
16 31.36 0.05 6.55 0.24
17 31.85 0.06 11.62 0.33
18 32.52 0.04 5.15 0.15
19 32.82 0.05 5.15 0.19
20 33.32 0.05 4.10 0.09
21 33.53 0.05 3.50 0.24
22 34.06 0.04 3.56 0.13
23 34.41 0.05 2.21 0.17
24 34.90 0.05 3.06 0.13
25 35.26 0.05 2.34 0.15
26 35.74 0.05 3.09 0.23
27 36.09 0.06 2.41 0.14
28 36.27 0.06 4.17 0.23
29 37.02 0.05 2.74 0.17

30 37.34 0.06 3.37 0.14
31 37.81 0.06 4.55 0.22
32 38.50 0.06 2.00 0.09
33 39.00 0.06 1.25 0.06
Rainbow Trout Oil

1 33.47 0.06 0.11 0.02
2 34.03 0.06 0.13 0.02
3 34.37 0.06 0.20 0.02
4 34.90 0.06 0.93 0.13
5 35.37 0.06 2.42 0.07
6 36.58 0.06 4.42 0.16
7 37.16 0.05 6.12 0.39
8 37.94 0.06 8.07 0.12
9 38.72 0.07 9.60 0.08
10 39.42 0.06 10.81 0.85
11 ** 40.15 0.06 10.33 0.70

Page 88
TABLE 2.37.
Peak No. tR* (min) SD Proportion (%) SD
12 40.69 0.15 10.31 0.14
13 41.43 0.06 10.56 0.18
14 ** 42.08 0.07 8.94 0.33
15 42.67 0.07 6.68 0.39
16 43.22 0.07 4.45 0.31
17 43.76 0.07 3.44 0.12
18 44.26 0.06 1.45 0.05
19 44.73 0.05 1.04 0.04

* Five replicates.
** Duplicate peak integrated as one peak.
esters. After development, the spots were detected by spraying the plates with 5% phosphomolibdic
acid in ethanol and heated at 110-120°C for 5-10 min. Qualitative evaluation was performed with a
Shimadzu densitometer. The Rf values of the lipids in the corresponding eluent mixtures were
cholesterol (sterols), 0.37; oleic acid (free fatty acids), 0.43; triolein (triacylglycerols), 0.74; and
cholesterol oleate (cholesterol esters), 0.67. The lipid content of samples are compiled in Table 2.40.
Statistical analysis proved that only the amount of free fatty acids shows a significant difference
between regular and lowfat eggs, while the concentration of the other lipid classes was the same in both
types of eggs. It has been stated that the accuracy and precision of the method make it adequate for use
in routine analysis.
TLC combined with an FID was used for the analysis of cereal lipids [ 116,117]. Quinoa (Chenopodium
quinoa Willd.) seeds were ground, and 10 g were extracted twice with 40 mL of boiling 2-propanol and
twice with 40 mL of chloroform:methanol:water (1:2:0.8 v/v); then 20 mL of chloroform and water
were added to the second extract, and the organic phase was recovered and combined with the 2-
propanol extract. Water was removed from the combined extract by adding 20 mL of benzene. Samples
were evaporated to dryness under nitrogen. Neutral lipids (NL), galacolipids (GL), and polar lipids (PL)
were separated on a sintered silica gel quartz rod (Iatroscan Mark IV, Japan) using the following eluent
systems: dichloroethane:chloroform:acetic acid (70:12:1.7:0.4) for NL; acetone:acetic acid:water
(70:1.2:1.5) for GL; and chloroform:methanol:water:acetic acid (50:27:3.0:0.5) for PL. Dry extract was
also separated into lipid classes on an aminopropyl silica cartridge using the eluents listed in Table 2.41.
Some characteristic TLC-FID chromatograms are shown in Figure 2.43. Nearly baseline separation was
achieved for each lipid component, proving the good separation characteristics of the method. This
sequential development technique uses a fairly low amount of total lipids (5-10 µg) and is suitable for
the detection and quantitative evaluation of a few dozen nanograms
Page 89
TABLE 2.38. Distribution (mol%) of Triglyceride (TG) Species with Common Carbon
Numbers (CN)* in Bands 2 and 3 of Fractions from TLC Plates of Milk Fat from Cows
Given Various Diets.
Diet: A ** B C D
Band: 3 2 3 2 3 2 3 2
CN Fatty acids
36 4+14+18 30.0 18.6 46.3 — 55.2 26.8 31.1 15.9
4+16+16 59.6 30.1 40.0 16.1 36.2 19.0 61.6 43.0
6+12+18 — — 0.5 4.9 2.9 13.7 0.4 1.5
6+14+16 9.0 37.8 8.5 35.5 5.3 26.7 5.9 25.1
8+10+18 0.5 4.9 — 19.3 0.3 3.3 — 1.1
8+12+16 — 2.8 0.4 — — 1.4 — —
8+14+14 0.1 — — 5.6 — 2.4 — 4.5
10+10+16 — 2.1 0.8 1.3 — 2.2 0.2 1.3
10+12+14 — — 1.4 8.6 — 4.1 — 4.0
12+12+12 — 3.5 1.8 8.0 — — 0.5 2.1
38 4+14+20 0.8 — — — — — — —
4+16+18 88.5 26.7 86.8 16.5 91.8 49.6 93.4 57.6
6+14+18 2.3 16.8 5.8 20.7 4.8 26.3 1.4 7.8
6+16+16 7.9 28.2 3.8 20.9 3.1 13.5 5.1 24.0
8+12+18 — — — 12.1 — — — 1.2
8+14+16 — 13.5 — 4.4 — 4.3 — 4.0
10+10+18 — — — 2.8 — — — 0.2
10+12+16 — 10.2 2.4 4.3 — 5.8 — —
10+14+14 — 3.5 — 17.6 — — — 3.8
12+12+14 — 0.9 1.1 — — — — 1.2
40 4+16+20 5.0 — — — — — — —
4+18+18 89.4 3.7 94.0 12.5 94.9 48.4 87.9 29.4
6+14+20 — — — — 0.5 — — —
6+16+18 4.3 28.4 5.6 38.5 4.2 36.3 5.3 42.7
8+14+18 — 10.9 — 11.1 — 4.8 — 2.2
8+16+16 — 14.2 — 9.6 — 3.6 0.9 11.3
10+12+18 — — — 6.4 — 1.5 1.1 —
10+14+16 0.4 33.1 — 16.0 — 4.1 2.6 11.2
12+12+16 — 9.5 — 1.9 — 0.7 0.6 0.3
12+14+14 — — — 3.0 — — 1.1 2.7
*Data given only for those CN-TG common to both bands and recovered in sufficient
quantity for complete analyses.
** See text for details of diets.
Reprinted from Reference [ 113] with permission from VV-GmbH Volkswirtschaftlicher

Verlag.
of lipid. The recovery from the extracts varied between 88.4% and 98.6%, indicating the reliability of
the method. The analysis time was less than 1 h, and the solvent consumption was very low compared
with other analytical methods.
Solid-phase extraction (SPE) using aminopropyl silica support was employed for the determination of
lipid composition in anhydrous milk fat and
Page 90
TABLE 2.39. Distribution (mol%) of TG Species with Comon CN * in Bands 1 and 2 of

Fractions from TLC Plates of Milk Fat from Cows Given Various Diets.
Diet: A ** B C D
Band: 2 1 2 1 2 1 2 1
CN Fatty acids
44 6+18+20 0.4 — — — — — — —
8+16+20 0.4 — 1.2 — 1.4 — — —
8+18+18 4.7 3.4 15.6 11.4 20.4 11.8 13.2 10.8
10+16+18 40.7 36.3 42.4 42.8 40.5 40.0 47.5 47.3
12+12+20 — — — — — — 0.9 —
12+14+18 14.3 15.7 16.1 18.6 17.7 24.3 9.6 16.1
12+16+16 19.7 21.6 11.0 11.2 9.3 9.1 13.6 12.3
14+14+16 19.5 22.6 13.6 15.5 10.3 14.6 14.3 13.1
46 8+18+20 — — 1.0 — — 0.8 — —
10+16+20 1.4 0.8 — 0.4 1.4 — — —
10+18+18 13.2 9.9 23.9 24.5 34.8 30.5 22.5 23.3
12+16+18 33.1 33.6 33.0 31.8 28.6 29.9 34.7 34.7
14+14+18 17.5 20.5 23.3 21.2 20.1 21.3 14.9 14.0
14+16+16 34.5 34.7 16.8 21.9 14.9 17.1 27.0 27.1
48 10+18+20 1.3 1.2 1.2 0.6 — 2.0 — —
12+16+20 0.4 — — 0.5 0.6 — — —
12+18+18 14.1 14.3 21.0 18.6 28.8 27.2 16.6 22.5
14+14+20 — — 0.3 0.1 — — — —
14+16+18 69.0 68.8 69.3 71.8 65.4 65.6 68.1 62.5
16+16+16 14.9 15.5 7.9 7.9 4.9 4.8 14.9 14.4
50 12+18+20 1.1 — — — 1.5 — 0.4 —
14+16+20 3.1 2.6 1.8 1.6 — 2.0 0.6 —
14+18+18 34.3 37.7 48.9 50.0 61.4 57.0 33.2 40.6
16+16+18 61.4 59.6 48.5 47.7 36.8 40.0 65.1 58.8
*Data given only for those CN-TG common to both bands and recovered in sufficient
quantity for complete analyses.
** See text for details of diets.
Reprinted from Reference [ 113] with permission from VV-GmbH Volkswirtschaftlicher

Verlag.
dry whey products such as dry whey powder (WP) and whey protein concentrates (WPC) containing
34% and 75% protein (WPC34%, WPC75%) [ 118]. Before extraction, WP75%, WP34%, and WP were
hydrated with water at the weight ratio of 2:1, 2:1.5, and 2:2.5, respectively. Samples of 3 g, 4 g, and
5.4 g of hydrated solutions were used for extraction. An aliquot of 35 mL of chloroform:methanol (1:1)
was added to the samples, and the mixture was homogenized and then centrifuged. The supernatant was
decanted, and the pellet was extracted again with 35 mL of chloroform:methanol 2:1 v/v. The combined
extract was evaporated to dryness at 35°C. A Sephadex G-25 column hydrated with water:methanol of
1:1 was used for the removal of nonlipid contaminants. The column (150 × 20 mm I.D.) was
equilibrated with chloro-
Page 92
Figure 2.43.
TLC-FID chromatograms of quinoa lipid separation. (A) Neutral lipids;
1 = wax esters (WE) + esterified sterols (STE); 2 = triglycerides
(TG); 3 = free fatty acids (FFA); 4 = 1,3-diglycerides (1,3DG);
5 = 1,2-diglycerides (1,2DG); 6 = free sterols (ST). (B) Monoglycerides
and galactolipids: 7 = monoglycerides (MG); 8 = monogalactosyl
diglycerides (MGDG); 9 = digalactosyl diglycerides (DGDG).
(C) Phospholipids: 10 = phosphatidic acid (PA); 11 = phosphatidyl
ethanolamine (PE); 12 = phosphatidyl serine (PS); 13 = phosphatidyl
inositol (PI); 14 = lysophosphatidyl ethanolamine (LPE);
15 = phosphatidyl choline (PC); 16 = lysophosphatidyl
choline (LPC); SP, sample application area.
(Reprinted from Reference [ 117], copyright © 1994. With kind
ether:acetic acid (70:30:1, vol/vol/vol) eluent, and 10% cupric sulfate in 8% phosphoric acid as
detection reagent. The lipid compositions of anhydrous butter fat and dry whey products determined by
the SPE method are compiled in Table 2.43. The high percent of recovery indicates that the method is
also suitable for the extraction of total lipids from products containing a high concentration of protein
and can be used for any products with a low moisture content. SPE separates lipid classes rapidly and
efficiently with a minimum exposure to air; therefore, the fractions can be used for subsequent GC or
HPLC analyses for the evaluation of the individual lipid components.
Page 93
TABLE 2.42. Solvents Required for Solid-Phase Extraction with Mega Bond Elute* in
Isolation and Purification of Lipid Classes.
Amount Lipid Class
Name Solvents P ** (mL) *** Eluted****
A 2:1 (vol/vol) Chloroform/2- 4.07 18 NL

propanol
B 2%(vol/vol) Acetic acid in diethyl 2.86 18 FA
ether
C Methanol 5.1 18 FA
D Hexane 0.01 12 CE
E 1%(vol/vol) Diethyl ether,
10%(vol/vol) methylene chloride

in hexane 0.437 36 TG
F 5%(vol/vol) Ethyl acetate in hexane 0.315 36 C
G 15%(vol/vol) Ethyl acetate in 0.616 36 DG

hexane
H 2:1(vol/vol) Chloroform/methanol 4.43 18 Mg
* Varian Sample Preparation Products (Harbor City, CA).
** Solvent strengths.
***Solvent volumes represent about 80 mg or 60 mg of total lipids from whey protein
concentrate or anhydrous milk fat, respectively.
**** NL = Neutral lipids; FA = free fatty acids; PL = phospholipids; CE = cholesterol
ester; TG = triacylglycerols; C = cholesterol; DG = diacylglycerols; MG =
monoacylglycerols.
TABLE 2.43. Lipid Composition of Anhydrous Butter Fat and Dry Whey Products Using Amino Propyl
Solid -Phase Extraction.
Total Lipids (%w/w)*
Anhydrous
Lipid Classes Milk Fat WPC-75 WPC-34 WP
Free fatty acids 0.22 ± 0.03 2.29 ± 0.10 3.36 ± 0.52 4.95 ± 0.26
Phospholipids 1.30 ± 0.14 23.64 ± 1.15 17.53 ± 1.63 18.04 ± 2.55
Neutral lipids 98.48 ± 0.21 74.08 ± 1.45 79.11 ± 1.12 77.01 ± 2.69
Cholesterol ester 0.22 ± 0.05 1.52 ± 0.11 1.70 ± 0.37 1.84 ± 0.57
Triacylglycerols 95.44 ± 0.22 61.12 ± 2.44 64.10 ± 3.86 60.82 ± 2.74
Cholesterol 0.22 ± 0.05 2.41 ± 0.20 2.54 ± 0.30 3.21 ± 0.53
Diacylglycerols 2.17 ± 0.06 5.46 ± 0.16 4.87 ± 0.49 5.32 ± 0.38

Monoacylglycerols 0.07 ± 0.01 3.30 ± 0.04 5.70 ± 0.05 5.83 ± 0.10
% Total lipid recovery 99.64 ± 1.22 99.74 ± 1.42 99.80 ± 0.97 99.01 ± 1.51
* Reported values are means ± standard deviations.

Page 94
Many HPLC methods have been developed for the separation of triglycerides in a wide variety of foods.
A laser light scattering detector was used for the detection of triglycerides in oils and fats [ 119].
Isocratic GP -gel permeation chromatography was successfully used for the analysis of triglyceride
methanolysis mixture containing mono-, di-, and triglycerides and fatty acid methyl esters in
transesterified vegetable oils using density detection [ 120], and a silver ion HPLC column was used for
the separation of triglycerides in sheep adipose tissue, sunflower seed oil, linseed oil, and fish oil [ 121].
To overcome the difficulty arising from the lack of linear response and the need of response factors for
UV detection of triacylglycerols, a transport FID has been used frequently in the HPLC analyses of
these solutes [ 122,123]. Silver ion HPLC combined with an FID has also been employed in the analysis
of triacylglycerols in vegetable oils [ 124]. Separation of triacylglycerides of vegetable oils was carried
out on a chromosphere lipids column (250 × 4.6 mm I.D., 5 µm particle size; Chrompack International,
Middleburg, The Netherlands) using isocratic elution (0.5 vol% acetonitrile in hexane, flow rate 1
mL/min, elution time 120 min). The fatty acid composition of the individual triacylglycerol fractions
was determined by gas chromatography of the fatty acid methyl esters. Some characteristic silver ion
chromatograms are shown in Figures 2.44 and 2.45. The chromatograms show the efficacy of the silver
ion HPLC combined with FID. The separation of triacyglycerols (TAGs) is based on the number of
double bonds in the TAG molecule, which makes the method suitable for the analysis of margarine-
based stocks produced from corn and soybean oils. The composition of TAGs in various vegetable oils
is given in Table 2.44. The data prove that the triacylglycerol composition of the various vegetable oils
differs considerably. The results of reversed-phase ion silver ion HPLC show good agreement,
indicating that both methods can be successfully used for the determination of the triacylglycerol
composition of these oils.
HPLC has also been used for the detection of the adulteration of olive oil with linoleic acid-rich oils
using either octadecyl [ 125] or octyl-bonded silica columns [126]. In the last case the separation of
triacylglycerols was carried out on two 150 × 4.5 mm I.D. octyl-bonded silica columns. The isocratic
eluent was acetone:acetonitrile 70:30, v/v, and the flow rate was 1 mL/min. The triacylglycerol
fractions were detected with a refractive index detector. Oils were dissolved in the eluent and injected
without any pretreatment. Triacylglycerols are separated according to their equivalent carbon number
(ECN) as shown in Figure 2.46. Since the highest differences were found in the fractions containing 42
equivalent carbon numbers, this fraction was used for the detection of the adulteration of olive oil with
the other oils. The authenticity factor (Au) was calculated by equation (23):
Page 95
Figure 2.44.
Triacylglycerol analyses by Ag-HPLC-FID of corn oil
and randomized corn oil, blend of corn oil and cottonseed
oil stearine, and its interesterified product.
(Reprinted from Reference [ 124], by courtesy of Marcek Dekker, Inc.)
The triacylglycerol composition and the authenticity factors of olive, corn, soybean and sunflower oils
are presented in Table 2.45. The ratio of added oils can be calculated by the following equations:
The use of slightly different constants was motivated by the different characteristics of added oils. It
was concluded from the data that the method is
Page 96
Figure 2.45.
Triacylglycerol analyses by Ag-HPLC-FID of soybean oil and randomized
soybean oil, blend of soybean oil and cottonseed
oil stearine, and its interesterified product.
rapid (analysis time less than 15 min), reliable, and suitable for the detection of 1% of linoleic acid-rich
vegetable oils in olive oil.
HPLC has also been used for the separation of neutral lipid classes in green beans [ 127]. Green beans
(Phaseolus vulgaris, L. v Helda) were homogenized and liophilized; then 2 g of sample were extracted
in a Soxhlet apparatus for 4 h. The extract was evaporated to dryness under a nitrogen stream, and the
rest was redissolved in 15 mL of hexane. An aliquot of 5 mL of the hexane solution was evaporated to
dryness and redissolved in 1 mL of HPLC eluent for injection. A silica column (250 × 4.6 mm I.D.,
particle size 5 µm) was employed for the separation. The isocratic eluent was n-hexane:2-
propanol:acetic acid (100:0.14:0.07 v/v), and the flow rate was set to 1.5 mL/min. NL fractions were
detected with a refractive index detector. A typical chromatogram of the
Page 98
TABLE 2.44. (continued).
Quantitation (area percent)*

Resolution Pumpkinseed Linseed Soybean Canola
I II III IV V IV V IV V IV V
SDD 4 LLS, LLP 20.6 20.7 2.1 4.8 16.9 15.5 3.9 5.1
MMD 4 LOO 9.6 10.5 1.9 5.2 11.1 9.4 22.4 21.7
DDM, TMM 5 LLO, LnOO 17.7 18.9 — — — — — —
DDM, 4-5 LLO, LnOO, — — 12.1 14.2 17.7 21.2 20.12 1.2
TMM, SMT LnOP
DDD, 5-6 LLL, LnLO, — — 14.9 17.9 21.8 20.5 11.7 9.8
TDS, TDM LnLP
DDD 6 LLL 18.9 16.7 — — — — — —
TDD 7 LnLL 0.3 0.4 16.8 12.4 6.8 6.7 3.5 3.1
TTM 7 LnLnO — — 6.5 5.7 — — 0.1 0.0
TTD 8 LnLnL — — 12.7 11.7 1.0 0.9 0.6 0.8
TTT 9 LnLnLn — — 22.2 19.2 — — — —
Unidentified 0.8 0.4 0.3 0.0 0.2 0.9 0.8 1.1

* Area percent standard deviation ± 0.1 % to 0.2%. I = Triacylglycerol saturated (S), monoenoic (M), dienoic (D),
trienoic (T) fatty acid species. II = Number of double bonds. III = Triacylglycerol molecular species, reversed -phase;
S, P, O, L, and Ln arestearic, palmitic, oleic, linoleic and linolenic acids, respectively, attached to the triacylglycerol
moiety. IV = Reversedphase. V = Silver ion.
separation of green bean neutral lipids is shown in Figure 2.47. It was established the the column
temperature has a marked effect on the separation efficiency, and separation has to be carried out below
24°C. The validation parameters (precision of measurement, precision of method, and detection limit)
are compiled in Table 2.46. The precision of the measurement was determined by injecting 10 times the
same sample, and the precision of the method was calculated by subjecting 10 aliquots of the same
green bean homogenizate to the complete preparation procedure before HPLC analysis. The mean and
standard deviation of the neutral lipid classes in various green bean samples (mg/100 g dry sample)
were sterol esters, carotenoids, and waxes: 150 ± 20; triglycerides: 28 ± 5; saturated and
monounsaturated fatty acids: 87 ± 16; diunsaturated fatty acids: 28 ± 7; and triunsaturated fatty acids:
26 ± 5.
HPLC has also been successfully employed for the detection of pork fat (lard) in processed foods [ 128].
The objective of the development of this method was the detection of adulteration of meat products
with pork meat. Lipids were extracted from the food products with CHCl3: CH3 OH (2:1 v/v), and
triglycerides were isolated by column chromatography on 6 g of silica support. Lipids dissolved in n-
hexane were applied to the column and were eluted with 20 mL of n-hexane followed with 5% ethanol
in n-hexane.
Page 99
Figure 2.46.
Separation of triacylglycerols on Supelcosil-LC 8 with
acetone acetonitrile (70:30, v/v) as the mobile phase and
refractive index detection. Flow rate, 1.0 mL/min.
(a) Olive oil; (b) soybean oil; (c) sunflower oil; (d) corn oil.
(Reprinted from Reference [ 126], copyright © 1995.
With kind permission from Elsevier Science—NL, Sara
Page 100
TABLE 2.45. The Triacylglycerol Composition (%) (± SD, n = 9) and the

Authenticity Factors (Au) of Olive, Corn, Soybean, and Sunflower Oils Separated
by RP-HPLC.
Oil
Triacylglycerol
ECN Corn Sunflower Soybean Olive
38 nd * nd 1.2 ± 0.04 nd
40 nd nd 7.0 ± 0.02 nd
42 24.2 ± 0.04 22.4 ± 0.10 24.9 ± 0.11 1.0 ± 0.02
44 38.2 ± 0.40 35.2 ± 0.16 30.8 ± 0.32 5.7 ± 0.51
46 23.6 ± 0.21 25.0 ± 0.16 21.6 ± 0.54 23.2 ± 0.37
48 9.2 ± 0.04 12.7 ± 0.03 10.5 ± 0.13 60.1 ± 1.25
50 1.4 ± 0.01 1.1 ± 0.09 2.8 ± 0.22 6.8 ± 0.19
52 1.5 ± 0.10 2.2 ± 0.11 0.9 ± 0.12 0.9 ± 0.13
Au 3.2 ± 0.05 3.5 ± 0.03 3.1 ± 0.03 100.0 ±

2.73
Reprinted from Reference [ 126], copyright © 1995. With kind permission from
Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The
Netherlands.
Separation was carried out on an octadecylsilica column (125 × 4 mm I.D., particle size µm) using an
isocratic eluent (CH3CN:CH2Cl2 58:42, v/v) at a flow rate of 1 mL/min and a refractive index detector.
The chromatograms of beef, lard, mutton, chicken, and turkey fats are shown in Figure 2.48. It was
concluded from the measurement that this HPLC method is easy, simple, rapid, and suitable for the
detection of 5% lard in processed food products.
Because of the high number of triglycerides in natural fats and oils, their identification is difficult and
subjected to considerable errors. The use of mass spectrometry combined with HPLC considerably
facilitates the identification of individual triacylglycerol species [ 129]. RP-HPLC, coupled with fast
atom bobardment mass spectrometry, was employed for the identification of polyunsaturated fish
triacylglycerols [ 130]. Refined fish oil was selectively hydolyzed with Lipase OF (Candida
cylindracea); the resulting products (free fatty acids, mono-, di-, and triacylglycerols) were separated on
a Florisil column, and the TG fraction containing polyunsaturated fatty acids (PUFA-TG) was used for
further HPLC investigations. Separation of PUFA-TG was achieved on three octadecylsilica columns
connected in series (two columns of 200 × 21 mm I.D.; one column 100 × 2.1 mm I.D.; particle size of
each column 5 µm). Eluent composition was acetonitrile:acetone (65:35) at a flow rate of 0.15 mL/min
(column temperature 40°C). After passing through a UV detector, the eluent was further analyzed with
positive and negative fast atom bombardment mass spectrometry (FAB-MS). The chromatogram of
PUFA-TGs detected at 210 nm is shown in Figure 2.49. It was assumed that the separation of PUFA-
TG species was not complete and one peak may contain more than one TG species. The triacylglycerol
components of selectively
Page 101
Figure 2.47.
Chromatogram of neutral lipid classes of a green bean
sample analyzed by HPLC using refractive index detection.
Peaks: 1 = sterol esters, carotenoids and waxes;
2 = triglycerides; 3 = saturated and monounsaturated
fatty acids; 4 = diunsaturated fatty acids; and
5 = triunsaturated fatty acids.
Preston Publications, A Division of Preston Industries,
Inc. and the corresponding author.)
hydrolyzed fish oil determined by HPLC/FAB-MS are given in Table 2.47. The measured and
calculated retention time values showed good agreement, indicating that the relative retention potential
index theory can be successfully used for the prediction of the RP -HPLC retention of this class of
triacylglycerols. It was concluded from the data that the combination of HPLC with FAB-MS facilitates
the identification of triacylglycerols co-eluted from the HPLC column and can be employed for the
analysis of the individual triacylglycerol fractions present in natural oils.
TABLE 2.46. Precision of Measurement, Precision of Method, and Limits of Detection for
Neutral Lipid Classes in Green Beans by HPLC.
Sterol Esters, Fatty Acids
Carotenoids,
and Waxes Triglyglycerides A B C
Precision of 0.79 1.44 0.97 1.02 1.51

measurement (%)
Precision of method 2.42 2.68 1.09 1.71 2.66
(%)
Limit of detection 0.082 0.019 0.075 0.121 0.098
(mg/mL
A = Saturated and monounsaturated; B = Diunsaturated; C = Triunsaturated.
Reprinted from Reference [ 127] with permission from Preston Publications, a Division of
Preston Industries, Inc.
Page 102
Figure 2.48.
Component triacylglycerols of genuine beef, mutton,
chicken, and lard.
(Reprinted from Reference [ 128], by courtesy of
Marcel Dekker, Inc.)
RP-HPLC, coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry, was used
for the analysis of triacylglycerols in soybean oils [ 131]. Separation of triacylglycerols by HPLC -MS
was carried out on two octadecylsilica columns (250 × 4.6 mm I.D., particle size 10 µm, carbon load
30%; 250 × 4.6 mm I.D., particle size 5 µm, carbon load 12%). The eluent gradient was 100%
propionitrile (PrCN) for 65 min, linear from 65-75 min to 75% PrCn and 25% n-hexane, and held for 15
min. The flow rate was 1 mL/min. The detection of triacylglycerols was carried out simultaneously with
an evaporative light scattering detector (ELSD) and an APCI mass spec-
Page 103
Figure 2.49.
Separation of triacylglycerols enriched with polyunsaturated fatty acids from
selectively hydrolyzed fish oil by HPLC with octadecylsilica (ODS) columns and
UV detection. The chromatographic conditions are described in the text.
The peak numbers are the same as in Table 2.47.
(Reprinted from Reference [ 130] with permission from The Japan
Society for Analytic Chemistry, Tokyo.)
trometry. RP -HPLC-FID was used for the quantitative determination of triacylglycerols using two
octadecylsilica columns connected in series (500 × 4.9 mm I.D., particle size 5 µm). The 120-min linear
gradient elution was 70:30 to 40:60 acetonitrile:methylene chloride (v/v) at 0.8 mL/min flow rate. The
fatty acid composition was determined by the capillary GC analysis of fatty acid methyl esters. Typical
chromatograms obtained by both RP -HPLC methods are shown in Figures 2.50 and 2.51. The elution
order of triacylglycerols was similar in both RP-HPLC sytems, although the separation capacity of RP-
HPLC-FID was considerably higher than that of RP -HPLC-ELSD. The similarity between the
chromatograms also allowed the identification of peaks on the chromatograms of HPLC-FID. The
quantitative evaluation of triacylglycerol composition determined RP-HPLC-FID is listed in Table 2.47.
The results of the GC analysis of fatty acids agree well with the fatty acid composition calculated from
the RP-HPLC measurements, confirming the accuracy and reliability of the HPLC analyses. The data
indicate that APCI mass spectrometry, coupled with RP-HPLC, is suitable for the identification of
triacylglycerols in vegetable oils. HPLC, coupled with a light scattering detector, has been used for the
analysis of triglycerides in avocado oil [ 132] and in almond, corn, cotton seed, and olive oils [133].
Page 104
TABLE 2.47. Triacylglycerol Analyses via RP-HPLC with Flame-Ionization Detection of

Normal, Palmitic, and Stearic Acid Containing Soybean Varieties. *
Soybean Variety
''Normal" Soybean High Stearic High Palmitic
TAG ** Oil
LnLnLn 0.2*** 0.1*** 0.4***
LnLnL 1.1 1.2 2.0
LLLn 6.0 4.8 1.5
LnLnO 1.4 1.3*** 4.8***
LnLnP 0.5 0.4*** 2.3
LLL 17.3 9.0 6.8

LnLO 5.1 4.9 2.3
PLLn 3.1 3.8 13.6
LLO 17.2 9.2 4.5
LnOO 1.3*** 1.1*** 0.0
LLP 12.1 10.6 19.4

LnOP 1.4*** 1.6*** 2.6***
PPLn 0.3*** 0.3*** 2.5
OOL 8.4 3.0 1.2

LLS 3.0 9.7 0.0
POL 8.3 7.3 12.4
(POL and SOLn)
PPL 1.5 2.2 16.0
OOO 2.9 1.4*** 0.6***
SOL 2.6 8.3 0.5***
OOP 2.2 1.0*** 0.9
PSL(LnSS)**** 1.0(SLP)**** 5.6**** 2.6(PSL)****

(LnSS and PSL)
PPO 0.3 0.6*** 1.4
PPP 0.1*** 0.4*** 0.1
OOS 0.2 1.2 0.1

SLS 0.9*** 5.8 0.2***
SOP 0.4*** 1.2*** 0.4***
PPS 0.2*** 0.7*** 0.4***

SOS 0.2*** 1.5 0.1***
PSS 0.1*** 0.3*** 0.2***
SSS 0.1*** 0.1*** 0.1***
Unidentified 0.6 1.4 0.2

* Determined by RP-HPLC with flame-ionization detection.
** TAG confirmed by RP-HPLC quadrupole mass spectrometer analysis.
*** TAG confirmed by HPLC retention with respect to standard TAG.
**** TAG PLS and LnSS have the same HPLC retention times. These TAGs were confirmed
in the respective soybean varieties by HPLC quadrupole mass spectrometry analysis.

Page 105
Figure 2.50.
Chromatogram of normal soybean oil triacylglycerols by an
RP-HPLC-ELSD. Effluent also introduced into a quadrupole mass
spectrometer via atmospheric pressure chemical ionization.
Triacylglycerol fatty acids S: stearic; P: palmitic; O: oleic; L: linoleic;
Ln: linolenic. (B) Chromatogram of high -palmitic soybean oil
triacylglycerols. (C) Chromatogram of high-stearic
soybean oil triacylglycerols.
Page 106
Figure 2.51.
RP-HPLC with FID chromatogram of normal, high stearic, and high palmitic
soybean oil triacylglycerols. Triacylglycerol fatty acids (S: stearic;
P: palmitic; O: oleic; L: linoleic; Ln: linolenic).
It has been established many times that fats and oils having similar or identical fatty acid composition,
but different triacylglycerol structures, can show different biological activities. These findings made
necessary the development of methods suitable for the stereospecific analysis of TGs in various oils and
fats. The number of studies dealing with this interesting field of chromatographic analysis is
surprisingly low, thus the stereospecific analysis of monounsaturated triacylglycerols in cocoa butter
[134]. Cocoa butter was extracted from unroasted cocoa beans with chloroform, and the TG fraction was
separated from the raw cocoa butter on a silica column using chloroform as eluent. TG fraction was
further purified by preparative TLC on silica plates with n-hexane:diethyl ether 85:15. The spots were
detected with 2, 7-dichlorofluorescein solution and extracted with diethyl ether. Argentation HPLC was
used for the separation of the monounsaturated TG fraction (eluent: n-hexane/diethyl ether 350:1 v/v;
flow rate 0.5 mL/min, detection wavelength 220 nm). The monounsaturated TG fraction was further
separated into three fractions by RP -HPLC (octadecylsilica column 250 × 4.6 mm I.D.; acetoni-
Page 107
TABLE 2.48. Stereospecific Analysis of Cocoa Butter Triacyglycerols and its

Monounsaturated Fraction.
Composition(mol%)*
Acyl Group Total sn-1 sn-2 sn-3
Total triacylglycerols
16:0 26.0 36.9 2.5 40.2
16:1n-9 0.2 0.2 0.2 0.3
18:0 35.3 52.8 1.9 52.4
18:1n-9 34.3 9.1 87.5 3.9
18:2n-6 3.0 0.7 7.4 0.6
18:3n-3 0.2 ND 0.4 ND
20:0 1.0 0.4 ND 2.7
Monounsaturated fraction**
16:0 28.7 42.1 3.8 41.7
18:0 37.6 54.8 3.3 54.4
18:1n-9 33.3 3.2 92.9 2.8
20:0 0.4 ND ND 1.1

*Wt% was calculated fom peak area percent using the calibration factors obtained from
GLC of reference mixtures. Mol% was calculated from wt% and the molecular weight of
each component.
** Separated from total triacylglycerols by argentation TLC and HPLC (yield 81.38).
trile/tetrahydrofuran 7:3 v/v; flow rate 1 mL/min; detection wavelength 220 nm). The partial hydrolysis
of the fractions of RP -HPLC analysis was carried out with ethyl magnesium bromide, and the
hydrolyzed mixture was reacted with 3,5-dinitrophenyl isocyanate. The di-3,5-dinitrophenylurethane
(3,5-DNPU) derivatives of the 1- and 2-monoacyglycerols (MG) were purified with preparative TLC on
silica layers using chloroform:acetone (96:4 v/v). The chiral separation of 1-MG derivatives was
achieved on two Sumichiral OA-4100 columns (250 × 4 mm I.D.) connected in series. The eluent was
n-hexane/1,2-dichloroethane/ethanol (40:12:3 vol/vol/vol); the flow rate was 0.5 mL/min at -10°C.
Detection wavelength was 254 nm. Fatty acid analysis was carried out on the methyl esters using
capillary GC. The results of the stereospecific analysis of total and monounsaturated triacylglycerols are
compiled in Table 2.48. The data indicate that the fatty acid distribution in cocoa butter is relatively
simple; palmitic and stearic acids are primarily located at the sn-1 and sn-3 positions. The results of the
stereospecific analysis of the monounsaturated triacylglycerol fractions separated with RP-HPLC are
presented in Table 2.49. As the triacylglycerols are separated in RP-HPLC according to the number of
carbon atoms, the three fractions represent TGs with 48, 50, and 52 equivalent carbon numbers,
respectively. The calculated composition of monounsaturated TGs is shown in Table 2.50. It was
Page 108
TABLE 2.49. Stereospecific Analysis of Each Fraction Separated from Total

Monounsaturated Fraction. *
Acyl Group mL% and SD
Acyl Group Total sn-1 sn-2 sn-3
Peak 1 (18.2%) **
16:0 66.4 95.8 ± 1.4*** 7.3 ± 0.2 96.1 ± 1.8
18:0 0.4 0.5 ± 0.0 0.4 ± 0.0 0.4 ± 0.0
18:1n-9 33.2 3.2 ± 0.3 92.3 ± 1.5 3.4 ± 0.1
Peak 2 (46.8%)
16:0 33.1 48.7 ± 1.0 3.2 ± 0.1 47.9 ± 0.8
18:0 33.5 47.6 ± 0.9 3.4 ± 0.1 48.9 ± 1.2
18:1n-9 33.4 3.6 ± 0.1 93.4 ± 1.7 3.1 ± 0.1
Peak 3 (35.0%)
16:0 1.6 4.0 ± 0.2 0.4 ± 0.0 0.3 ± 0.0
18:0 64.5 93.5 ± 1.2 6.9 ± 0.2 92.9 ± 1.5
18:1n-9 32.4 2.5 ± 0.1 92.7 ± 1.8 3.0 ± 0.1
20:0 1.5 ND ND 3.8 ± 0.2

* Fractions 1-3 were separated from the monounsaturated farction by RP-HPLC.
** Yields (wt%) of each fraction by weighing.
*** Mean value and SD in triplicate analyses.
established that acyl migration was negligible during the analytical procedure; that is, the method is
reliable and the data reflect the true composition of monounsaturated triacylglycerols in cocoa butter.
A similar method was used for the stereospecific analysis of TGs containing τ-linolenic acid in Evening
Primrose and borage oils (EPO and BO) [ 135]. TGs were first separated from the other components in
the oils on silica TLC
TABLE 2.50. Estimated Composition of the Monounsaturated Triacylglycerols.
Peak* Composition (mol%)
Triacylglycerol Number mol% Each Peak Total

POP-TG 1 18.9 100 19
POS-sn-TG ** 2 47.1 51 24
SOP-sn-TG 49 23
SOS-TG 3 34.0 96.2 32.7
POA-sn-TG *** 3.8 1.3
* Peaks separated by RP-HPLC.
** POP-TG, 1,3-dipalmitoyl-2-oleoyl-glycerol; POS-sn-TG, 1-palmitoyl-2-oleoyl-
3-stearoyl-sn-glycerol; SOP-sn-TG, 1-stearoyl-2-oleoyl-3-palmitoyl-sn-glycerol;
SOS-TG, 1,3-distearoyl -2-oleoyl-glycerol; POA -sn-TG, 1-palmitoyl-2-oeoyl-3-
eicosanoyl-sn-glycerol.
*** A, eicosanoyl.

Page 109
plates using a hexane:diethyl ether:formic acid (80:20:2, v/v) mixture as eluent. After development, the
TGs were extracted with chloroform:methanol (1:1, v/v) and further fractionated with RP-HPLC. The
individual fractions were selectively hydrolyzed with ethyl magnesium bromide and then reacted with
(S) - (+) - 1 - (1-naphthyl) ethyl isocyanate. The resulting sn-1,2(2,3)-DG urethane derivatives were
separated on two silica columns connected in series. The isocratic eluent was 0.3% 1-propanol
(containing 2% water) in isooctane. The flow rate was 1 mL/min and the detection wavelength 280 nm.
The fatty acid composition of the fractions was determined by capillary GC. Control experiments with
pure standard indicated that the transmigration of fatty acids causes about 4% absolute error. The fatty
acid compositions of the individual TG fractions isolated from EPO and BO are compiled in Table 2.51.
A characteristic chromatotogram is shown in Figure 2.52. It was established that the various sn-DG
naphthylethyl urethane derivatives are well separated under these HPLC conditions. However, in the
case of samples containing long-chain fatty acids, the peak of the sn-1,2-DG derivatives with long fatty
acid chains overlaps with the peak of sn-2,3-DG derivatives containing fatty acids of normal length.
TABLE 2.51. Positional Fatty Acid Analysis of the Individual TG Fractions Isolated from EPO
and BO (expressed as mole %).
Fatty Acid * TG Species ** All sn-1*** sn-2**** sn-3 §
EPO
LGG
18:2 35.2 59.8 25.5 20.5
18:3 64.8 39.5 75.3 79.6
LGL
18:2 66.8 79.8 68.9 51.7

18:3 33.2 20.2 32.4 47.0
OGL
18:1§§ 34.4 36.8 32.6 33.8

18:2 35.8 46.8 41.8 18.8
18:3 29.9 16.7 25.3 47.7
PGL
16:0 28.5 59.1 1.5 24.9

18:2 36.8 24.6 52.0 33.8
18:3 34.6 16.2 49.6 38.0
OLL
18:1§§ 33.6 27.2 41.4 32.2

18:2 66.4 72.8 58.6 67.8
PLL
16:0 30.0 48.2 6.4 35.4
18:1§§ 2.0 ND ND ND
18:2 67.0 42.8 103 55.6
18:3 1.0 ND ND ND

Page 110
TABLE 2.51.
Fatty Acid * TG Species ** All sn-1 *** sn-2 **** sn-3 §
Borage oil
LGG
18:2 33.9 88.8 5.6 7.2

18:3 66.1 12.0 93.5 92.8
LGL
18:2 65.4 92.1 60.8 43.3

18:3 34.8 8.4 38.8 57.2
OGG
18:1§§ 27.5 65.4 2.8 14.2

18:2 12.2 14.5 47.1 79.7
18:3 60.4 20.2 101 6.1
PGG
16:0 30.4 57.4 38.4 –4.6

18:2 3.3 3.9 9.3 –3.3
18:3 66.3 38.5 53.0 107
OGL
18:1§§ 33.0 20.2 23.4 55.4
18:2 33.9 68.1 19.5 14.1
18:3 33.0 11.2 57.4 30.4
PGL
16:0 31.3 43.7 27.3 22.9
18:2 33.4 44.0 27.6 28.6
18:3 35.3 12.5 44.9 48.5
OLL
18:1§§ 33.6 15.4 21.0 64.4
18:2 66.4 84.6 30.0 35.5
SGL and PGO
16:0 18.6 36.5 11.3 8.0

18:0 13.5 2.4 22.2 15.6
18:1§§ 19.1 8.5 24.3 24.5
18:2 15.5 32.9 1.7 11.9
18:3 33.4 19.8 40.6 39.8
* Unless otherwise indicated, the unsaturated fatty acids are from the n-6 family.
** Abbreviations: P = palmitic; S = stearic; O = oleic; L = linoleic; G = τ-linolenic acid. Three
letters in column TG Species indicate the three fatty acids composing the triglyceride.
*** 3 × [TG] – 2 × [sn-2,3-DG urethanes].
**** 3 × [TG] – [sn-1] – [sn-3].
§ 3 × [TG] – 2 × [sn-1,2-DG urethanes].
§§ n-7 and n-9.
Science—NL, Sara Burgerhartstraat 25, 1055 KV Amdsterdam, The Netherlands.
Page 111
Figure 2.52.
HPLC profiles of the sn-1,3-, sn-1,2-, and sn-2,3-DG naphtylethyl urethane
derivatives obtained from BO (left) and EPO (right). The peaks under the letter
A represent the sn -1,3-DG derivatives; under the letter B, the sn -1,2-DG derivatives;
and under the letter C, the sn-2,3-DG derivatives. Conditions: two silica columns in
series (150 × 4.6.mm I.D., 3 µm nd 250 × 4.6 mm I.D., 3 µm); mobile phase,
iso -octane:2-propanol (containing 2% water) (99.7:0.3, v/v)
at 1 mL/min and UV detection at 280 nm.
2.2.3—
Steroids
Although the main constituents of both fats and oils are triglycerides, some minor components such as
sterols and tocopherols also have a considerable impact on nutritional value and can be used for the
detection of adulteration. Because of their volatility, various gas chromatographic methods have been
used frequently for the separation and quantitative determination of steroids in fats and oils. Thus, TLC
combined with GC was used for the identification and quantification of cholesterol oxides in fresh and
stored butter, butter cake, butter cookie, whole egg powder [ 136], and meat products [ 137]. It is generally
accepted that blood cholesterol increases when dietary cholesterol does and may result in
atherosclerosis illnesses. Because of the paramount importance, many chromatographic methods have
been developed for the quantitative determination of cholesterol in various foodstuffs [ 138], in
unirradiated and irradiated meats [139], and so on. As the public interest in cholesterol levels of various
food products increased considerably, the rapidity of the analysis markedly influences the output of
control laboratories, with the time-consuming sample preparation frequently being the limiting step
before GLC analysis was successfully automated [ 140].
The block diagram of the automated sample preparation system is shown in Figure 2.53. The sample
size depended on the type of food product, as
Page 112
Figure 2.53.
Cholesterol robot diagram.
demonstrated in Table 2.52. Saponification of the samples was carried out with ethanolic potassium
hydroxide solution. Saponification time can be specified according to the character of the sample. After
saponification, 1 mL of sample was removed and mixed for 10 s with 0.3 mL 6.4 M HCl and 4.0 mL of
6 ± 1% potassium hydrogen phthalate solution. Then the samples were transferred into C18 SPE
cartridges conditioned with 3 mL of methanol and 5 mL of water. Cartridges containing the samples
were dried for 13 min and eluted with 15 mL 2-propanol: hexane (15:85 v/v). The eluate was dried by
adding 1 g Na2SO 4. Samples were evaporated to dryness, derivatized with bis (trimethylsilyl)
trifluoracetamide/trimethylchlorosilane (BSTFA/TMS), and analyzed with capillary GLC. Separation
was carried out on a fused-silica column DB 1 (30 m × 0.25 mm I.D., film thickness, 0.25 µm).
Hydrogen was used as the carrier gas. The temperature of the injector and FID detector were 280°C and
300°C, respectively. The temperature program of the column was from 245°C at 5°C/min, raised to
285°C, and held for 18 min. It has been established that the recovery of the automated sample
preparation method described above is over 97%, and the results of the traditional manual sample
preparation method and the new automated procedure did not differ at the 95% significance level.
The removal of interfering triglycerides from edible oils and fats before the GC determination of
cholesterol has also been automatized [ 141]. The block scheme of the continuous transesterification
system is shown in Figure 2.54.
Page 113
TABLE 2.52. Examples of Optimum Sample Size as a Function of Matrix.

Sample Type Recommended Sample Weight (mg)
Egg yolk 100–150
Animal oils 100–200
Vegetable oils 200–400
Butter fat 250–300
Soybean oil 400–500
Viscous dressings 300–400
Natural cheese 300–500
Process cheese 300–400
Pourable dressing 400–500
Sour cream 400–600
Ice cream 400–600
Ice milk 500–600
Milk 700–900
Cream cheese 300–400
Pasta 400–500
Frozen dinner 400–500
Luncheon meats 300–400
Breakfast sausage 200–300
Seafood 200–400
A 10- 250-mg aliquot of oils or fats and 250 µ of 5 α -cholestane internal standard were dissolved in n-
hexane and passed through the systems at a flow rate of 0.4 mL/min. It was mixed with a methanolic
solution of 0.1 M potassium methylate and 20 mg/mL ascorbic acid (flow rate 0.4 mL/min). Fatty acid
methyl esters were formed in a reaction tube (400 cm × 0.5 mm I.D.) at 65°C. The mixture was cooled
in a 100-cm coil in cooling bath. The mixture was washed with a water stream (0.7 mL/min) to remove
excess reagents and reaction products. Water traces were retained by the desiccating column prior to
injection. A dimethyl polysiloxane-coated fused-silica capillary column (10 m × 0.53 mm I.D., film
thickness 2.65 µm) was used for the separation. The carrier gas was nitrogen; the injector and detector
(FID) temperatures were both 250°C. The temperature program was 210°C for 10 min, raised 2.5°
C/min to 235°C, and held for 15 min.
The method exposed good validation parameters: the coefficients of regression between the
solute/internal peak ratio and solute concentration (1-80 µg/mL) were 0.9991, 0.9993, and 0.9990 for
cholesterol, α -tocopherol, and α -tocopherol acetate, respectively. The detection limits were 0.5, 0.7,
and 0.8 in the same order. The relative standard deviations determined from the analysis of 11 samples
were 1.9%, 2.2%, and 3.1% for cholesterol, α -tocopherol and α -tocopherol acetate, respectively.
Typical chromatograms are shown in
Page 114
Figure 2.54.
Setup used for the continuous transesterification of triglycerides and determination
of cholesterol, α -tocopherol and α-tocopheryl acetate. P: pump; CB: cooling bath;
PS: phase separator; W: waste; DC: desiccating column;
IV: injection valve; GC: gas chromatograph.
Elsevier Science—NL, Sara Burgerhartstraat 25,
Figure 2.55. The fatty acid methyl esters are eluted before the internal standard, not interfering with the
determination of the sterols. The chromatograms indicate that the oils contain different sterols in
different quantities, which can be used for their identification. The mean value and the standard
deviation of the sterol contents of some oils and fats are given in Table 2.53. The data indicate that the
cholesterol and α-tocopherol content of oils and fats is highly different and depends considerably on the
type of the oil or fat. It has been stated that the method eliminates the difficulty caused by the high
triglyceride content of the samples. It is suitable for the quantitative determination of cholesterol and α-
tocopherol in samples with high triglyceride content, and it is rapid and relatively easy to carry out.
A continuous separation module was also developed for the GC determination of cholesterol,
campesterol, β-sitosterol, and stigmasterol in vegetable oils and lard [ 142]. The method separates sterols
and 25% of the triglycerides on a silica column and transesterify the remaining triglycerides with acetyl
chloride-methanol to fatty acid methyl esters, which elute before the sterols in GC. The scheme of the
module is depicted in Figure 2.56.
Samples of 9-460 mg were dissolved in 5 mL of n-hexane and was introduced into the sorbent column
(40 mm × 2.5 mm I.D., silica sorbent) at a flow rate of 0.4 mL/min for 3 min by pump 1. Sterols and
approximately 25% of triglycerides were eluted from the column with methanol, mixed with 0.7 M
acetyl chloride in n-hexane, heated in the TEC coil (500 cm × 0.5 mm I.D.) at 70°C, and cooled in the
cooling bath. Excess acetyl chloride was decomposed
Page 115
Figure 2.55.
Chromatograms obtained for a sample solution containing 150 mg of (A) sunflower
seed oil, (B) wheat-germ oil, and (C) butter in 5 mL of n-hexane. Peaks:
1 = cholesterol; 2 = α-tocopherol; 3 = α-tocopherol acetate; IS = internal standard.
by a water stream of 0.6 mL/min, and the extract was desiccated in a 50 mm × 3 mm I.D. column filled
with sodium aluminosilicate pellets. A fused-silica capillary column (10 m × 0.53 mm I.D., film
thickness 2.65 µm of 100% cross-linked polydimethylsiloxane) was employed for the separation.
Nitrogen was used as the carrier gas, and the injector and detector (FID) temperatures were 300°C. The
column temperature increased from 230°C (11 min) to 235°C (35 min) at 10°C/min. The validation
parameters of the method are compiled in Table 2.54. The good validation parameters (recovery being
between 91.4% and 103.9%) indicate that the use of the method results in increased precision and
sampling frequency, and it is suitable for the detection of the adulteration of oils, as demonstrated in
Table 2.55.
Not only capillary, but also packed column, GC has been used for the separation and quantitative
determination of cholesterol. Thus, the cholesterol content of milk, cream, and butter was measured by
employing a packed GC column [143]. Cholesterol was extracted from the samples with diethyl ether
and separated on a glass column [160 cm × 4 mm I.D., packed with 3% OV on 80/100 mesh
Chromosorb W(HP)]. Argon was used as the carrier gas. The injector and detector temperatures were
325°C and 350°C, respectively. The column temperature increased from 230°C to 340°C at a rate of 5°
C/min and was kept constant to the end of the separation. The variation coefficient and the mean
recovery value were 4.32% and 94.30%, respectively. The cholesterol levels determined in milk, cream,
and butter fat samples are given in Table 2.56. It was stated that the method is suitable for the
quantitative
Page 116
TABLE 2.53. Concentrations (µ g/g) of Cholesterols, α-Tocopherol, and α-Tocopherol

Acetate in Oils and Fats as Determined by the Proposed Method.
Oil/Fat Cholesterol α-Tocopherol α-Tocopherol Acetate
Olive oil — 175.8 ± 2.7 —
Sunflower-seed oil — 473.0 ± 13.0 —

Corn oil — 130.8 ± 4.3 —
Wheat-germ oil 82.4 ± 2.0 409.8 ± 10.1 2772.4 ± 70.8 *

Grape-seed oil 45.6 ± 1.7 — —
Castor-bean oil 91.6 ± 2.3 — —
Sweet almond oil 58.1 ± 1.5 164.8 ± 5.3 —
Margarine — 126.4 ± 3.1 —
Butter 1979.5 ± 62.8 — —
Lard 953.7 ± 16.8 — —

* Added to samples.
determination of cholesterol in dairy products. A collaborative study was conducted on the

determination of cholesterol in various food products by direct saponification and capillary gas
chromatography [ 144]. Food samples were saponified at the boiling point with ethanolic KOH solution
for 70 ± 10 min. The unsaponified fraction (containing cholesterol and other sterols) is extracted with
toluene. The toluene fraction is washed with water and dried with Na2SO4. The dry toluene solution is
dried, dissolved in dimethylformamide and derivatized by adding hexamethyldisilane and
trimethylchlorosilane. The separation and quantitative determination of cholesterol was carried out on a
capillary column (25 m × 0.32 mm I.D., film thickness of cross-linked 5% phenylmethyl silicone or
methyl silicone gum 0.17 µm). Helium was used as the carrier gas, and the injector and FID
temperatures were 250°C and 300°C, respectively. The temperature program was: 190°C for 2 min;
raised 20°C/min to 230°C, and held 3 min; and an increase of 40°C/min to 255°C and held 25 min. The
validation parameters of the method are presented in Table 2.57. It has been stated that this direct
saponification-GC method is suitable for the determination of cholesterol in various food products.
Not only cholesterol, but also other sterols have been determined in foods by GC. Thus, oxysterols in
spray dried eggs were separated by GC using and comparing three different preparation methods [145].
Lipids were extracted from a 0.65-g aliquot of powdered egg with 15 mL of chloroform:methanol (2:1
v/v). After 30 min of stirring, the solution was filtered, the solid residue extracted again with 10 mL of
chloroform:methanol (2:1 v/v) for 10 min and rinsed with 5 mL of the same mixture. The liquid extract
was washed with 5 mL of distilled water and evaporated to dryness. Cold saponification was carried out
by adding 10 mL of methanolic 1 M KOH to the extract and held
Page 117
Figure 2.56.
Experimental setup for isolation-preconcentration of sterols and
transesterification of triglycerides in oils. The broken line represents the isolation
and preconcentration of sterols, while the solid lines correspond to elution,
transesterification of triglycerides and determination of sterols (P: pumps;
IV: injection valves; C: sorbent column; PS: phase separator; W: waste; TEC:
transesterification-extraction coil; CB: cooling bath; DC: desiccating column;
GC:gas chromatograph.) Flow conditions: sample, eluent, and extractant flow
rates at 0.4 mL/min; water flow rate, 0.6 mL/min; injection volume, 5 µm.
Elsevier Science—NL, Sara Burgerhartstraat 25,
at room temperature for 20 h. After the saponification step, 10 mL of diethyl ether and 10 mL of
distilled water were added to the reaction mixture and shaken, and the organic phase was separated. The
aqueous phase was extracted twice with 10 mL of diethyl ether. The collected organic phase was
washed with 5 mL of 0.5 M KOH followed by 2 × 5 mL of distilled water, dried over anhydrous
sodium sulphate, and evaporated at 30°C. The flow diagram of the three methods applied is shown in
Figure 2.57. Silanization was identical for each method: oxysterols were dissolved in anhydrous
pyridine and silanized with N, O-bis(trimethylsilyl)-acetamide-trimethylchlorosilane-N-
trimethylsilylimidazole (3:2:3). A fused-silica capillary column (25 m × 0.25 mm I.D., film thickness of
methyl silicone stationary phase 0.13 µm) was used for the separation of sterols. Helium was the carrier
gas, and injector and FID temperatures were 290°C and 350°C, respectively. The temperature gradients
were (I) from 210°C to 240°C at 6°C/min, from 240 to 270°C at 4°C/min, from 270 to 290°C at 2°
C/min, and 5 or 82 min at 290°C; (II) from 210°C to 264°C at 2°C/min, from 264 to 290°C at 5°C/min,
and 5 or 80 min at 290°C. Typical chromatograms of methods are shown in Figure 2.58. Both the
chromatograms and the validation parameters (recovery and linearity of response) indicated that method
III, including saponification and silica cartridge
Page 118
TABLE 2.54. Analytical Figures of Merit of the Determination of Sterols.

Correlation Linear Range
Sterol Regression Equation* Coefficient** (mg/100 g oil)
Cholesterol A = –0.012 + 0.534. X 0.997 0.1–7.6

Campesterol A = –0.012 + 0.306. X 0.995 0.2–7.6
Stigmasterol A = –0.014 + 0.477. X 0.997 0.1–7.6
β-Sitosterol A = –0.010 + 0.388. X 0.994 0.1–7.6
Detection Limit Preconc.
Sterol (mg/g oil) RSD (%) *** Fraction ****
Cholesterol 0.04 3.6 2.5
Campesterol 0.08 2.7 2.7
Stigmasterol 0.06 3.4 2.4
β-Sitosterol 0.08 1.6 2.3
* A = Analyte/internal standard peak ratio; X = concentration (mg/100 g oil or fat.
** n = 8.
*** n = 11 at 20 mg/L.
**** Preconcentration factor for 3 min sampling.
Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amdsterdam, The
Netherlands.
TABLE 2.55. Sterol Profile of Edible Oil Samples (g/100 g total sterols).*
Oil Cholesterol Campesterol Stigmasterol β-Sitosterol Other **
Olive 1 — 2.9 ± 0.1 — 97.1 ± 0.6 —
Olive 2 — 1.9 ± 0.1 — 98.1 ± 0.5 —
Sunflower seed 1 1.0 ± 0.1 6.7 ± 0.2 9.3 ± 0.3 70.9 ± 0.8 12.1 ± 0.3
Sunflower seed 2 1.1 ± 0.1 7.0 ± 0.3 9.8 ± 0.2 68.3 ± 0.7 13.8 ± 0.3
Maize — 22.3 ± 0.4 1.1 ± 0.1 67.3 ± 0.9 9.3 ± 0.3
Wheat germ — 37.1 ± 0.7 — 62.9 ± 0.8 —
Coconut 3.2 ± 0.2 24.0 ± 0.8 35.9 ± 0.9 36.9 ± 1.1 —
Lard 100 ± 0.0 — — — —
Adulterated olive*** — 4.7 ± 0.2 3.8 ± 0.28 7.7 ± 0.9 3.8 ± 0.1
Adulterated olive 2**** 20.5 ± 0.5 2.0 ± 0.1 — 77.5 ± 1.1 —
* Each value represents the mean of three measurements ± standard deviation.

** This column includes sterol not included in the above columns.
*** Olive oil adulterated with sunflower seed oil.
**** Olive oil adulterated with lard.
Reprinted from Reference [ 142], copyright © 1995. With kind permission from Elsevier Science—NL,
Sara Burgerhartstraat 25, 1055 KV Amdsterdam, The Netherlands.
Page 119
TABLE 2.56. Content of Cholesterol mg/100 g Fat in Milk -, Cream-, and Butter-Isolated Fat.
Sample Milk Cream Butter
1 276.47 281.14 291.55
2 276.63 231.56 264.00
3 225.78 225.77 213.91
4 243.55 217.29 244.24
5 173.77 261.36 212.50
Average 239.24 243.42 245.24
Standard deviation (mg/100 g fat) 42.63 26.83 33.73
Variation coefficient (%) 17.18 11.02 13.75
Reprinted from Reference [ 143] with permission from the Polish Journal of Food and Nutrition.
purification, is the most reliable for the determination of oxysterols in dried egg powders.
The gas chromatographic profile of sterols has been used for the detection of adulteration of candied
fruits (substitution of the fruits of Cucurbitaceae with the roots of Cruciferae) [ 146]. Samples were
lyophilized, and the lipidic substances were extracted with diethyl ether. The lipid fraction was
saponified with ethanolic KOH under reflux for 3 min. The unsaponified fraction was extracted three
times with diisopropyl ether, then washed with water to neutrality and dried with anhydrous sodium
sulfate. Ether was removed in vacuum at 40°C and the residue dissolved in 0.5 mL of chloroform. The
extract was further purified with TLC. Silica plates were impregnated by immersing 0.2 N methanolic
KOH solution for 10 s, dried for 2 h at room temperature, and activated at 103°C for 2 h. Sterols were
separated by n-hexane-diethyl ether (2:1 v/v) eluent and were detected with dichlorofluoresceine. The
part of the plates containing sterols were scraped off, extracted with methylene chloride, and dried
under vacuum. Sterols were silanized by adding pyridine, 1,1,1,3,3,3-hexamethyldisilazane and
trimethylchlorosilane to the residue. After 5 min reaction time, the samples were dried under nitrogen
stream and dissolved in heptane. A fused-silica capillary column (30 m × 0.3 mm I.D., film thickness of
methylphenyl silicone 0.15 µm) was employed for the separation of steroids. The carrier gas was
hydrogen, and injector, FID, and column temperatures were 295°C, 295°C, and 260°C, respectively. A
slightly different GC/MS system was employed for the identification of the individual sterols.
Characteristic chromatograms obtained from Cucurbitaceae and Cruciferae are shown in Figure 2.59.
The chromatograms clearly show that Cucurbitaceae and Cruciferae differ considerably in their sterol
composition; that is, the determination of the sterol profile can be used for the identification of the raw
materials used for the preparation of candied fruits. The concentrations of sterols, being the most
characteristic for the detection of adulteration, are
TABLE 2.57. Method Performance for Determination of Cholesterol in Foods by Direct Saponification Method.
Mean rec. sr sR RSDr RSDR r*
(mg/100 g) (mg/100 g)
Sample (mg/100 g) (mg/100 g) (%) (%)
Butter cookies 28.1 0.75 0.83 2.69 2.95 2.11
Vegetable-bacon baby
food 3.71 0.36 0.47 9.60 12.6 1.00
Vegetable-chicken baby
food 4.69 0.32 1.00 6.82 21.3 0.89
Skinless wieners 76.7 2.79 5.98 3.63 7.80 7.80
NIST egg powder (SRM

No. 1845)*** 1965 60.0 82.1 3.05 4.18 168
Commercial powdered
eggs 1195 65.7 66.6 5.50 5.57 184
Cheez Whiz ® 66.9 1.61 3.78 2.40 5.64 4.50
sr = within-laboratory variance; RSD r = relative standard deviation of within-laboratory variance (repeatability); sR = between laboratory
variance; RSD R = relative standard deviation of between laboratory variance (reproducibility).
* r = 2.8 × sr.
** R = 2.8 × sR.
*** NIST certified value at 1900 ± 20 mg/100 g.

Page 121
Figure 2.57.
Diagram of the three methods compared.
Page 122
Figure 2.58.
Chromatograms of silanized extracts obtained from a dried egg sample (temperature
program II). (A) method II and (B) Method III. α -CE = cholesterol-5 α, 6 α-epoxide;
7β-HC = 7 β-hydroxycholesterol; CT = cholestanetriol; 7 -KC = 7 -ketocholesterol;
25-HC = 25 -hydroxycholesterol.
Page 123
Figure 2.59.
Sterol chromatograms of (A) Cucurbitaceae and (B) Cruciferae:
1 = cholesterol; 2 = brassinolide; 3 = campesterol; 4 = not identified;
5 = 24-ethylcholesta-8,22 -dienol; 6 = 24 -methylcholest-7-enol and
24-methylenecholest-7-enol; 7 = β-sitosterol; 8 = α-spinasterol;
9 = not identified; 10 = ∆ -5-avenasterol; 11 = not identified;
12 = ∆-7-stigmasterol and 24 -ethylcholesta-7,22-dienol;
13 and 14 = not identified; 15 = ∆-7-avenasterol.
(Reprinted fom Reference [ 146] with permission
from Lavoiser Abonnements.)
given in Table 2.58. The method—completed with light and electron microscopic investigations—was
proposed for the analysis of the botanical origin of the components in various candied fruits.
Using a more sophisticated GLC method, it was established that the sterol composition of vegetable oils
is more complex than it was previously assumed [ 147]. Silanized sterols were separated by two GC
methods. Method A: fused-silica capillary column (25 m × 0.32 mm I.D., film thickness of LAP 0.1
µm). Temperatures of injector, column, and FID were 300°C, 300°C, and 220°C, respectively.
Hydrogen was employed as the carrier gas. Method B: two fused-silica capillary columns (100 m total
length × 0.25 mm I.D., film thickness of CP Sil 88 0.2 µm). Temperatures of injector, column, and FID
were 260°C, 200°C, and 300°C, respectively. Hydrogen was employed as the carrier gas. The gas
chromatographic profiles of sunflower, soybean, peanut, and corn oils determined with methods A and
B are shown in Figures 2.60 and 2.61, respectively. The chromatograms prove that method B is superior
to method A in
Page 124
TABLE 2.58. Variation in Sterol Contents (%) According to Botanical Origin.

Campesterol β-Sitosterol α–Spinasterol ∆-7-Stigmasterol
Cucurbitaceae 0–1.6 0–0.1 21 –48 42–66
Cruciferae 11–21 77 –86 <0.1 0–0.5
Reprinted from Reference [ 146] with permission from Lavoiser Abonnements.
separating fractions 2 and 3. Mass spectrometry indicated that the molecular masses of peaks 2 and 3
are identical. It was concluded that peak 2 is 22,23-dihydribrassicasterol, an epimer of campesterol
(peak 3).
Thin-layer chromatography has also been employed in the separation and identification of the various
oxidation products of cholesterol as a prepurification step. Thus, the oxidation products of cholesterol in
butter [ 148] and eggs [ 149] were separated first with various TLC techniques, and the fractions were
further analyzed either with GC or with HPLC. The oxidation products of cholesterol in milk and dairy
products have also been separated by TLC [ 150]. Cholesterols were separated from butter by SPE: 2 g of
butter were dissolved in 3 mL of hexane at 40°C and applied to an aminosilica cartridge preconditioned
with 3 mL of hexane. Triacylglycerols and cholesterol were eluted with 8 mL of hexane-ethyl acetate
(9:1), and then oxysterols were removed from the cartridge with 8 mL of acetone. Acetone was
removed in vacuum, and the residue was dissolved in 500 µL of acetone. Separation was carried out on
silica plates using two eluent systems. Eluent I: hexane:ethyl acetate (9:1) and eluent II: methylene
chloride:acetone:toluene (2:1:1). Plates were dried between the application of the two consequent
eluents. After development, the spots were detected by spraying the plates with sulfuric acid (500 g/kg)
and heated at 120°C for 10 min; then the color of spots were evaluated in visible and UV lights (366
nm). The Rf values and the colors of standards are presented in Table 2.59. This method was used for
the analysis of butters; however, it was established that the probable presence of polar impurities such
as mono- and dialkylglycerides considerably decrease the sensitivity of the method.
Not only GC and TLC, but also HPLC, have been frequently used for the determination of cholesterol.
Thus, an RP -HPLC method was developed for the determination of cholesterol in eggs [151] and in
many other food products. Cholesterol was also quantitatively determined in butter by HPLC [152]. A
0.2-g aliquot of butter oil was dissolved in 10 mL of 96% ethanol, and the solution was saponified by 2
mL of KOH (50% w/v) at 70°C for 8 min. The sample was cooled at room temperature, extracted with
50 mL of hexane-diethyl ether (1:1 v/v), and washed with 30 mL of water. An aliquot of 25 mL was
evaporated to dryness and redissolved in 10 mL of HPLC eluent. Sterols were separated on a µ-Porasil
column (300 × 3.9 mm I.D.) using 7%
Page 125
Figure 2.60.
Gas chromatogram of the sterols in (A) sunflower,
(B) soybean, (C) peanut, and (D) corn oils using method
A: 1 = α-cholestanol; 2 = 22,33-dihydro-brassicasterol;
3 = campesterol; 4 = not identified; 5 = stigmasterol;
6 = ∆-7-campesterol; 7 = sitosterol; 8 = ∆-5-avenasterol;
9 = ∆-7-stigmastenol; 10 = ∆-7-avenasterol.
(Reprinted from Reference [ 147] with permission from Stazione
Sperimentale Oli e Grassi and the corresponding author.)
Page 126
Figure 2.61.
Gas chromatogram of the sterols in (A) sunflower,
(B) virgin olive, (C) and corn oils using method
B. 1 = α -cholestanol; 2 = 22,33 -dihydrobrassicasterol;
3 = campesterol; 4 = stigmasterol; 5 = sitosterol;
6 = ∆-5-avenasterol; 7 = ∆-7-stigmastenol;
8 = ∆-7-avenasterol.
Page 127
TABLE 2.59. R f Values and the Colors of Standards in Visible and UV Lights (366 nm).
Color
Standard Rf Visible 366 nm
Cholestane-3β ,5 α.6 β-triol 0.05 Yellow-brown White
Cholestane-3β,5α,diol-6-one 0.20 Yellow Blue
7 α-Hydroxycholesterol 0.23 Blue Brown
7 β-Hydroxycholesterol 0.27 Blue Brown
Cholestane-3β-ol,5α,6α-epoxyde 0.39 Brown Brown

7-Cetocholesterol 0.42 * *
25-Hydroxycholesterol 0.44 Brown Brown

20α-Hydroxycholesterol 0.54 Brown Green
Cholesterol 0.58 Mallow Rose-
mallow
4-Cholestene-3-one 0.72 * Blue
* Can be detected under UV light at 254 nm.
2-propanol in hexane (v/v) as a mobile phase. The flow rate was 1 mL/min, and the detection
wavelength was set to 208 nm. Cholesterol and the internal standard pregnenolone are well separated
from each other and from the other constitutuents in butter oil, as demonstrated in Figure 2.62. The
validation parameters of the method are compiled in Table 2.60. The detection limit of the method was
0.2 mg cholesterol/gram of sample. The good validation parameters indicate that the method is accurate
and precise and can be used for the quantitative determination of cholesterol in animal fats. Since it has
been established that phytosterols coelute with cholesterol, the method cannot be employed for the
determination of the cholesterol content in samples containing vegetable oils or fats.
An RP-HPLC method was developed for the determination of cholesterol in muscle and adipose tissue
and in offal [ 153]. Samples were homogenized prior to extraction of cholesterol, and the stigmasterol
internal standard is added to 2 g of homogenate. The amount of added stigmasterol depends on the
probable cholesterol concentration of the sample. Extraction is carried out with 50 mL of n-hexane:2-
propanol (3:2 v/v) for 1 min in a mixer. The extract was filtered, and 0.5-2 mL (depending on the fat
content of the sample) was saponified with 10 mL of sodium methylate solution (4 g of NaOH
dissolved in 60 mL of methanol and 40 mL of diethyl ether, and 15 mg of phenolphtalein added).
Saponification was carried out at room temperature for 30 min, and then the mixture was acidified with
5 mL of methanolic HCl (10 mL of cc.HCl + 45 mL of methanol). The acidified mixture is extracted
with 20 mL of n-hexane, filtered through sodium sulfate, evaporated to dryness, and redissolved in 1
mL of 9% of diethyl ether in n-hexane. The solution was put on a
Page 128
Figure 2.62.
HPLC chromatogram of cholesterol and
(A) internal standard mix and of
(B) butter oil: 1 = cholesterol; 2 = pregnenolone
(internal standard). Eluting solvent: 7%
2-propanol in hexane; flow rate: 1 mL/min;
column: Waters µ Porasil (300 × 3.9 mm I.D.)
VV-GmbH Volkswirtschaftlicher Verlag.)
silica column (250 × 7 mm I.D.) and washed with 20 mL of 9% of diethyl ether in n-hexane and 3 mL
of dichloromethane. Cholesterol and the internal standard are eluted with 20 mL of dichloromethane,
and the solution is evaporated to dryness and dissolved in 200 µL of eluent. Cholesterol was separated
on an octadecylsilica column (220 × 4.6 mm I.D., particle size 5 µm) with acetonitrile/2-propanol
(80:20 v/v) eluent. The flow rate was 1.5 mL/min, and the detection wavelength was set to 210 nm.
Cholesterol and the stigmasterol internal standard are well separated from each other and from the other
impurities present in the samples, as shown in Figure 2.63. It was established that the recovery of the
method is between 92% and 105%, and it is linear between 20 mg and 450 mg cholesterol/100 g.
Because of its simplicity and reliability, the method was proposed for the routine determination of
cholesterol in meats and meat products.
Page 129
TABLE 2.60. Validation Parameters of the Method for the Determination of

Cholesterol.
Peak Area
Concentration
(mg/mL) Mean SD CV%
Within-Batch Precision Data. Analysis Carried Out in Triplicate
Cholesterol 0.01 83283 1311.5 1.6
Pregnenolone 0.0225 218001 2045.7 0.9
Cholesterol 0.025 212888 2156.2 1.0
Pregnenolone 0.0225 234027 2854.7 1.2
Cholesterol 0.05 398420 6025.8 1.5
Pregnenolone 0.0225 219494 2880.1 1.3
Cholesterol Concentration (mg/100 g)
Sample Mean SD CV%
Precision of the Results Obtained on Three Butter Oil Batches
Sample A (n = 292.5 5.75 1.96

13)
Sample B (n = 297.5 5.64 1.8

5)
Sample C (n = 299.8 4.34 1.4

5)
Cholesterol Concentration (mg/100 g)
GLC HPLC GLC HPLC
Data Obtained on Butter Oil and on Butter Oil at Lower Cholesterol Level Using
Two Different Analytical Methods
301.4 294.4 182.3 202.2
219.0 220.9 158.2 164.3
214.2 219.6 52.0 58.9

n = number of replicates.
Reprinted from Reference [ 152] with permission from VV-GmbH
Volkswirtschaftlicher Verlag.
Since the principles of separation in GC and HPLC are different, the combination of both methods
offers considerable advantages in the analysis of complicated mixtures. GC combined with HPLC has
also been employed for steroid analysis in foods. A combined method was used for the determination of
free and esterified sterols and of wax esters in olive, rapeseed, and sunflower oil and in butter fat [154]
and for the measurement of sterols and other minor components in olive oils [ 155]. Because GC on
polar capillary column is not suitable for the separation of 2,4- and 3,5-stigmastadiene, a combined
HPLC-GC method was developed for their separation [ 156]. As stigmastadienes are formed during
processing of oils, their determination may indicate the degree of heat treatment and bleaching of oils.
A 20% solution of the oil sample in n-hexane is separated from the triacylglycerols and squalene on a
silica column. After the elution of this fraction to the second column, the first
Page 130
Figure 2.63.
Chromatogram of a sample of (1) poultry meat and of a
(2) cholesterol/stigmasterol standard. A = cholesterol, B = stigmasterol.
(Reprinted from Reference [ 153] with permission from the
Redaction of Flieschwirtschaft.)
column was backflushed with dichloromethane. The fraction was further separated on a second silica
column with the same dimensions, using n-hexane as eluent (flow rate 300 µL/min, detection
wavelength 235 nm).
The HPLC separation profile of a refined olive oil is shown in Figure 2.64. The GC analysis of the
HPLC fractions was carried out on two different GC systems (System I: column: 25 m × 0.25 mm I.D.,
coated with 0.2 µm layer of dimethylpolysiloxane; temperature program from 170°C to 260°C at 2°
C/min. System II: column: 30 m × 0.25 mm I.D., coated with polyethylene glycol; temperature program
from 200°C to 245°C at 2°C/min). The separations of HPLC fractions by GC systems I and II are
shown in Figures 2.65 and 2.66, respectively. It can be concluded from the comparison of the LC-LC-
GC chromatograms that the polar polyethylene glycol stationary phase separates well the 2,4- and 3,5-
dienes whereas the separation capacity of the apolar methylpolysiloxane stationary phase is unsuitable
for this purpose.
A similar combined HPLC-GC method was developed for the determination of 3,5-stigmastadiene in
edible oils [ 157]. For the traditional GC method, the oils were saponified, extracted with n-hexane,
concentrated, separated on a silica column, and analyzed on a fused-silica capillary column (25 m ×
0.25 mm I.D., film thickness of methylphenyl silicone coating 0.25 mm). Combined
Page 131
Figure 2.64.
LC-LC-UV (235 nm) chromatogram of a refined olive oil.
The peak labeled ''3,5-dienes" contains (among others) the
3,5-isomers of campestadiene and stigmastadiene, that labeled
"2,4-dienes" those isomers that are coeluted with the
3,5-dienes from apolar GC columns. Bars numbered
1-3 indicate fractions analyzed by GC.
Sperimentale Oli e Grassi.)
Figure 2.65.
LC-LC-GC -FID chromatogram of the HPLC fraction comprising the
two peaks labeled in Figure 2.64. The methylpolysiloxane stationary
phase (PS-255) does not resolve the dienes separated by HPLC.
Stazione Sperimentale Oli e Grassi.)
Page 132
Figure 2.66.
LC-LC-GC-FID chromatogram of a refined olive oil, using a Carbowax-like
GC stationary phase; LC fraction of 1000 µL corresponding to 3 in
Figure 2.64. 2,4- and 3,5-dienes are fairly well separated.
(Reprinted from Reference [ 156] with permission
from Stazione Sperimentale Oli e Grassi.)
HPLC-GC analysis was carried out by injecting of the oil sample dissolved in n-hexane into a
Spherisorb S5W column (100 × 2 mm I.D.). Eluent was n-hexane at a flow rate of 200 µL/min, and the
detection wavelength was 235 nm. The eluted conjugated dienes were further separated on a fused-
silica capillary column (25 m × 0.32 mm I.D., 0.15 µm film thickness of methylphenyl silicone)
equipped with a 2 m × 0.32 mm I.D. uncoated precolumn and a 3 m × 0.32 mm I.D. retaining
precolumn. Dienes were transferred at 115°C column temperature; after 6 min the temperature was
increased to 235°C at 30°C/min, held for 5 min, and then increased to 260°C at 1.5°C/min.
Triacylglycerols were removed from the HPLC column by blackflushing with 1.6 mL of
dichloromethane. The HPLC and GC profiles of an olive oil sample are shown in Figures 2.67 and 2.68,
respectively. The chromatograms prove that the method separates well 3,5-stigmastadiene from the
internal standard (cholesta-3,5-diene) and from the other impurities present in the oil samples. The 3,5-
stigmastadiene contents of various oils determined by the official GC method and the combined HPLC-
GC method are given in Table 2.61.
Page 133
Figure 2.67.
HPLC trace of an olive oil sample showing the transferred peak.
Figure 2.68.
GC trace of the transferred fraction (2.10 min) of the same olive
oil sample, showing the 3,5-stigmastadiene and a low amount of
saturated hydrocarbons. Peak identification: A = C29; B = 31;
1 = internal standard; 2 = 3,5 -stigmastadiene.
Page 134
TABLE 2.61. Contents of 3,5-Stigmastadiene, Analyzed by the Official and the

On -line HPLC -GC Procedure.
3,5-Stigmastadiene (mg/kg)
Oil Official Method HPLC-GC Method
1) "Extra -virgin" olive 0.10 0.09
3) "Extra -virgin" olive ND ND
6) "Pure" olive * 1.38 1.02
7) Olive 2.60 2.50
8) Cold pressed 0.09 ND

Sunflower
10) Peanut 2.70 2.80

* Definition referred to the Argentinian olive oil.
The "t" probe proved that the 3,5-stigmastadiene contents determined by both methods did not differ
significantly from each other; that is, the combined HPLC-GC method can be used as a substitute of the
official analytical procedure. The limit of quantitation is 0.1 ppm; the reproducibility varies between
5.7% and 15%, depending on the concentration of 3,5-stigmastadiene in the oil sample. A combined
HPLC-GC method has also been employed for the analysis of the sterol content of olive oil [ 158]. The
internal standard cholestanol was added to the oil sample before hydrolyzing it with sodium methylate
in tert -butylmethyl ether. After hydrolyzation (ambient temperature, 20 min), it was extracted with n-
hexane. The hexane phase was used for further analysis. The extract was first separated by HPLC on
silica column (200 mm × 2.1 mm I.D.) using hexane: 2-propanol (99:1). The flow rate was 0.2 mL/min,
and the detection wavelength was set to 200 nm. GC separation was carried out on a fused-silica
capillary column system, the first deactivated with hexamethyldisilane (3 m × 0.32 mm I.D.), and the
second being identical with the analytical column (4 m × 0.32 mm I.D.). The analytical column (25 m ×
0.32 mm I.D.) was coated with 5% phenyl and 95% dimethylsilane (film thickness 0.4 µm). The
injector and the detector (FID) temperature were 115°C and 340°C, respectively. Initial temperature of
the column was 115°C for 6 min, elevated to 260°C at 40°C/min, and then further elevated to 290°C at
1°C/min. The methyl esters of the fatty acids are eluted before the sterol fraction in HPLC, allowing the
separation of both fractions (Figure 2.69). The method separated well 14 sterols present in virgin olive
oil, as demonstrated in Figure 2.70. The HPLC-GC method was compared with the official GC method
by analyzing 60 different virgin olive oil samples. The data proved that the com-
Page 135
Figure 2.69.
HPLC chromatogram of a virgin olive oil. Column:
Hypersil Silica 5 µm. Mobile-phase n-hexane:2-propanol
(99:1). Flow rate 0.2 mL/min; detection 200 nm.
bined HPLC-GC method produced identical results as the official method for 55 samples from 60. It
was stated that the method is rapid and precise and can be employed for the analysis of the sterol
composition of virgin olive oils of different origin.
2.2.4—
Other Lipoid Compounds
Many other lipid compounds were found in various food products and analyzed with different
chromatographic techniques. Thus, the determination of the isomers of diacylglycerols (DGs) in
vegetable oils contains information on
Page 136
Figure 2.70.
LC-GC -FID chromatogram of the sterol fraction of a virgin olive oil.
1 = cholesterol; 2 = brassicasterol; 3 = campesterol; 4 = campestanol;
5 = stigmasterol; 6 = ∆-7-campesterol; 7 = ∆-5,23-stimastadienol; 8 = clerosterol;
9 = β-sitosterol; 10 = sitostanol; 11 = ∆ -5-avenasterol; 12 = ∆ -5,24-stigmastadienol;
13 = ∆-7-stigmastenol; 14 = ∆-7-avenasterol; IS = internal standard (α-cholestanol).
Temperature program: 115°C, 6 min, to 260°C at 40°C/min, to 290°C
at 1 °C/min, FID 340°C.
the quality and the treatment of the oil. A combined method (SPE followed by GC) was developed for
the separation and quantitative determination of DG isomers in vegetable oils [ 159]. Oil samples (0.2
mg/mL) were dissolved in n-hexane and 500 µL of sample, and 200 µL of 1,3-dipalmitin or 1,3-
dimiristin (1 mg/mL) standard solution was applied to a SPE column (3 mL diol-bonded phase
equilibrated with n-hexane). The column was washed with 6 mL of hexane:methylene chloride:ethyl
ether (89:10:1, v/v) (fraction I) and subsequently with 4 mL of chloroform:methanol (2:1, v/v) (fraction
II). The purity of both fractions was checked by TLC [silica plates; hexane:diethyl ether:acetic acid
(79:20:1 v/v); detection 50% H2SO 4 and heating]. The 1,2-and 1,3-DGs separated from fraction II by
TLC were scraped off and extracted with chloroform. Fraction II of SPE was evaporated to dryness and
silylated before GC analysis. Silylation was carried out by adding 200 µL of a silylating agent (3 mL of
hexamethyldisilazane + 1 M1 of trimethylchlorosilane +
Page 137
Figure 2.71.
Gas chromatogram of the polar fraction isolated from fresh virgin olive oil.
Peaks: 1 = β-sitosterol; 2 = 24-methylenecycloartanol (L: linoleic acid, cis,
cis-9,12-octadecanoic acid; Ln: linolenic acid, cis-cis-cis-9,12,15-octadecatrienoic
acid; M: miristic acid, teradecanoic acid; O: oleic acid, cis-9-octadecanoic
acid; P: palmitoleic acid, cis-9-hexadecenoic acid; S: stearic acid, octadecanoic acid.
9 mL of anhydrous pyridine) to the dry residues. A fused-silica capillary column (25 m × 0.25 mm I.D.,
coating agent 65% methylphenyl silicone, film thickness 0.1 µm) was employed for the separation of
DGs. Hydrogen was used as the carrier gas. The injector and FID temperatures were 300°C and 325°C,
respectively. The temperature program was as follows: initial hold at 270°C for 4 min, raised to 295°C
at 1°C/min, and final hold for 1 min.
The gas chromatograms of fresh virgin olive oil, crude palm oil, and refined soybean oil are shown in
Figures 2.71-2.73. The SPE-GC method showed excellent separation capacity; it separated DGs
according to the number of carbon atoms, the 1,2- and 1,3-isomeric structure, and the number of double
bonds. No interfering compounds were observed because monoacylglycerols and alkanols are eluted
with the solvent peak and sterols elute before DGs. The relative standard deviation and the recovery of
1,3-dipalmitoyl-glycerol (PP) were 3% and 104%, respectively. The data concerning the repeatability of
the
Page 138
Figure 2.72.
Gas chromatogram of the polar fraction isolated from a crude palm oil
(L: linoleic acid, cis, cis-9,12-octadecanoic acid; Ln: linolenic acid,
cis-cis-cis-9,12,15-octadecatrienoic acid; M: miristic acid, tetradecanoic
acid; O: oleic acid, cis-9-octadecanoic acid; P: palmitoleic acid,
cis-9-hexadecenoic acid; S: stearic acid, octadecanoic acid.
analysis are presented in Tables 2.62 and 2.63. The data show the good repeatability of the method. It
was further established that the isomerization is fairly slow under chromatographic conditions, making
the method suitable for the separation and quantitative determination of DGs in vegetable oils.
Phospholipids (PLs) are standard constituents of living tissues, being the main structural components of
cell membranes. Because they show considerable surfactant activity, they markedly influence the
physicochemical parameters of food products. Silica support has been frequently used for the HPLC
separation of PLs present in pig muscle [ 160], soybean [ 161], and rapeseed oil [ 162]. RP-HPLC was not
extensively used for the separation of PLs in food science [ 163]. A silica column was also employed for
the separation of phospholipids from cooked beef [ 164]. Patties were prepared by six different methods.
The conditions of the treatments are listed in Table 2.64. Lipids were extracted from 30 g of patties; 250
mg of extract was dissolved in 250 µL of
Page 139
Figure 2.73.
Gas chromatogram of the polar fraction isolated from a refined soybean oil
(L: linoleic acid, cis, cis-9,12-octadecanoic acid; Ln: linolenic acid,
cis-cis-cis-9,12,15-octadecatrienoic acid; M: miristic acid, teradecanoic acid;
O: oleic acid, cis-9-octadecanoic acid; P: palmitoleic acid,
cis-9-hexadecenoic acid; S: stearic acid, octadecanoic acid.
chloroform and applied to an NH2 bonded-phase SPE column equilibrated with hexane. PLs were eluted
by adding 2.5 mL of chloroform:2-propanol (2:1 v/v) two times, 2.5 mL of 2% acetic acid:diethyl ether
two times, and 1 mL of methanol four times. Methanolic fraction was analyzed by HPLC. Silica
columns (250 × 4.6 mm I.D., particle size 10 µm) were used for the separation. The binary gradient
system is listed in Table 2.65. The main PL fractions were collected and transmethylated, and the
methylated fatty acids (FAMEs) were separated by capillary gas chromatography. Fused-silica capillary
column (25 m × 0.25 mm I.D., thickness of cyanopropyl methyl silicone film 0.25 µm) was employed
for the separation of FAMEs using helium as the carrier gas. The injector and detector (FID)
temperatures were 260°C. Column temperature varied from 150°C to 240°C at 3°C/min.
A characteristic chromatogram is shown in Figure 2.74. The main PL classes were well separated under
the chromatographic conditions proving the
Page 140
TABLE 2.62. Repeatability Data of Diacylglycerol Analysis by SPE-GC of a Virgin Olive Oil Using a New
Column Each Time.
Diacylglycerols (%)
OP OO LO
LP OS LL Total
1,2- 1,3- 1,2- 1,2- 1,2- 1,3- 1,2- 1,3- * 1,2- (mg/g)
Mean (n = 9.47 0.86 1.63 3.20 61.50 1.57 18.10 2.10 0.79 13.41
6)
SD 0.075 0.053 0.016 0.025 0.449 0.051 0.174 0.054 0.015 0.208
RSD (%) 0.79 6.20 0.98 2.25 0.69 3.23 0.96 2.59 1.12 1.51
* There is a contribution of 1,2-LnO.
adequacy of the method. The phospholipid contents of beefs subjected to different cooking treatments
are compiled in Table 2.66. It was concluded from the data that cooking treatments exert a negligible
influence on the PL content of beef. The decrease of the sphingomyelin concentration was explained by
the supposition that Sph is less strongly bonded to the membrane and is lost with the fat during the
cooking procedure. The fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in
different samples is given in Tables 2.67 and 2.68, respectively. Also, the cooking methods were
different, but the FAME profiles of the samples were similar. This observation indicates that cooking
exerts a negligible influence on both the composition of phospholipids and the fatty acid profile of PLs.
Phospholipids were also separated on a β-cyclodextrin-bonded silica support [ 165]. The phospholipid
fraction of various soybean oil samples was separated on a silica column and further analyzed by two
different HPLC methods (method I: β-cyclodextrin bonded silica column of 250 × 4.6 mm I.D.; particle
size 5 µm; eluent hexane:2-propanol:ethanol:aqueous 5 mm teramethyl ammonium phosphate
(35:32.7:26.8:5.5); detection wavelength 208 nm; flow rate 1 mL/min. Method II: silica column of 250
× 4 mm I.D.; particle size 3 µm; gradient elution was from 100% A to 100% B in 30 min, hold for 10
min, A = chloroform:tert-butyl methyl ether 75:15, B = methanol:chloroform:ammonium hydroxide
92:1;7; evaporative light scattering detector; flow rate 0.5 mL/min). PLs were well separated on the β-
cyclodextrin-bonded silica ( β-CDS) column using isocratic elution, as demonstrated in Figure 2.75. The
analysis time was shorter than that of the gradient elution on the silica column, and the retention order
of PLs was different on a β-cyclodextrin and silica column. This result indicates that the retention
mechanism is different on both columns although typical direct-phase mobile phases were employed in
both instances. It was proposed that the method can be successfully used for
Page 141
TABLE 2.63. Repeatability Data of Diacylglycerol Analysis by SPE-GC of a Crude Palm Oil Using the Same Column Each
Time.
Diacylglycerols (%)
PP OP LP OO LO
Total
1,2- 1,3- 1,2- 1,3- 1,2- * 1,3- 1,2- 1,3- 1,2-** 1,3- (mg/g)
Mean (n = 0.91 15.02 11.18 32.12 2.50 9.43 4.22 14.31 2.74 5.57 95.42
6)
SD 0.083 0.178 0.604 0.199 0.219 0.205 0.207 0.351 0.269 0.086 1.389
RSD.(%) 9.51 1.18 5.35 0.61 8.53 2.15 4.85 2.42 9.67 1.53 1.46
* There is a contribution of 1,3-PS.
** There is a contribution of 1,3-OS.
Reprinted from Reference [ 159], copyright © 1996. With kind permission from Elsevier Science—NL, Sara Burgerhartstraat 25,
1055 KV Amdsterdam, The Netherlands.
Page 142
TABLE 2.64. Cooking Times and Temperatures for Six Cooking Treatments.
Cooking Treatments Cooking Time (min) Cooking Conditions
BO 20 100°C
R 30 225°C
M 6 1000W *
BA 18 200°C
MP 15 180°C
COMB 10 225°C + 1000W *

* Power of microwave heating.
Abbreviations: BO = boiling; R = roasting in an oven; M = microwave heating; BA
= barbecue cooking style; MP = roasting over a metal plate without oil; COMB =
combination roasting-microwave heating.
Netherlands.
the separation of phospholipids from soybean oils and other industrial lecithin products too. The results
of quantitative analyses are shown in Table 2.69. The data indicate that the phospholipid composition of
genetically modified soybeans differs considerably from that of unmodified soybean. The low values of
the coefficient of variation prove again the good reproducibility and accuracy of the method.
Lipids can be decomposed during the irradiation of various foodstuffs such as bacon [ 166], shrimp [ 167],
frog legs [ 168], chicken [169,170], and pork and beef [ 171]. The volatile decomposition products are
generally determined by GC [ 172,173] or by combined LC-GC methods [174,175]. Capillary gas
chromatography was also employed for the detection of volatile hydrocarbons in τ-irradiated raw-milk
Camembert cheeses [ 176]. Samples of 50 g of cheese were melted for 30 min at 50°C, and 50 mL of
pentane:2-propanol (3:2 v/v) were gradually added under continous shaking. The mixture was
centrifuged, and the supernatant was evaporated to 5 mL. The pentane fraction was dried
TABLE 2.65. HPLC Solvent Program for a Binary Gradient.

Time (min) A (%) B (%)
0 100 0
8 45 55
15 40 60
20 40 60
35 100 0
A = chloroform:methanol:ammonium hydroxide 30% (80:19.5:0.5, v/v); B =
chloroform: methanol:water:ammonium hydroxide 30% (60:34:5.5:0.5, v/v).
Netherlands.
Page 143
Figure 2.74.
HPLC trace of phospholipids from roasted ground
beef (A: free fatty acids; PE: phosphatidylethanolamine;
PC: phosphatidylcholine; SPH: sphingomyelin.
with 5 g of anhydrous sodium sulfate. The fat was remelted and filtered. An aliquot of 1 g fat was
dissolved in 1 mL of pentane containing eicosane as the internal standard and applied on a florisil
column (200 × 20 mm I.D., partially inactivated with 3% of water). Hydrocarbons were eluted with 60
mL of pentane and evaporated to 5 mL. Then 0.8 mL of isooctane was added, the rest of the pentane
was evaporated, and the sample was filled up to 1 mL with isooctane. A Hewlett-Packard free fatty acid
column (HP -FFAP) capillary column (25 m × 0.20 mm I.D., 0.33 µm film thickness) was employed for
the hydrocarbon analysis. Nitrogen was the carrier gas. The injector and FID temperatures were 230°C
and 250°C, respectively. The column was held at 50°C for 2 min, and then the temperature was
increased to 200°C at 4°C/min and held
Page 144
TABLE 2.66. Effect of Cooking Treatments on the Phospholipid Contents in Ground

Beef (expressed in mg/g fat).
Phosphatidyl - Phosphatidyl -
Treatment ethanolamine choline Sphingomyelin Total
RM 15.36 11.36 9.36 36.08
BO 15.84 16.80 7.76 40.40
R 17.36 15.44 8.16 40.96
M 17.44 15.20 7.92 40.56
BA 17.68 18.80 7.20 43.68
MP 14.24 11.52 6.48 32.24
COMB 16.96 13.28 6.64 36.88
Abbreviations: RM = raw; BO = boiling; R = roasting; M = microwave heating; BA =
barbecue cooking style; MP = roasting over a metal plate without oil; COMB =
combination roasting-microwave heating.
Netherlands.
TABLE 2.67. Effect of Cooking Treatments on FAME Content (%) in Ground

Phosphatidylcholine.
Fatty Acids %
Treatment 14:0 15:0 16:0 16:1t 16:1 17:0 17:1 18:0 18:1t 18:1
RM 0.7 0.3 24.0 0.2 2.1 0.7 0.7 10.0 0.6 30.9
BO 0.1 0.3 20.5 0.7 1.3 0.5 0.8 7.1 0.6 27.4
R 0.1 0.2 21.9 0.6 1.3 0.5 0.5 7.3 0.8 27.7
M 0.1 0.2 23.0 0.7 1.3 0.5 0.5 7.7 0.8 29.3
BA 0.2 0.3 22.9 0.7 1.5 0.5 0.7 7.1 0.4 27.4
MP 0.2 0.3 23.2 0.6 1.3 0.5 0.6 7.2 0.5 27.3
COMB 0.2 0.3 23.1 0.7 1.5 0.6 0.9 7.3 0.6 26.6
Fatty Acids %
Treatment 18:1 18:3 20:1 20:3 20:4 20:5 22:4 22:5 22:6 Others
RM 22.6 1.2 0.5 0.7 3.1 0.3 0.2 0.6 0.1 0.5
BO 29.4 1.0 0.2 1.6 5.3 0.5 0.4 1.4 0.4 0.6
R 28.9 0.9 tr 1.5 4.5 0.8 0.3 0.9 0.2 0.5
M 25.4 1.0 tr 1.6 4.8 0.9 0.3 1.0 0.2 0.5
BA 28.5 0.2 tr 1.4 4.6 0.4 0.7 1.3 0.2 0.6
MP 28.3 0.8 0.2 1.1 4.4 0.3 0.4 0.8 tr 0.5
COMB 28.0 1.0 0.2 1.5 4.8 0.4 0.5 1.1 0.2 0.6
Abbreviations: RM = raw; BO = boiling; R = roasting; M = microwave heating; BA = barbecue
cooking style; MP = roasting over a metal plate without oil; COMB = combination roasting-
microwave heating; t = trans; tr = trace.
NL, Sara Burgerhartstraat 25, 1055 KV Amdsterdam, The Netherlands.
Page 145
TABLE 2.68. Effect of Cooking Treatments on FAME Content (%) in Ground Beef Phosphatidylethanolamine.
Fatty acids %
Treatment 16:0 16:1 17:0 17:1 18:0 18:1t 18:1 18:2 18:3n6 18:3n3
RM 3.2 0.6 0.2 0.2 18.9 0.2 15.8 26.9 tr 0.5
BO 1.7 0.6 0.3 0.3 18.7 0.8 15.1 28.2 0.1 0.7
R 2.0 0.8 0.2 0.2 20.4 0.5 15.7 31.3 0.2 0.7
M 5.4 1.0 0.3 3.2 20.7 0.9 14.5 29.5 0.2 0.5
BA 2.0 0.7 0.3 0.6 24.0 0.3 12.4 21.9 0.2 1.0
MP 3.2 1.1 0.6 1.3 17.6 0.1 13.5 26.7 0.1 1.2
COMB 2.9 0.7 0.4 0.2 19.5 0.1 14.5 27.1 0.1 0.7
Fatty Acids %
Treatment 20:1 20:3 20:4 20:5 22:4 22:5 22:6
RM 0.2 3.2 22.5 1.2 2.0 3.8 0.8
BO 0.5 3.3 22.7 1.4 1.8 3.3 0.6

R 0.4 3.5 19.5 1.0 1.1 2.3 0.4
M 0.2 3.4 24.3 1.6 2.0 4.3 0.4
BA 0.4 2.5 25.0 1.4 1.8 3.5 2.0
MP 0.3 2.2 25.1 0.8 1.3 3.7 1.0

COMB 0.2 4.4 22.2 1.3 2.0 3.2 0.5
Abbreviations: RM = raw; BO = boiling; R = roasting; M = microwave heating; BA = barbecue cooking style;

MP = roasting over a metal plate without oil; COMB = combination roasting-microwave heating; t = trans; tr
= trace.
for 10 min. Typical chromatograms of irradiated and on-irradiated samples are shown in Figure 2.76.
The chromatograms indicate that the quantity and composition of hydrocarbons in Camembert cheese
considerably depend on the dose of irradiation. It was established that tridecane, 1-dodecene, 1-
tetradecene, and 1-hexadecene were present only in the irradiated samples; that is, the determination of
these hydrocarbons can prove the irradiation of the cheese. The concentration of hydrocarbons did not
change during the ripening and depended only on the dose of irradiation, as proven by the quantitative
data in Table 2.70. Since a fairly good linear correlation was found between the amount of
hydrocarbons and the dose of irradiation, it was suggested that the determination of the hydrocarbon
concentration can be used for the approximate calculation of an irradiation dose.
The performance of florisil column chromatography and on-line coupled LC-GC for the detection of
radiation-induced hydrocarbons in Camembert cheese has recently been compared [ 177]. Fat was
extracted by mixing 6 g of a homogenized cheese sample with 40 g of anhydrous sodium sulfate and by
Page 146
Figure 2.75.
HPLC-UV-CDS separations of phospholipid components
in selected samples derived from genetically modified
soybean oils (A, B, C, and D) along with a control,
unmodified soybean oil samples (E). HPLC-ELSD-SI
separation (C') of PL components was obtained from
sample C. For chromatographic conditions, see text.
(Reprinted from Reference [ 165], by courtesy of
Marcel Dekker, Inc.
Page 147
TABLE 2.69. Quantitative Analyses of Phospholipids in Selected Samples of Soybean

Oil by HPLC-UV with a β-Cyclodextrin Silica Column.*
Soybean Phospholipid Component
PE PC PI PA
Sample
No.** A B A B A B A B
1 17.9 30.3 26.0 44.0 11.8 20.0 3.40 5.70
(CV) (3.5) (4.0) (2.3) (3.3) (3.7) (3.9) (5.8) (6.3)
2 19.8 28.9 32.0 46.7 13.5 19.7 3.15 4.61
(CV) (4.1) (3.7) (1.9) (1.8) (3.3) (5.3) (5.0) (6.5)
3 31.9 28.7 47.8 42.9 25.5 22.9 6.10 5.50
(CV) (3.6) (4.5) (2.0) (3.0) (2.7) (2.3) (4.9) (5.8)
4 31.9 35.2 32.3 35.6 22.0 24.2 4.53 5.00
(CV) (4.5) (4.4) (2.9) (3.1) (4.3) (3.6) (6.0) (6.7)
5 39.7 27.0 72.1 48.9 32.3 21.9 3.20 2.20
(CV) (4.0) (3.9) (2.3) (1.9) (5.2) (1.9) (6.6) (5.5)
* A = amount (mg) of a phospholipid component present in 10 g of each soybean oil
sample.
B = percent composition of a PL component in each oil sample. These values are mean
values of three determinations.
**Samples 1 -4 represent genetically modified soybean oil; sample 5 represents control
unmodified soybean oil.
extracting the mixture with 100 mL of n-hexane for 2 min. After centrifugation, the supernatant was
evaporated to dryness. Hydrocarbons were separated from fat on a florisil column deactivated with 3%
water with n-hexane in method I and were further analyzed with GC. In the combined LC-GC method
(method II), a lipid solution containing 1-hexene, 1,5-hexadiene, and n-eicosane internal standards was
injected into a silica column (50 mm × 4 mm I.D.), and the hydrocarbons were eluted at a flow rate of
200 µL/min with n-hexane. The detection wavelength was set to 200 nm. The LC fraction marked in
Figure 2.77 was directly transferred to GC. GC analysis of the fraction eluted from the florisil column
was carried out on a fused-silica capillary column (30 m × 0.2 mm I.D.). The temperature gradient was
2 min at 55°C, then 12°C/min to 155°C, and 5°C/min to 230°C. For on-line LC-GC, a different
capillary column was employed (30 m × 0.25 mm I.D., DB 5 film thickness 0.25 µm). The initial
column temperature was 78°C for 3 min and raised to 240°C at 5°C/min. Typical gas chromatograms
are shown in Figure 2.78. The hydrocarbon profiles of samples irradiated before and after maturing
were similar; that is, the ripening process exerts no significant influence on the formation of
hydrocarbons. The amount of hydrocarbons increased with an increasing dose of irradiation. It was
stated that both the florisil and the LC-GC combined methods produce similar hydrocarbon profiles;
that is, both
Page 148
Figure 2.76.
Gas chromatographic analyses of volatile hydrocarbons
present in (A) unirradiated and irradiated [(B) 1.21 kGy,
(C) 1.91 kGy, and (D) 3.97 kGy)] Camembert cheeses after
21 days of ripening (e.g., 6 days after irradiation).
Burgerhartstraat 25, 1055 KV Amsterdam,
The Netherlands.)
Page 149
TABLE 2.70. Evolution of the Amount of Hydrocarbons Found in τ-Irradiated and

Nonirradiated Control Camembert Cheeses During the Second Phase of Ripening (after
irradiation) at 10°C (17 days) and Then Storage (43 days) at 4 °C. *
nmol of Hydrocarbons/g Fat
Dose Days after
(kGy) Irradiation C12:1 C13:0 C14:0 C15:0 C16:1C C17:1
0 6 0.00 0.00 0.00 0.42 0.00 1.09
10 0.00 0.00 0.00 0.41 0.00 1.05
13 0.00 0.00 0.00 0.43 0.00 1.17
24 0.00 0.00 0.00 0.41 0.00 1.20
39 0.00 0.00 0.00 0.39 0.00 1.18
60 0.00 0.00 0.00 0.40 0.00 1.25
1.20 6 1.40 0.98 2.49 2.03 0.98 2.00
10 1.14 0.89 2.31 2.01 1.00 2.08
13 1.21 0.82 2.06 1.77 0.88 1.53
24 1.39 1.08 2.56 2.16 0.99 1.95
39 1.37 1.04 2.59 2.17 1.04 1.88
60 1.36 1.05 2.44 2.21 0.98 1.85
1.91 6 2.41 1.74 4.22 3.45 1.83 2.97
10 2.59 1.92 4.67 3.76 1.78 2.54
13 2.24 1.56 3.82 3.13 1.56 2.25
24 2.34 1.80 4.20 3.58 1.75 2.70
39 2.07 1.63 3.74 3.30 1.50 2.25
60 2.16 1.88 4.12 — 1.76 2.83
3.97 6 5.10 3.90 8.34 7.52 3.64 3.48
10 4.55 3.22 7.73 6.20 3.12 3.37
13 4.95 3.05 8.12 6.53 2.96 3.21
24 4.42 3.44 7.59 6.62 3.05 —
39 4.25 3.23 7.38 6.20 3.00 3.30
60 4.46 3.64 7.78 7.35 3.35 3.77

* The hydrocarbon concentration has an experimental error of approximately ± 15%; all
the determinations were carried out in triplicate.
methods can be employed for the detection of irradiation, the detection level being at 250 Gy. The LC-
GC method was superior to the traditional florisil method because it is more rapid and because sample
volume and solvent consumption is markedly lower then in the florisil method.
Another GC -MS method was employed for the detection of irradiated eggs in processed bakery
products [178]. Biscuits were prepared with irradiated and unirradiated eggs, and the fat content was
extracted with pentane:2-propanol (3:2 v/v). Hydrocarbons were isolated from the fat on a florisil
column and were separated on a fused-silica capillary column (30 m × 0.2 mm I.D., film thickness of
SE-54 0.25 µm). Helium was used as the carrier gas. Characteristic
Page 150
Figure 2.77.
LC-UV chromatogram of a Camembert
sample marked by the standards 1-hexene
(6:1) and 1,5-hexadiene (6:2). The fraction
transferred, including alkanes, alkenes, and
alkadienes, is indicated by the bars.
(Reprinted from Reference [ 177] with
permission from the publisher and the
corresponding author, copyright © 1995
American Chemical Society.)
chromatograms of hydrocarbon and cyclobutanon fractions are shown in Figures 2.79 and 2.80. Also in
this instance, a good linear relationship was found between the amount of hydrocarbons and the dose of
irradiation, indicating that the concentration of hydrocarbons is a good indicator of the irradiation dose.
Because of its rapidity and lower solvent consumption, supercritical fluid extraction (SFE) has also
been employed in the analysis of irradiated foods [179]. The pretreatment of samples highly depended
on the character and composition of the sample. Peanut oil was extracted from peanuts by treating 10 g
of crushed peanut with 100 mL of methylene chloride overnight. The extract was dried over anhydrous
sodium sulfate and evaporated to dryness. Instant soup mix powder was sonificated with 200 mL of
methylene chloride for 30 min and then treated as peanuts. Roasted pistachio nuts were crushed and 200
mL of methylene chloride:acetone (2:1 v/v) was added to the 10-g sample. The extract was treated as
the peanut extract. Duck breast and pork meat were homogenized, and 80 mL of heptane:2-propanol
(3:2 v/v) was added to 30-50 g of the sample. The mixture was sonificated for 30 min and centrifuged,
and the supernatant was dried with anhydrous sodium sulfate and evaporated. German caviar was mixed
with anhydrous CaCl2 (50:50 g), and then 60 mL of heptane: 2-propanol
Page 151
Figure 2.78.
Gas chromatograms of the hydrocarbon fraction of Camembert analyzed
by on-line coupled LC-GC -MS. The cheese was treated before (top, center)
and after (bottom) maturing. (Top) Control; (center and bottom) irradiated with 500 Gy.
(Reprinted from Reference [ 177] with permission from the publisher and the
corresponding author, copyright © 1995 American Chemical Society.)
(3:2 v/v) was added, sonificated three times for 30 min, left overnight, dried over Na 2 SO4 , centrifuged,
and evaporated. Solid-phase extraction was carried out by homogenizing 1 g of extracted fat or oil with
10 g of nonporous glass beads (dp = 25 50 µm); then it was transferred into the extraction chamber.
Extraction conditions were 78°C, 150 bar (r = 0.45 g/mL), for 40 min, flow rate 1.0 mL/min (liquid
carbon dioxide at 4°C, r = 0.92 g/mL). Hydrocarbons were collected by n-hexane, the n-hexane was
evaporated, and the residue was dissolved in methylene chloride for GC-MS analysis. The capillary
column was 12 m × 0.22 mm I.D. coated with 0.33 µm methylpolysiloxane. Typical chromatograms of
irradiated and nonirradiated instant soup powder and duck breasts are shown in Figures 2.81 and 2.82,
respectively. It was established that SFE alone is suitable for the extraction of hydrocarbons from
foodstuffs in one step; however, the use of organic solvent extraction prior to SFE results in more clean
chromatograms. Roasting also produces hydrocarbons. As previously stated, irradiation can be
Page 152
Figure 2.79.
Gas chromatogram of hydrocarbons isolated from biscuits prepared
with irradiated eggs (3 kGy).
corresponding author.)
Figure 2.80.
Gas chromatogram of cyclobutanon fraction isolated from biscuits prepared
with irradiated eggs (5 kGy). DCB: 2-dodecylcyclobutanone; TenCB:
2-teradecenylcyclobutanone; TCB: 2-teradecylcyclobutanon; CHCH:
2-cyclohexylcyclohexanone (internal standard).
publisher and the corresponding author.)
Page 153
Figure 2.81.
GC analysis of irradiated (A, top) and nonirradiated (B, bottom) instant soup
powder after SFE. Conditions: 78°C, 152 bar, density 0.45 g/mL; fluid flow 1.0
mL/min, extraction time 40 min. Hydrocarbons: (1) 14:1, (2) 14:2, (3) 15:0,
(4) 2 -alkylcyclobutanone, (5) 16:2, (6) 16:1, (7) 17:1, (8) 17:0, (9) 16:0.
detected in products that were roasted, by using 2-alkylcyclobutanone as a marker. SFE, combined with
GC-MS, has been proposed for the characterization of irradiated foods because it is selective and rapid,
the solvent consumption is low, and hydrocarbons and 2-alkylcyclobutanone can be extracted in one
step.
Not only irradiation, but also heat treatment and oxidation, can cause the decomposition of lipids
[180,181]. Because the lipid decomposition products caused by heat treatment or oxidation are also
volatile, GC is the method of preference for the monitoring of lipid oxidation and thermal
decomposition. Thus, headspace GC was employed for the evaluation of the flavor defects in dried milk
products caused by oxidation [182]. An aliquot of 5.00 g dry sample was dissolved in 10 mL of water
and steam-distilled (9 mL of distillate collected); 2 mL of distillate and 1.0 g of sodium chloride were
added to a headspace vial thermostatted at 60°C. Separations were carried out on a
Page 154
Figure 2.82.
GC analysis of (A) nonirradiated and (B) irradiated fat from duck breasts.
Conditions: 78°C, 152 bar, density 0.45 g/mL; fluid flow 1.0 mL/min, extraction time
40 min. Hydrocarbons: (1) 12:0, (2) 14:1, (3) 14:0, (4) 15:0, (5) 2 -alkylcyclobutanone,
(6) 16:2, (7) 16:1, (8) 16:0, (9) phosphoric ester, (10) 17:2, (11) 17:1, (12)
17:0, (13) carboxylic acid or ester, (14) presumably octadecanal.
fused-silica capillary column (25 m × 0.32 mm I.D., film thickness of SE-54 1.0 µm). The initial
column temperature was 38°C for 3 min and increased to 170°C at 6°C/min. Hydrogen was the carrier
gas. GC -MS was employed for the identification; the GC conditions were the same as above, but
helium carrier gas was used. The gas chromatograms of the volatiles of whole milk powder and a milk-
based baby formula are shown in Figures 2.83 and 2.84, respectively. The method separated the
volatiles produced by lipid autooxidation well; therefore, it was proposed as a rapid technique for
quality control of dried milk products.
The formation of volatiles during the heating of triolein and trilinolein was followed by static headspace
capillary gas chromatography/infrared
Page 155
Figure 2.83.
Headspace volatiles of whole milk powder
stored in air at 40°C (peroxide value = 1.01):
1 = pentanone; 2 = pentanal; 3 = methyl butyrate
(internal standard), 4 = hexanal;
5 = heptanone; 6 = heptanal; 7 = octanal;
8 = octanone; 9 = nonanal.
(Reprinted from Reference [ 182], copyright
© 1995. With kind permission from Elsevier
Science—NL, Sara Burgerhartstraat 25, 1055 KV
Amsterdam, The Netherlands.)
spectroscopy-mass spectrometry [ 183,184]. Triolein was heated at 190°C and samples were taken every
12 h. Samples were heated at 150°C in the headspace vial, and then the volatiles were transferred and
collected at the capillary column at -50°C. The fused-capillary column (50 m × 0.32 mm I.D., 0.52 µm
film thickness of cross-linked 5% phenyl methyl silicone) was held at -50°C for 1 min, heated to 60°C
at 20°C/min, to 120°C at 10°C/min, and to 220°C at 20°C/min. The injector and detector temperatures
were 200°C and 250°C, respectively. Helium was used as the carrier gas. The concentration of volatiles
in heated triolein are given in Table 2.71. It was established that the concentration of the volatile
aldehydes considerably decreased during heating. The results of the analysis of volatile formed during
the heating of trilinolein are shown in Table 2.72. The amount of volatile oxidation products increased
considerably during heating. The quantity of volatile compounds from trilinolein is higher than that
from triolein, indicating the better heat stability of the latter. The volatile profiles of triolein and
trilinolein differ considerably, suggesting different oxidation mechanisms.
Page 156
Figure 2.84.
Headspace volatiles of a milk-based baby formula
stored in air at 40°C (peroxide value = 1.23): 1 = pentanone;
2 = pentanal; 3 = methyl butyrate (internal standard),
4 = hexanal; 5 = heptanone; 6 = heptanal; 7 = octanal;
8 = octanone; 9 = nonanal.
With kind permission from Elsevier Science—NL,
Sara Burgerhartstraat 25, 1055 KV Amsterdam,
The Netherlands.)
Page 157
TABLE 2.71. Effect of Heating on Concentration of Individual Volatiles Formed by

Triolein.
Concentration (ppm)
Volatile Compound 0h 12 h 24 h
Propanal 0.07 ± 0.03 * 0.25 ± 0.00 ND
Pentanal ND 3.07 ± 0.41 2.68 ± 0.22

Acetic acid 1.95 ± 0.73 ND ND
1-Heptene 1.02 ± 0.03 1.02 ± 0.19 0.68 ± 0.15

Heptane 16.36 ± 1.99 30.17 ± 1.19 26.14 ± 0.97
1-Octene 1.12 ± 0.08 ND ND
Octane 24.48 ± 0.21 40.77 ± 1.68 33.93 ± 0.87

Heptanal 21.68 ± 1.49 9.11 ± 3.96 7.52 ± 1.39
1-Heptanol 7.82 ± 1.29 3.96 ± 0.00 2.67 ± 0.00
Nonane ND 1.86 ± 0.09 1.22 ± 0.14
Octanal 35.53 ± 2.13 36.64 ± 0.53 13.19 ± 1.81
1-Octanol 6.91 ± 0.30 4.89 ± 0.00 3.72 ± 0.38
Nonanal 68.95 ± 2.81 43.16 ± 4.74 41.23 ± 4.04
(E)-2-Nonenal 2.28 ± 1.39 ND ND
(E)-2-Decenal 135.00 ± 8.00 89.00 ± 13.00 74.50 ± 7.00

(E)-2-Undecenal 88.50 ± 4.00 61.00 ± 11.00 50.00 ± 7.50
36h 48 h 60 h
Propanal 0.13 ± 0.00 ND 0.25 ± 0.21

Pentanal 1.95 ± 0.34 2.68 ± 0.00 1.17 ± 0.27
Acetic acid ND ND ND
1-Heptene 0.42 ± 0.00 ND ND
Heptane 13.18 ± 0.81 10.55 ± 0.18 5.16 ± 0.97

1-Octene ND ND ND
Octane 17.96 ± 1.07 15.10 ± 0.21 4.39 ± 1.12
Heptanal 9.01 ± 0.84 11.49 ± 1.19 5.45 ± 0.77
1-Heptanol 3.37 ± 0.10 5.25 ± 0.10 1.09 ± 0.30
Nonane 0.83 ± 0.00 ND ND
Octanal 15.21 ± 0.45 18.18 ± 2.23 7.02 ± 2.34

1-Octanol 3.83 ± 0.45 4.68 ± 0.62 1.91 ± 0.68
Nonanal 40.53 ± 0.00 49.47 ± 6.32 18.07 ± 6.14
(E)-2-Nonenal 4.74 ± 0.00 6.84 ± 2.11 0.61 ± 0.00
(E)-2-Decenal 65.00 ± 7.00 89.0 ± 10.50 12.50 ± 0.50
(E)-2-Undecenal 45.50 ± 3.55 79.00 ± 13.00 6.00 ± 2.55
* Mean concentration ± standard deviation; n = 3.

Page 158
TABLE 2.72. Effect of Heating on Concentration of Dissolved Individual Volatiles

from Trilinolein.
Concentration (ppm)
Volatile Compound 0h 12 h 24 h
Pentane 33.4 ± 0.7* 67.6 ± 2.3 398.7 ± 94.5
Acrolein 4.4 ± 0.2 ND ND

Butanal 1.7 ± 0.1 2.3 ± 0.3 ND
Formic acid ND 2.2 ± 0.0 18.7 ± 0.0
Pentanal 11.7 ± 2.1 11.9 ± 0.6 73.5 ± 0.6
1-Pentanol 5.2 ± 0.1 7.3 ± 0.6 46.7 ± 10.0
Hexanal 67.6 ± 2.2 70.8 ± 2.53 346.1 ± 91.4
(E)-2-Hexenal 12.9 ± 2.2 18.6 ± 0.5 87.6 ± 25.9
Heptanal 2.5 ± 0.5 4.3 ± 0.3 29.5 ± 8.4
2-Heptanone ND ND 5.2 ± 0.7
(E)-2-Heptenal 309.6 ± 24.9 268.7 ± 19.3 894.1 ± 106.4
1-Octen3 -ol 35.2 ± 2.0 31.2 ± 0.5 150.2 ± 33.3
Hexanoic acid ND ND 83.8 ± 27.1
2-Pentylfuran 29.2 ± 2.0 15.4 ± 0.5 146.9 ± 37.6
(E)-2-Octenal 26.4 ± 0.0 36.0 ± 6.0 347.2 ± 83.2
(E)-2-Nonenal 31.6 ± 0.0 43.4 ± 2.0 163.2 ± 34.2
(E, Z)-2,4-Decadienal 15.0 ± 1.9 63.8 ± 11.3 320.0 ± 10.0
(E, E)-2,4-Decadienal 43.1 ± 11.3 322.5 ± 45.01 1137.5 ± 140.0
* Average concentration ± standard deviation; n = 3.
2.3—
Separation of Amino Acids, Amines, Peptides and Proteins
2.3.1—
Amino Acids
Free amino acids and other nitrogen compounds can markedly influence not only the nutrition value of
foods, but also the organoleptic characteristics. Amino acids are the building blocks of proteins
determining their importance for human and animal health. In contrast to the beneficial effects of
protein amino acids, nonprotein amino acids can be toxic to humans and animals.
2.3.1.1—
Because of its versatility, high separation capacity, and reliability, HPLC is the method of preference
for the analysis of amino acids. Since the over-whelming majority of amino acids has absorbance
neither in the visible nor in the UV region, they cannot be detected by the visible and UV detectors used
most frequently in HPLC. Many efforts have been devoted for the development
Page 159
TABLE 2.73. Linear Solvent Gradient for Elution.

Time (min) Solvent A (%) Solvent B (%) Solvent C (%)
0 35.0 62.8 2.2
3 35.0 62.0 3.0
15 35.0 62.0 3.0
16 35.0 58.0 7.0
30 35.0 50.0 15.0
45 35.0 42.0 23.0
50 0.0 0.0 100.0
60 0.0 0.0 100.0
65 35.0 62.8 2.2

Solvent A = aqueous solution containing 0.1 M sodium acetate, 0.68
mL/L triethylamine and 0.2 mg/mL sodium pentanesulphonate. pH
was adjusted to 5.27 with glacial acetic acid; Solvent B = water;
Solvent C = acetonitrile.
Reprinted from Reference [ 188], copyright © 1994. With kind
of derivatization procedures, making the easier detection of free amino acids possible. The derivatizing
agent has to comply with the following requirements:
1. The product has to show considerable absorbance in the visible and UV region, or it can be detected
by fluorescence detectors.
2. The derivatization process has to be relatively rapid, and the yield has to be as high as possible
(preferably 100%).
3. The product has to be reasonably stable during the separation and detection process.
Phenylisothiocyanate (PITC) has been extensively used to form UV, absorbing phenylthiocarbamyl
derivatives of amino acids [ 185,186]. The advantages and disadvantages of the use of PITC have been
previously discussed [ 187]. PITC has been successfully used for the separation and quantitative
determination of amino acids and ethanolamine in musts and wines by HPLC without any
prepurification of the samples [ 188]. A sample of must or wine (40 or 100 µL) was mixed with 20 µL of
norleucine internal standard (400 mM in 0.1 M acetic acid) and with 20 µL of triethylamine (TEA)
solution. The composition of the TEA solution was TEA:water:ethanol (1:2:2). The mixture was dried
under vacuum. The derivatization agent [ethanol:TEA:water:PITC (7:1:1:1)] was freshly prepared each
day. Amino acids and ethanolamine were derivatized by adding 20 µL of reagent to the dried samples,
sealing them in vacuum vials for 20 min. Then the reagents were removed under vacuum. Separation
was carried out on an octadecylsilica column of 250 × 4.6 mm I.D., particle size 5 µm. The flow rate
was set to 1 mL/min, and the detection wavelength was 254 nm. The column was thermostatted at 35°
C. The composition of the linear solvent gradient is shown in Table 2.73. PITC amino acids and
Page 160
Figure 2.85.
Chromatogram of Paradella (P1) grape must.
Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV
ethanolamine were successfully separated both from must (Figure 2.85) and wine samples (Figure
2.86). The good separation of amino acids and ethanolamine from each other and from the other
compounds present in must and wine indicates that the method is a valuable tool for the determination
of the amino acids in must and wines without the time-consuming and sometimes complicated
pretreatment of the samples. The validation parameters related to the repeatability and reproducibility of
the method are presented in Table 2.74.
Figure 2.86.
Chromatogram of Paradella (P1) grape wine.
from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV
Page 161
TABLE 2.74. Repeatability of Retention Time (tR), Precision, Recovery, Linearity, Limit
of Detection (LD) and Limit of Quantification (LQ) for Amino Acids and Ethanolamine.
Compound Repeatability, t R(%) Precision (%) Recovery (%) (N = 4)
Cy * 3.00 9.85 100.1 ± 7.5

Asp 1.83 6.87 100.8 ± 2.7
Hyp 1.72 2.06 100.7 ± 1.8
Glu 2.71 5.98 103.1 ± 4.4
Ser 1.16 4.23 99.4 ± 1.8
Asn 1.89 4.63 94.0 ± 2.8
Gly 1.78 5.69 102.7 ± 10.4
Gln 1.52 3.66 100.6 ± 4.8
Thr 1.07 5.75 97.9 ± 7.9
Ala 1.05 3.18 102.9 ± 8.9
His 0.96 6.15 95.4 ± 2.8
Pro * 1.24 2.16 99.9 ± 0.8

GABA 0.88 2.46 98.2 ± 4.8
Arg 0.94 6.94 102.5 ± 3.5
ETA ** 0.84 3.22 107.1 ± 1.8

Tyr 0.77 4.09 96.5 ± 3.3
Val 0.45 2.79 100.6 ± 4.3
Met 0.49 9.34 103.3 ± 5.1
Ile 0.21 4.08 102.3 ± 2.9
Leu 0.11 5.82 99.6 ± 3.4
Phe 0.15 4.79 95.0 ± 4.7
Orn 0.18 3.56 93.7 ± 3.2
Trp 0.31 6.97 95.2 ± 5.2
Lys 0.51 6.71 95.6 ± 5.1
Compound Linearity (r) LD (nmol) LQ (nmol)
Cy * 0.9999 0.020 0.052
Asp 0.9989 0.25 0.063

Hyp 0.9995 0.015 0.039
Glu 0.9991 0.018 0.046
Ser 0.9992 0.016 0.046
Asn 0.9992 0.016 0.041
Gly 0.9997 0.016 0.040
Gln 0.9997 0.016 0.041
Thr 0.9989 0.017 0.042
Ala 0.9999 0.015 0.039
His 0.9999 0.018 0.045
Pro * 0.9990 0.013 0.033
GABA 0.9995 0.021 0.054

Arg 0.9997 0.017 0.044
ETA ** 0.9998 0.017 0.044
Tyr 0.9996 0.017 0.044

Val 0.9994 0.021 0.053
Met 0.9999 0.020 0.052

Page 162
TABLE 2.74.
Compound Linearity (r) LD (nmol) LQ (nmol)
Ile 0.9998 0.021 0.053
Leu 0.9999 0.017 0.043
Phe 0.9999 0.018 0.047
Orn 0.9997 0.011 0.027
Trp 0.9995 0.014 0.035
Lys 0.9992 0.012 0.031
* n = 3.
** Ethanolamine.
The repeatability of the retention time was calculated from six parallel determinations of the sample of
Parellada wine. The linearity values are the average of three independent calibration plots, and the
recovery was determined by adding different amounts of amino acids and ethanol amine (ETA) to the
same sample. The values of the limit of detection [189] and limit of quantitation [ 190] were calculated by
where LD is the limit of detection, X is the noise, t is the student's t value for a 99.99% significance
level, S is the standard deviation, and LQ is limit of quantitation. The values of the validation
parameters prove again that the method is reliable, sensitive, reproducible, and suitable for the
separation and quantitative determination of amino acids in must and wine.
The stability data of dry and dissolved phenylthiocarbamyl (PTC) amino acids are given in Table 2.75.
The temperature of storage was -20°C and +4°C for dry and dissolved PTC amino acids, respectively.
The data in Table 2.75 indicate that the majority of PTC amino acids are stable at -20°C in dry state for
4 weeks. Only the slight decomposition of PTC Gln and Trp was observed after the third week. The
stability of dissolved PTC amino acids is considerably lower, indicating that they have to be used
within 1 hour of preparation. The average amino acid concentration in must and wine is shown in Table
2.76. The amino acid content of musts and wines differs considerably, indicating that not only the type
of variety, but also the other conditions may have a marked impact on the quantity of these
microcomponents. The fact that the amino acid content is lower in wines than in musts suggests that
they are utilized or decomposed during the fermentation process.
Page 163
TABLE 2.75. Stability of Dry and Solution PTC -Amino Acids (mg of amino
acids/L of wine).
Solution Time (min)
Compound 0 15 45 60 90
Cya 10.2 9.9 9.5 10.1 10.2
Asp 17.5 17.8 17.4 17.3 17.2
Hyp 14.3 14.2 14.7 14.0 13.9
Glu 20.1 19.7 20.4 19.8 20.2
Ser 12.3 11.9 12.5 12.4 12.0
Asn 10.1 9.9 10.0 9.8 10.2
Gly 12.5 12.4 12.7 12.8 12.8
Gln 11.1 11.0 11.4 11.2 10.5
Thr 8.4 8.2 8.0 7.9 8.3
Ala 12.1 12.5 11.9 12.3 12.5
His 9.1 8.9 9.0 9.1 8.9
Pro 287.3 280.1 279.1 290.4 285.4
GABA 11.8 11.5 11.4 11.9 11.7
Arg 10.2 10.3 10.6 9.9 10.3
ETA 21.6 21.5 21.8 21.4 21.5
Tyr 12.5 12.4 12.6 12.4 12.0
Val 7.5 7.2 7.3 7.9 7.3
Met 8.4 8.2 8.4 8.1 8.2
Ile 6.9 6.3 6.8 6.4 6.2
Leu 12.0 12.4 11.8 11.9 12.0
Phe 9.5 9.2 9.1 9.6 9.3
Orn 5.4 5.2 5.3 5.9 5.4
Trp 18.4 18.2 18.1 18.5 17.2
Lys 2.8 2.6 2.9 2.8 2.6
Dry Time (min)
Compound 0 1 2 3 4
Cya 10.2 12.0 10.7 10.9 11.6
Asp 17.5 17.2 17.7 17.0 17.1
Hyp 14.3 14.0 13.8 14.7 14.6
Glu 20.1 20.5 18.0 20.5 20.5
Ser 12.3 12.9 12.3 13.1 12.9
Asn 10.1 10.3 10.8 10.2 10.9
Gly 12.5 13.1 12.9 13.9 13.0
Gln 11.1 11.4 10.2 10.8 7.5
Thr 8.4 7.9 8.2 8.7 8.4
Ala 12.1 12.1 13.3 13.5 12.6
His 9.1 9.0 9.1 9.6 9.0
Pro 287.3 302.2 310.6 313.4 298.3
GABA 11.8 11.6 11.7 11.0 11.6
Arg 10.2 11.2 10.0 11.7 10.8
ETA 21.6 20.5 22.3 22.4 22.0

Page 164
TABLE 2.75.
Dry Time (min)
Compound 0 1 2 3 4
Tyr 12.5 13.9 13.6 12.9 13.8
Val 7.5 7.9 8.0 8.9 7.6
Met 8.4 8.1 8.6 8.4 8.5
Ile 6.9 6.7 7.1 7.3 6.5
Leu 12.0 13.0 12.0 12.5 12.4
Phe 9.5 9.7 9.1 10.1 9.9
Orn 5.4 5.6 5.2 5.8 5.6
Trp 18.4 17.8 19.3 20.3 15.0
Lys 2.8 3.1 2.7 2.7 2.6
Reprinted from Reference [ 188], copyright © 1994. With kind permission

Amdsterdam, The Netherlands.
TABLE 2.76. Results for Must and Wine Samples.

Must* (mg/mL)
Compound M1 M2 P1 P2
Cy * ND ND ND ND
Asp 40.4 6.8 12.4 6.6
Hyp 1.6 0.7 12.4 6.6
Glu 51.1 13.2 11.2 11.2
Ser 28.8 11.8 21.0 16.3
Asn 8.8 4.7 3.9 2.4
Gly 3.3 2.9 2.7 2.0
Gln 73.4 29.9 39.9 37.0
Thr 27.6 11.9 28.1 18.3
Ala 39.6 29.4 27.3 27.3
His 12.0 8.5 8.8 12.3
Pro 125.1 92.7 103.7 57.5
GABA 25.3 27.9 16.4 21.2

Arg 249.6 81.3 241.3 172.4
ETA 13.4 7.0 14.2 10.5
Tyr 20.9 8.8 21.8 18.2
Val 8.6 5.3 8.9 7.1
Met 2.8 1.2 2.8 1.2
Ile 6.5 3.5 7.1 5.7
Leu 15.0 5.1 11.9 11.0
Phe 15.0 7.5 19.6 15.7
Orn 5.3 2.1 3.3 5.9
Trp 8.9 4.7 17.4 12.9
Lys 5.0 2.2 3.9 6.5

Page 165
TABLE 2.76.
Wist* (mg/mL)
Compound M1 M2 P1 P2
Cy * 1.0 0.1 0.1 0.1
Asp 6.2 2.8 9.3 5.5
Hyp 2.1 2.3 3.2 2.9
Glu 10.2 5.6 13.3 10.1
Ser 2.1 1.3 2.5 1.6
Asn 2.5 1.5 8.3 1.0
Gly 1.4 2.4 2.2 2.5
Gln 3.8 1.3 6.1 12.3
Thr 2.8 0.8 1.3 1.2
Ala 8.3 5.3 9.5 15.8
His 1.6 1.3 1.8 2.0
Pro 167.5 162.6 354.0 251.6
GABA 3.9 2.8 5.0 6.6
Arg 6.1 4.4 7.3 6.5
ETA 35.8 33.2 82.0 70.4
Tyr 3.4 3.1 5.0 3.7
Val 4.2 2.3 3.1 2.1
Met 2.4 1.3 2.7 1.8
Ile 5.1 2.8 4.5 2.0
Leu 8.2 7.5 10.3 3.9
Phe 4.4 2.9 5.8 6.4
Orn 1.9 1.3 6.3 6.1
Trp 2.5 3.3 2.9 1.8
Lys 6.7 7.3 6.8 4.3

* M1 and M2 = Macabeo variety; P1 and P2 = Parellada variety.
The PITC derivatization method has also been used for the determination of the free amino acid profile
of infant formulas (vegetables, meat, fish, and fruits) [ 191]. Samples were prepared by adding 50 mL 0.6
N perchloric acid to 5 g infant formula. The solution was homogenized, centrifuged at 3500 rpm for 20
min, and filtered through a 0.45-mm filter, and pH was adjusted to 7 ± 0.2 with 30% potassium
hydroxide. Derivatization was carried out as described above. PTC amino acids were separated on an
octadecylsilica column (250 × 4.6 mm I.D., 5 µm particle size) using gradient elution. The
characteristics of gradient elution are compiled in Table 2.77. Except for Cys, PTC amino acids were
adequately separated with this RP -HPLC method (Figure 2.87). The free amino acid contents of infant
foods are presented in Tables 2.78-2.81. It has been stated that this RP-HPLC method can be
successfully used for the reliable, rapid, and reproducible determination of the free amino acid profiles
in infant foods.
Page 166
TABLE 2.77. Gradient Table. The Gradient was Linear in all Cases.
Time (min) A (%) B (%) Flow (ml/min)
0 100 0 1.00
14.5 54 54 1.00
15.0 0 100 1.00
17.0 100 0 1.50
20.0 100 0 1.50
20.5 100 0 1.00
A = 19 g sodium acetate was dissolved in 1 L water, 500 µL TEA was added,

the pH was adjusted to 6.40 with glacial acetic acid, and then 760 mL of this
solution was mixed with 60 mL acetonitrile.
B = acetonitrile:water (6:4).
Although PITC has been proven to be an excellent derivatizing agent, its use needs high vacuum and a
fairly long time to remove the free PITC entirely. The application of butylisothiocyanate (BITC)
overcomes the disadvantages of PITC: it is more volatile, and moreover, it is suitable for the separation
of cysteine and cystine [ 192]. The results obtained by butylthiocarbamyl (BTC), PTC derivatization, and
automated amino acid analyzer have been recently compared [ 193]. The samples of standard amino
acids of protein hydrolysates (25 µL of each) were dried with nitrogen at 50°C, then 50 µL of coupling
Figure 2.87.
Elution profile of fish sample showing resolution of
PTC-amino acids by liquid chromatography.
(Reprinted from Reference [ 191].
Page 167
TABLE 2.78. Means and Coefficient of Variation of Amino Acids in

Vegetable Samples Determined by LC Methods.
Amino Acid Concentration Found* CV (%)
Asp 220.39 ± 10.30 4.67
Glu 360.34 ± 5.90 1.91
Ser 91.62 ± 3.65 3.98
Gly 70.92 ± 2.46 4.17
His 80.27 ± 3.59 4.47
Arg 79.46 ± 2.07 2.60
Thr 62.31 ± 3.29 5.28
Ala 76.91 ± 3.32 4.32
Pro 19.12 ± 1.12 5.86
Tyr 63.03 ± 3.49 5.53
Val 66.88 ± 3.40 5.08
Met 38.64 ± 2.04 5.28
Cys 11.38 ± 0.78 6.75
Ile 115.02 ± 2.40 2.09
Leu 83.34 ± 1.93 2.32
Phe 144.17 ± 2.90 2.01
Trp 39.39 ± 1.77 4.49
Lys 81.57 ± 7.85 9.62
* Mean ± SD (n = 6).

Meat Samples Determined by LC Methods.
Asp 137.77 ± 7.16 5.19
Glu 351.54 ± 12.75 3.63
Ser 56.65 ± 2.92 5.15
Gly 67.35 ± 3.28 4.87
His 85.36 ± 2.21 2.59
Arg 140.12 ± 5.62 4.01
5.24
Thr 65.45 ± 3.43
Ala 96.40 ± 4.25 4.41
Pro 138.61 ± 5.68 4.09
Tyr 53.19 ± 3.16 5.95
Val 46.34 ± 2.11 4.55
Met 45.27 ± 2.36 5.21
Cys 7.22 ± 0.35 4.85
Ile 64.41 ± 2.02 3.14
Leu 65.44 ± 3.04 4.65
Phe 92.77 ± 2.55 2.75
Trp 60.18 ± 2.91 4.83
Lys 317.07 ± 16.12 5.09

* Mean ± SD (n = 6).
Page 168

Fish Samples Determined by LC Methods.
Asp 234.60 ± 9.88 4.21
Glu 271.78 ± 15.73 5.79
Ser 110.69 ± 3.72 3.36
Gly 80.33 ± 3.84 4.79
His 126.96 ± 3.95 3.11
Arg 131.13 ± 1.61 1.23
Thr 57.50 ± 3.75 6.52
Ala 122.58 ± 7.32 5.97
Pro 22.11 ± 1.21 5.47
Tyr 65.44 ± 2.60 3.97
Val 52.72 ± 2.27 4.30
Met 40.89 ± 2.26 5.52
Cys 5.21 ± 0.13 2.49
Ile 84.14 ± 2.23 2.65
Leu 43.05 ± 1.41 3.27
Phe 160.37 ± 4.05 2.52
Trp 78.92 ± 3.96 5.02
Lys 214.94 ± 8.69 4.04
* Mean ± SD (n = 6).

Fruit Samples Determined by LC Methods.
Asp 103.13 ± 3.87 3.75
Glu 45.62 ± 1.27 2.79
Ser 29.35 ± 1.34 4.56
Gly 28.14 ± 1.22 4.33
His 54.96 ± 2.06 3.75
Arg 19.08 ± 0.90 4.72
3.51
Thr 10.25 ± 0.36
Ala 68.19 ± 3.15 4.62
Pro 32.06 ± 1.16 3.62
Tyr 11.15 ± 0.86 7.71
Val 13.79 ± 0.47 3.41
Met 17.37 ± 0.73 4.20
Cys 5.07 ± 0.21 4.14
Ile 12.24 ± 0.24 1.96
Leu 39.91 ± 1.97 4.94
Phe 19.60 ± 0.87 4.44
Trp 10.05 ± 0.19 1.89
Lys 24.74 ± 1.34 5.44

* Mean ± SD (n = 6).
Page 169
buffer [acetonitrile:methanol:triethylamine (10:5:2)] and 3 µL BITC were added to the dry residue. It
was sonificated for 1 min and thermostatted at 40°C for 30 min. The sample was dried again with
nitrogen gas (about 10 min), and then 100 µL acetonitrile were added and dried again. The BTC
derivatives were dissolved in 1 mL aqueous ammonium acetate and filtered through a 0.25-µm
membrane filter. Proteins (soybean, whole egg, and bovine serum albumin) were hydrolyzed by 6 M
HCl at 145°C for 4 h and then cleaned on a cation-exchange column. Separation of BTC amino acids
was carried out on a dimethyloctadecylsilyl-bonded amorphous silica column (300 × 3.9 mm I.D., 4 µm
particle size). The gradient eluent system consisted of 0.05 M ammonium acetate, and the pH was
adjusted to 6.7 with phosphoric acid (eluent A); 0.02 M dibasic sodium phosphate containing 5%
methanol, 0.75% tetrahydrofuran, and 0.75% acetonitrile (eluent B); acetonitrile:water (7:3) (eluent C).
The gradient was: 0 min, 100% A; 8 min, 85% A-15% B; 14 min, 70% A-20% B-10% C; 20 min, 60%
A-20% B-20% C; 25 min, 30 A-20% B-50% C; 30 min, 10% A-20% B-70% C. The flow rate was 1
mL/min, and the detection wavelength was 240 nm. BTC cisteine and cystine were well separated;
however, BTC asparagine and serin were eluted as one peak in this gradient eluent system. The
detection limit for both BTC and PTC amino acids was in the range of 3.9 pmol. The chromatograms of
amino acids in soybean and whole egg hydrolysates are shown in Figures 2.88 and 2.89. The same
amino acids were detected by both derivatization methods, and the number of ghost peaks was low.
These results indicate that BTC and PTC are adequate reagents for the analysis of the amino acids
composition of various food proteins. The amino acid composition of soybean and whole egg
hydrolysates determined with three independent methods are given in Tables 2.82 and 2.83. The
similarities of the data clearly show that each method is suitable for the determination of the amino acid
composition of the hydrolysate of various food samples. Deviations were detected only in the case of a
few amino acids.
The new derivatizing agent, 6-aminoquinolyl-N-hydroxyzuccinimidyl carbamate (AQC) has been

recently synthesized and successfully used for the separation and quantitative determination of amino
acids in protein hydrolysates with both fluorescence [ 194] and ultraviolet detection [ 195]. It was
established that the derivatization is very rapid (only a few seconds) and simple; moreover, the products
are stable and the excessive AQC does not interfere with the separation process. The method has been
used for the determination of the amino acid composition of some food proteins [196]. Samples were
hydrolyzed with 6 N HCl for 22 h at 110 ± 2°C.
For the determination of Cys and Met, performic acid oxidation was used: 2 mL of chilled performic
acid was added to a 50- to 70-mg sample in a hydrolysis tube and was held for 16 h at 0°C. Then 0.3
mL of 48% HBr was added, and the mixture was incubated for 15 min at 0°C. The mixture was
Page 170
Figure 2.88.
Chromatogram of amino acids in soybean hydrolysate:
(A) BTC derivatives; (B) PTC derivatives.
dried on a rotary evaporator at <60°C, and the rest was hydrolyzed as described above. The
hydrolysates were filtered and dried at <50°C. Then α-aminobutyric acid (2.5 µmol/mL) as internal
standard and water were added to the sample to obtain <13 µmol/mL total amino acids. Derivatization
was carried out in a tube containing a sample of 10 µL, 70 µL 0.2 M borate buffer (pH 8.8) and 20 µL
of AQC solution of 3 mg/mL acetonitrile. The solution was shaken in a mixer, sealed, and heated for 10
min at 50°C. Separation of the amino acid derivatives was carried out on an octadecyl column of 150 ×
3.9 mm at the controlled temperatures of 37°C (for acid hydrolysates) and 47°C (for performic acid
hydrolysates). The flow rate was set to 1.0 mL/min, and the detection limit was 248 nm. Gradient
conditions listed in Table 2.84
Page 171
Figure 2.89.
Chromatogram of amino acids in whole egg hydrolysate: (A) BTC derivatives;
(B) PTC derivatives.
were the same for each hydrolysate. The influence of the concentration of sodium chloride in the
sample on the efficacy of the derivatization process has been studied in separate experiments, and it was
established that up until 10% w/w, the presence of sodium chloride did not influence the yield of
derivatization.
The chromatograms of corn and broiler starter samples are shown in Figures 2.90 and 2.91. Derivatized
amino acids are well separated even from feed samples (Figures 2.90 and 2.91). The peaks are
symmetric and a baseline separation was achieved in each instance. These data prove the good retention
characteristics of the RP-HPLC method. The results of the present method and those of the AOAC ion-
exchange chromatography (IEC) method are compiled in Tables 2.85 and 2.86. Because the results
were similar to those of the official AOAC method, it was assumed that the method described above is
comparable or better than ion-exchange chromatography.
Page 172
TABLE 2.82. Amino Acid Composition of Soybean Determined by the BTC Derivatives Method
Compared to the Use of PTC Derivatives and Ion-Exchange Chromatography (g/100 g dry
matter).
Amino Acid BTC Derivatives PTC Derivatives Ion-Exchange Chromatography
Asp 4.17 ± 0.24 4.64 ± 0.28 4.25 ± 0.32
Glu 6.96 ± 0.42 7.25 ± 0.34 7.19 ± 0.12
Ser 1.61 ± 0.12 1.59 ± 0.02 1.65 ± 0.01
Gly 1.59 ± 0.07 1.77 ± 0.10 1.62 ± 0.05
His 1.50 ± 0.06 1.52 ± 0.04 1.32 ± 0.05
Arg 3.38 ± 0.17 2.28 ± 0.08 3.50 ± 0.09
Thr 1.19 ± 0.04 1.79 ± 0.17 1.24 ± 0.01
Ala 1.72 ± 0.10 1.89 ± 0.35 1.80 ± 0.12
Pro 2.15 ± 0.12 2.06 ± 0.08 2.20 ± 0.06
Tyr 1.20 ± 0.04 1.29 ± 0.08 1.15 ± 0.05
Val 2.88 ± 0.24 2.47 ± 0.10 3.01 ± 0.14
Met 0.42 ± 0.08 0.54 ± 0.060 0.40 ± 0.03
Ile 2.46 ± 0.14 2.21 ± 0.06 2.25 ± 0.18
Leu 3.38 ± 0.18 3.03 ± 0.31 3.57 ± 0.21
Phe 2.27 ± 0.10 2.10 ± 0.10 2.19 ± 0.14
Lys 3.03 ± 0.13 3.15 ± 0.16 2.86 ± 0.19
All values are mean ± SD, n = 4.

TABLE 2.83. Amino Acid Composition of Whole Egg Determined by the BTC Derivatives Method
Compared to the Use of PTC Derivatives and Ion-Exchange Chromatography (g/100 g dry
matter).
Amino Acid BTC Derivatives PTC Derivatives Ion-Exchange Chromatography
Asp 4.20 ± 0.09 4.74 ± 0.07 4.23 ± 0.05
Glu 5.48 ± 0.05 5.85 ± 0.59 5.76 ± 0.07
Ser 3.50 ± 0.09 2.61 ± 0.42 3.54 ± 0.10
Gly 1.56 ± 0.09 1.54 ± 0.31 1.53 ± 0.02
His 1.90 ± 0.14 1.91 ± 0.15 1.79 ± 0.06
Thr 1.75 ± 0.13 2.15 ± 0.11 1.82 ± 0.15
Ala 2.71 ± 0.16 3.29 ± 0.19 2.70 ± 0.11
Arg 3.55 ± 0.10 3.43 ± 0.48 3.61 ± 0.16
Pro 1.95 ± 0.15 1.87 ± 0.24 1.98 ± 0.22
Tyr 2.36 ± 0.62 1.96 ± 0.20 2.23 ± 0.30
Val 3.64 ± 0.20 3.83 ± 0.36 3.59 ± 0.32
Met 1.68 ± 0.11 1.58 ± 0.18 1.63 ± 0.06
Ile 3.42 ± 0.16 2.92 ± 0.28 3.26 ± 0.21
Leu 4.63 ± 0.21 4.32 ± 0.42 4.35 ± 0.09
Phe 2.94 ± 0.14 2.65 ± 0.22 2.88 ± 0.15
Lys 4.16 ± 0.20 4.30 ± 0.33 4.18 ± 0.30
Cys 1.35 ± 0.16 0.40 ± 0.10 1.41 ± 0.23
All values are mean ± SD, n = 4.
Page 173
TABLE 2.84. Gradient Table for AQC Derivatized Amino Acid Separation. *
Common Amino Acids Sulfur-Containing Amino Acids
Time (min) A ** (%) B*** (%) A** (%) B*** (%)

0 100 0 100 0
17 93 7 92 8
21 90 10 83 17
32 66 34 73 27
34 66 34 50 50
35 0 100 50 50
37 0 100 0 100
38 100 0 0 100
45 100 0 100 0
* All gradient segments were linear.
**140 mM sodium acetate and 17 mM TEA was titrated to pH 4.95 with phosphoric acid,
and 0.1 g sodium azide was added to 1000 mL eluent.
*** 60% acetonitrile in water (v/v) containing 0.01% acetone.
Reprinted from Reference [ 196] with permission, copyright © 1995, by AOAC
International.
Dansyl (Dns) derivatization has also been used frequently in the analysis of amino acids [197,198].
Dansyl derivatization was used for the study of the influence of the microbial starter and breadmaking
step on the free amino acid profiles of wheat sours, doughs, and breads [ 199]. The study was motivated
by the fact that free amino acids are important sources of bread flavor precursors [ 200], influence the
metabolism of microorganisms in dough [ 201], and improve functionality [ 202]. The samples were
freeze-dried, and 10 g of fermented sourdough (FSD), unfermented bread dough (UBD), fermented
bread dough (FBD), and 5 g of bread (B) were extracted twice with 20 mL 40% ethanol (v/v). The
mixtures were centrifuged at 23,000 g for 20 min at 1-3°C, and then the supernatant was filled up to 50
mL. Aliquots were ultrafiltered (cut-off 10 kDa), the pH was adjusted to 2.0, and the acidified sample
was passed through a column containing 5 mL of Dowex 50W-X2 resin (50-100 mesh, H+ form,
previously equilibrated with 0.01 M HCl). Ammonia extracts were dried in a rotary evaporator.
Norvaline (69 mg/200 mL 0.01 M HCl) was used as the internal standard; 0.1 mL was added to the
sample extract containing about 50 µg of amino nitrogen. The sample was dried again and redissolved
in 0.5 mL of K2CO3 buffer, pH 10.5, and 0.2 mL dansyl chloride solution (0.5 g/25 mL dry acetone),
and 1.0 mL acetone was added. The mixture was heated at 100°C for 2 min, evaporated to dryness, and
redissolved in 1.7 mL of initial mobile phase. Separation of dansylated amino acids was carried out on
an octadecyl-bonded silica column (83 × 4.6 mm I.D., particle size 3 µm) preceded by a precolumn (30
× 4.6 I.D., particle size 3 µm). The mobile
Page 174
Figure 2.90.
Chromatogram of amino acid derivatives in (a) acid -hydrolyzed corn sample and
(b) performic acid oxidized corn hydrolysate.
phase consisted of an aqueous solution of 12 mM K2 HPO4pH 7 (A) and acetonitrile (B). The gradient
program was from 10% to 43.6% B in 42 min up to 70% B in 3 min. The flow rate was 1.50 mL/min,
and the detector wavelength was 254 nm. Columns were thermostatted at 30°C. The chromatograms of
the standard dansyl amino acid mixtures and a fermented dough sample are shown in Figure 2.92. The
chromatograms prove that this HPLC method results in the baseline separation of 23 dansylated amino
acid derivatives even from dough and bread extracts. It was established that the coefficient of variation
of the relative retention times in the samples was in the range 0.10-1.30%, and that of the response
factor relative to Nval was less than 5%, except Thr, Tyr, and Arg (<9%). The peak purity test indicated
99.5% purity for each amino acid except Cys and Met (90% peak purity).
Page 175
Figure 2.91.
Chromatogram of amino acid derivatives in (a) acid-hydrolyzed broiler starter
sample and (b) performic acid oxidized broiler starter hydrolysate.
The contents of free amino acids in the samples are presented in Table 2.87. It was established that Glu,
Ala, GABA, and Pro are present in considerable amounts in each sample whereas the content of Cys
and Met is fairly low in each instance. The amino acid content significantly depended on the type of
starter culture and on the fermentation and baking. The quantity of dicarboxylic amino acids, amides,
Ser, and hydrophobic amino acids decrease. This finding is in accordance with the results of Reference
[203]. The baking process generally increases the content of free amino acids. This result may be caused
by the thermal hydrolysis and enzymatic decomposition of proteins. It was concluded on the basis of
the results of factor and cluster analysis that
Page 176
TABLE 2.85. Comparison of Amino Acids Recovery in Corn Hydrolysate by Ion-

Exchange Chromatography (IEC) and Current Method.
AOAC Data by Data by Current Method** Data by IEC

Amino Acid IEC (mg/g *) mg/g RSD (%) (mg/g***)
Asp 0.54 0.56 0.94 0.55
Thr 0.29 0.28 0.53 0.29
Ser 0.39 0.33 1.4 0.36
Glu 1.51 1.57 0.74 1.56
Pro 0.73 0.76 0.48 0.75
Gly 0.33 0.34 1 0.34
Ala 0.61 0.62 0.68 0.62
Cys 0.18 0.16 1.7 0.18
Val 0.38 0.41 0.38 0.43
Met 0.18 0.16 0.01 0.18
Ile 0.28 0.32 0.67 0.34
Leu 0.99 1.01 0.73 1.05
Tyr nd **** 0.27 0.38 0.3
Phe 0.38 0.41 2.8 0.5

Lys 0.26 0.26 0.69 0.3
His 0.27 0.25 1.4 0.27
Arg 0.4 0.38 0.56 0.41
*Cited from 1992 AOAC collaborative study for amino acid analysis. Samples used
were acid hydrolysates oxidized by performic acid and with Na2S 2O5 as reducer.
** All data were obtained from the analysis of acid hydrolysates of corn powder, except
the data for Met and Cys, which were from the analysis of performic acid oxidized
hydrolysates (with HBr as the reducer). RSDs were calculated with data from five (for
Met and Cys) replicate derivatizations of the same hydrolysates with AQC.
*** Same hydrolysates were used for the IEC method like for the current method.

International.
almost every individual amino acid exerts a significant impact on the characterization of the
breadmaking processes. The same method has been previously used for the study of the biochemical
changes in free amino acids during wheat flour mixing and bread making [ 204].
Derivatization with o-phthalaldehyde (OPA) has also been used for the HPLC analysis of amino acids
from various matrices [ 205,206]. The free amino acid content of shrimp was determined by OPA
derivatization [ 207]. The study was motivated by the fact that free amino acids play a considerable role
in the osmoregulation in crustaceans and are important contributors to flavor quality. Samples were
prepared by mixing 1.0 g shrimp tissue with 2.0 mL trichloroacetic acid in a blender. The mixture was
centrifuged, and the supernatant was used for the analysis. The derivatizing solution for primary amino
acids was prepared by mixing 10 mg OPA dissolved in 250 µL methanol, 37.5 µL 30% Brij 35, 25 µL
of 2-mercaptoethanol, and 3 mL of 0.5 M potas-
Page 177
TABLE 2.86. Comparison of Amino Acids Recovery in Broiler Starter's Hydrolysate by

IEC and Current Method.
AOAC Data by Data by Current Method** Data by IEC

Amino Acid IEC (mg/g *) mg/g RSD (%) (mg/g***)
Asp 2.29 1.53 2.31 1
Thr 0.88 2.73 0.84 0.6
Ser 1.12 2.5 0.95 1.06
Glu 4.04 1.78 4 0.7
Pro 1.47 4.08 1.51 0.56
Gly 1.27 2.68 1.29 0.75
Ala 1.28 2.11 1.33 0.58
Cys 0.35 1.71 0.33 0.01
Val 1.11 1.71 1.2 0.46
Met 0.62 2.1 0.58 0.01
Ile 0.95 2 1.07 0.52
Leu 1.97 1.68 2 0.59
Tyr nd nd 0.72 0.57
Phe 1.12 2.23 1.16 2.9
Lys 1.35 2.37 1.33 0.58
His 0.65 2.77 0.61 0.54
Arg 1.57 2.68 1.54 0.52
*Cited from 1992 AOAC collaborative study for amino acid analysis. Samples used
were acid hydrolysates oxidized by performic acid and with Na2S 2O5 as reducer.
**All data were obtained from the analysis of acid hydrolysates of broiler starter, except
the data for Met and Cys, which were from the analysis of performic acid oxidized
hydrolysates (with HBr as the reducer). RSDs were calculated with data from five (for
Met and Cys) replicate derivatizations of the same hydrolysates with AQC. *** Same
hydrolysates were used for IEC method as for current method.
International.
sium borate buffer (pH 10.4) and diluted to 10 mL with the borate buffer. The sample of free amino
acids was diluted with 5 mL of sodium citrate buffer (pH 2.2) and filtered; 100 µL of extract was mixed
with 40 µL internal standard solution (2.5 µm α-aminobutyric acid/mL). Before injection, 0.5 mL of the
OPA solution was mixed with 0.5 mL of amino acid solution. The mixture was injected within 2 min.
Derivatization for secondary amino acids was carried out by mixing the amino acid extract, 0.4 M
borate buffer, and 4-chloro-7-nitrobenzofurazan solution [2 mg N-chloro-7-nitrobenzofurazan
(NBD)/mL methanol] in a volume ratio of 1:1:1, heated at 60°C for 5 min in a closed vial. Then the vial
was cooled to 0°C and the content injected.
Each derivative was detected with the monochromator set at 330 nm and a 418 nm cut-off filter. The
derivatives of primary amino acids were separated on 100 × 4.6 mm I.D. octadecyl dimethylsilane
column (particle size 3 µm) and a 30 × 4.6 I.D. precolumn filled with the same support. Derivatives of
Page 178
Figure 2.92.
Separation of dansylamino acids (Dns-AA) of a standard mixture
(a) and of fermented sourdough samples started with Lactobacillus
brevis, 25+ (b), by RP-HPLC. Concentration of Dns-CI [Dns-Cl] and molar
ratio [Dns-Cl]/AA = 8.5 mM, 4.6. Column: ODS (3 µm particle size, 8.3 cm
length, 4.6 mm I.D.). Mobile phases: A = 12 mM K 2 HPO4 pH 7.0;
B = ACN. Linear gradient, 10 to 43.6% B over 42 min; flow rate:
1.50 mL/min; injection volume: 20 µL; column temperature: 30 °C. Dns-AAs:
(D) Asp, (E) Glu, (N) Asn, (Q) Gln, (S) Ser, (T) Thr, (G) Gly, (A) Ala, (GABA)
τ-amino butyric acid, (P) Pro, (R) Arg, (V) Val, (M) Met, (I) Ile, (L) Leu,
(W) Trp, (F) Phe, (C) Cys, (O) Orn, (K) Lys, (H) His, and (Y) Tyr.
secondary amino acids were separated on a 300 × 4.6 I.D. octadecylsilica column (particle size 5 µm).
The excitation and emission wavelengths were 340 and 455 nm for primary amino acid derivatives and
220 and 370 nm for secondary amino acid derivatives, respectively. The columns were thermostatted at
23°C. The separations of the standards of primary and secondary amino acids are shown in Figures 2.93
and 2.94. It was established that the method
Page 179
TABLE 2.87. Individual and Total Amino Acid Content of Unstarted and Started Sours, Doughs, and
Breads.
Mean Content of Individual and Total Free Amino Acids*
Starter Sample Asp Glu Asn Gln Ser Thr Gly Ala GABA
A I 5.22 8.22 8.27 4.01 3.73 2.70 4.71 9.58 16.04
II 6.59 19.81 5.33 4.93 2.32 2.12 2.07 6.37 5.79
III 1.99 10.03 1.46 2.56 1.06 1.99 2.45 7.89 5.95
** ** ** ** **
IV 4.20 14.36 2.77 2.03 2.12 3.17 3.18 10.62 8.86
** ** ** ** ** ** **
B I 6.53 9.60 8.52 3.77 5.71 2.82 5.89 6.83 18.99
II 7.08 17.30 5.52 6.17 2.45 1.73 2.20 7.06 6.06
III 5.66 15.36 3.16 4.05 1.80 2.00 2.68 9.28 8.16
** ** ** ** ** **
IV 6.57 17.38 4.37 2.88 2.09 3.39 2.90 10.44 9.21
** ** ** ** ** **
C I 5.43 10.45 5.54 4.18 4.08 2.70 6.45 8.12 20.88
II 6.14 14.42 4.54 6.64 2.24 1.97 1.83 5.47 4.87
III 4.21 11.80 2.77 4.38 1.49 1.82 2.00 7.97 6.43
** ** ** ** **
IV 8.03 18.69 5.25 3.36 2.87 3.49 2.95 10.71 9.31
** ** ** ** ** ** ** ** **
D I 5.72 7.85 9.11 4.92 1.40 2.67 5.31 8.80 20.62
II 7.04 18.36 6.76 6.91 1.77 2.08 2.24 7.49 6.76
III 2.17 11.52 1.80 2.81 1.33 2.38 3.49 10.82 8.31
** ** ** ** ** ** **
IV 3.86 14.57 2.28 1.81 1.61 3.26 3.37 10.62 9.29
** ** **
Control II 7.88 20.25 6.31 5.82 2.13 1.86 1.55 5.74 4.31
III 4.98 9.46 0.66 3.53 1.24 1.80 2.03 3.96 5.39
** ** ** ** ** ** **
IV 3.49 13.65 1.14 1.47 1.75 2.88 2.32 5.95 7.48

** ** ** **
Mean Content of Individual and Total Free Amino Acids

Starter Sample Pro Arg Val Met Ile Leu Trp Phe Cys
A I 6.50 21.68 8.87 1.79 7.03 18.28 10.84 0.54 0.24
II 11.05 9.35 4.36 0.70 2.80 4.29 7.18 3.07 0.37
III 11.27 8.00 3.20 0.30 0.66 0.94 6.49 1.15 0.23
** ** **
IV 12.59 11.06 4.23 0.42 2.18 3.21 6.40 2.58 0.53
** ** ** ** **
B I 8.49 5.58 11.04 2.58 10.61 23.08 9.76 14.40 0.67
II 12.33 1.93 4.92 1.01 4.07 7.62 7.91 4.86 0.24
III 12.97 2.50 4.72 0.54 1.98 3.04 7.99 3.96 0.12
** ** **
IV 12.70 8.49 4.75 0.38 3.51 1.59 6.60 3.65 0.49

** ** ** ** **
C I 10.42 3.25 13.43 2.93 12.34 26.52 11.19 17.02 0.40
II 8.11 1.98 4.10 0.82 2.75 5.29 5.65 3.56 0.28
III 9.94 2.64 4.12 0.45 1.72 2.89 5.94 3.38 0.25
** ** ** **
IV 12.02 7.34 5.04 0.43 3.10 3.37 7.17 4.69 0.41
** ** ** ** **

Page 180

Starter Sample Pro Arg Val Met Ile Leu Trp Phe Cys
D I 10.72 25.98 12.15 2.95 9.87 30.33 13.83 17.98 0.53
II 13.00 10.35 5.48 1.15 3.61 8.13 8.45 5.15 0.34
III 15.51 8.18 3.51 0.18 0.75 0.98 8.01 1.96 0.27
** ** ** ** ** ** **
IV 12.26 13.02 4.80 0.51 0.98 1.75 6.44 2.59 0.47

** ** ** **
Control II 9.74 2.68 2.18 0.32 1.37 1.43 6.16 1.04 0.63
III 9.31 4.78 2.10 0.23 0.32 0.65 4.28 0.28 0.11
** ** **
IV 11.42 5.62 3.37 0.27 0.55 0.94 0.59 0.45 0.32

**
Starter Sample Orn Lys His Tyr TAA

A I 0.24 8.71 3.04 7.75 167.25
II 0.80 4.11 0.87 3.04 106.26
III 0.77 3.26 0.74 2.16 74.50
** ** **
IV 1.43 3.10 1.05 3.18 102.93

** ** **
B I 9.06 4.37 3.71 3.12 174.59
II 2.34 2.69 0.66 2.35 107.59
III 4.01 3.42 0.75 2.75 100.80

** ** **
IV 3.43 2.76 1.04 2.93 114.95
** **
C I 9.81 4.89 2.47 3.27 186.08
II 2.70 2.48 0.98 1.55 88.81
III 3.20 2.88 0.32 2.07 82.82
** ** **
IV 4.22 2.94 1.25 2.93 123.03
** ** ** **
D I 0.37 9.86 2.85 12.41 216.19
II 1.09 4.72 1.36 3.84 125.99
III 1.11 4.61 0.90 3.14 91.31

** **
IV 1.18 3.21 1.16 3.57 102.56

**
Control II 0.92 1.81 0.58 1.53 86.25
III 0.68 2.34 0.39 0.38 58.93

** **
IV 0.82 2.12 0.73 0.85 66.26
*mg amino acid/100 g sample, dry basis (d.b.); A = Lactobacillus plantarum B -39 (-); B = Lactobacillus
brevis 25a (-); C = Lactobacillus brevis 25a (+); D = Lactobacillus plantarum L-73 (+); I = fermented
sourdough; II = unfermented bread dough; III = fermented bread dough; IV = bread; TAA = total amino
acid content.
** Indicates a statistically significant change between mean values (p<0.05) for fermentation and baking
steps when second-order interactions (starter-breadmaking step) are significant.
Page 181
Figure 2.93.
Separation of OPA -amino acid standard. Operating conditions:
flow rate 1.2 mL/min. Solvent A: 0.1 M sodium acetate
buffer + 1% tetrahydrofuran, pH = 6.2. Solvent B:
methanol. Sample loop 10 µL. Excitation at 330 nm, emission
filter 418 nm. Peaks: 1 = aspartic acid, 2 = glutamic acid, 3 = serine,
4 = histidine, 5 = glycine, 6 = threonine, 7 = arginine, 8 = alanine,
9 = tyrosine, 10 = α -aminobutyric acid, 11 = methionine, 12 = valine,
13 = phenylalanine, 14 = isoleucine, 15 = leucine, 16 = lysine.
Figure 2.94.
Separation of NBD-amino acid standard. Operating
conditions: flow rate 1.2 mL/min. Solvent A: 0.1 M sodium
acetate buffer + 1% tetrahydrofuran, pH = 6.2. Solvent
B: methanol. Sample loop 10 µL. Excitation at 220 nm,
emission filter 370 nm. Peaks: 1 = hydroxyproline, 2 = proline.
Page 182
successfully separates the amino acids, and no interference from the shrimp tissues was observed. The
relative standard deviation of the retention time and peak were ±0.1 and ±3%, respectively, proving the
reliability and stability of the chromatographic system. The detection limit was found to be 100 fmol.
The average content of free amino acids found in shrimp samples is presented in Table 2.88. The data
clearly show that the most abundant amino acids in shrimp tissue are Gly, Arg, and Pro, whereas the
concentration of Asp, Phe, and hydroxypro is fairly low. It was further found that culture shrimps have
a higher concentration of free amino acids than the wild type does.
The determination of the whole amino acid profile of foods is not necessary in some instances. The
food scientist may be interested in the quantity of one or more amino acids that exert the highest impact
on the nutritional value and/or technological properties of the food sample. In these cases the analytical
procedure is considerably simplified, and isocratic HPLC methods can be used instead of the time-
consuming and sometimes less reliable gradient elution. Thus, an isocratic HPLC method has been
developed for the quantitative
TABLE 2.88. Free Amino Acids in White Shrimp (P. Vannamei) Wild and Cultured.*
Free Amino Acids Wild ** Cultured 1 ** Cultured 2 **
OPA-Amino Acids
Aspartic 2.43 ± 0.29 a 2.72 ± 0.13 a 4.05 ± 0.24 b
Glutamic 19.99 ± 1.38 a 19.54 ± 1.64 a 20.51 ± 1.28 a
Serine 20.86 ± 1.69 a 10.41 ± 0.39 b 19.54 ± 1.75 a
Histidine 68.27 ± 20.27 a 138.23 ± 22.42 b 101.10 ± 20.37 b
Glycine 367.55 ± 10.66 a 373.15 ± 22.11 a 575.14 ± 36.79
Arginine 439.13 ± 21.87 a 429.61 ± 34.14 a 389.84 ± 37.01 a
Taurine 71.51 ± 4.27 a 22.54 ± 1.94 b 88.57 ± 7.99 c
Alanine 73.23 ± 3.87 a 94.48 ± 8.07 b 82.42 ± 8.81 ab
Tyrosine 20.24 ± 3.62 a 24.84 ± 2.04 a 27.74 ± 3.35 a
Methionine 12.13 ± 0.78 a 5.56 ± 0.43 b 11.94 ± 1.03 a
Valine 18.91 ± 1.44 a 18.68 ± 0.52 a 22.93 ± 1.15 b
Phenylalanine 8.23 ± 0.86 a 4.76 ± 0.61 b 10.18 ± 0.41 a
Isoleucine 12.45 ± 0.78 a 8.92 ± 0.71 b 14.80 ± 0.96 a
Leucine 18.53 ± 1.48 a 13.52 ± 1.38 b 20.51 ± 1.87 a
Lysine 21.74 ± 124 a 17.49 ± 2.04 a 20.40 ± 2.84 a

NBD-Amino Acids
Proline 168.94 ± 12.61 a 490.29 ± 54.24 b 334.66 ± 31.67 c
Hydroxyproline 11.92 ± 1.27 a 7.23 ± 0.61 b 5.16 ± 0.56 c
Total free
amino acids 1344.14 a 1681.97 b 1749.40 c

* mg/100 g.
** a, b,c = significative differences (p < 0.05).

Page 183
determinaton of lysine, histidine, and tyrosine in various foods using dansylchloride as a derivatization
agent [ 208]. Protein samples were hydrolyzed with 6 M HCI at 110°C under nitrogen using 1 mL HCl
for 1 mg protein. The hydrolysate was filtered, and the volume was adjusted to 250 mL. Aliquots were
dried in a rotary evaporator at 40°C. The dry residue was dissolved in water to give a concentration of
0.15-0.25 mg protein/mL; 1 mL of protein solution was mixed with 2 mL of 40 mM LiCO 3 (pH 9.5)
and 1 mL of DnsCl solution (4 mg/mL, 14.83 mM) in this order. The mixture was kept at 60°C for 30
min. Then 50 µL methylamine solution was added to stop the reaction, and the sample was allowed to
stand for at least 15 min before injection.
Separation of dansylated amino acid derivatives was carried out on various octadecylsilica columns
thermostatted at 40°C. The detection wavelength was 254 nm, and the flow rate was 1.5 mL/min.
Isocratic eluent consisted of acetonitrile:0.01 M phosphate buffer (pH 7.0) (39:61 v/v). The
reproducibility of the retention times was found to be less than 1%. The chromatograms of protein
hydrolysates are shown in Figure 2.95. The chromatograms clearly show
Figure 2.95.
(A) Chromatogram of a casein sample on the µBondapak column.
(B) Chromatogram of a lysozyme sample on the Spherisorb column.
Page 184
TABLE 2.89. Recovery of Lysine, Histidine, and Tyrosine (mean

values of four replications).
Recovery (%) CV (%)
Lysine 100.21 4.18
Histidine 102.56 1.77
Tyrosine 95.06 5.41
Reprinted from Reference [ 208], copyright © 1996. With kind

that both columns are suitable for the baseline separation of dansylated Lys, His, and Tyr, although the
analysis time is longer on the Spherisorb column. The recovery and relative standard deviation of the
recovery of these amino acids from protein samples is given in Table 2.89. The data in Table 2.89
indicate that this HPLC method is precise and accurate and can be used as a valuable substitute for the
traditional ion-exchange chromatography. The results of protein analyses are compiled in Table 2.90.
It has been often established that the presence of D-amino acids in fermented foods and beverages is
caused by bacterial enzymes and may be an important indicator of food quality [ 209,210]. Because
proline is the most abundant free amino acid in wine [ 211], many efforts have been devoted to the
determination of the D- and L-proline content in various wines. An HPLC method using (1-fluoro-2,4-
dinitrophenyl)-5-L-alanine amide (FDAA) [ 212] was developed for the study of the distribution of
proline enantiomers in Italian wines [ 213]. Samples were prepared by mixing 50 µL of wine with 50 µL
0.2 M of sodium bicarbonate and 100 µL 4.0 µmol/mL of L-norleucine as the internal standard. The
mixture was dried in a rotation evaporator, and then 50 µL of water and 250 µL of 10 µmol FDAA/mL
acetone was added. The solution was thermostatted at 50°C for 60 min under weak stirring in a dark
vial and then dried under vacuum. The solid remainder was dissolved in 2 mL of water and was used
for the analysis. Separations were carried out on an octadecylsilica column (250 × 4 mm I.D.) guarded
by a precolumn (50 × 4 mm I.D.). Mixtures
TABLE 2.90. Lysine, Histidine, and Tyrosine Contents (g/16 g N)

in the Samples.
Amino Acid Casein Lysozyme Lentil Enteral Solution
Lysine 8.33 5.86 6.54 10.31
Histidine 3.91 1.23 3.07 2.42
Tyrosine 5.92 3.25 2.92 2.61
Page 185
of water and acetonitrile in various volume ratios were used as eluents, and the pH of the eluents was
adjusted to 3.0 ± 0.1 by using triethanolamine and o-phosphoric acid. A gradient elution was also used:
5 min at 10% acetonitrile and then a linear gradient to reach 40% acetonitrile after 45 min. The flow
rate was set to 1.2 mL/min, and the detection wavelength was 340 nm. The gradient elution successfully
separated the D- and L-enantiomers of proline present in the wine samples (Figure 2.96.). The average
content of D- and L-proline in Italian red and white wines is given in Table 2.91. The data prove again
that the proline content is higher in red than in white wine, and there is no relationship between the
alcohol and proline content of wines. It can also be observed that the proline content depends on the
vinification type [ 214], on the ripeness of the grape [ 215], and on the cultivar [ 216]. Red wines
originating in Sardinia show a lower proline content than the other red wines of similar age and alcohol
content [217]. It has been further established that older wines have a considerably higher content of D-
proline than the young wines do (the coefficient of variation of the isomeric ratio being 7.4%).
Since tryptophan is an essential amino acid not formed endogenously, it has to be added to infant
formulas to meet the requirements of newborn infants [ 218]. An isocratic HPLC method was developed
and optimized for the determination of the tryptophan content of infant formulas using phenyl
Figure 2.96.
Chromatogram of the wine sample Rosso del Salento
''Merum." The chromatographic conditions were first 5 min
of isocratic flow with 10% of acetonitrile solution and then a linear
gradient to reach 40% of acetonitrile solution after 45 min with a
constant flow of 1.2 mL/min.
HO-DAA = 1-hydroxy-24-dinitriphenyl-5-L-alanine;
FDAA = 1-fluoro -2, 4-dinitrophenyl-5-L-alanine amide.
Page 186
TABLE 2.91. Proline Isomers Relative Abundance in Italian Wines.*

Samples
L-Pro D-Pro
Red Wines (mg/mL) (mg/mL) D/(L+D)
Cabernet "Castello di Porcia" (Friuli Venezia Giulia rd) 12° 1992 637 ± 11 tr **
Chianti "Tenuta del Poggio" (Tuscany rd) 12° rd) 12° 1992 454 ± 5 ND **
Chianti "Piccini" (Tuscany rd) 12° 1992 571 ± 13 ND
Chianti "Mellini" (Tuscany rd) 12° 1987 1241 ± 1 13 ± 0.5 1.1

Chianti "Brolio" (Tuscany rd) 12° 1985 491 ± 17 3 ± 0.4 0.6
Corvo Rosso "Duca di Salaparuta" (Silicy rd) 12° 1991 667 ± 10 6 ± 0.6 0.9
Marsala intarcia" (Sicily rd) 18° 1992 999 ± 20 4 ± 0.5 0.4
Marsala "Vecchio Florio" (Sicily rd) 18° 1989 836 ± 20 2 ± 0.1 0.2
Marsala "Mirabella" (Sicily rd) 19° 1984 937 ± 21 9 ± 0.5 1.0
Merlo "Isonzo" (Friuli-Venezia Giulia rd) 12.5° 1992 1323 ± 2 3 ± 0.2 0.2
Merlot "La Delizia" (Friuli-Venezia Giulia rd) 12° 1992 1414 ± 2 tr
Merlot "Tami" (Friuli-Venezia Giulia rd) rd) 12.5° 1992 1352 ± 1 5 ± 0.4 0.4
Merlot "Formentini" (Friuli-Venezia Giulia rd) 12.5° 1992 1364 ± 9 3 ± 0.2 0.2
Monica de Sardegna (Sardinia rd) 12° 1970 411 ± 1 39 ± 3 8.7
Reciotto "Grasso" (Veneto rd) 12° 1968 463 ± 4 43 ± 1 8.4
Refosco Colle Vento" (Friuli -Venezia Giulia 12° 1993 815 ± 12 ND
Refosco "La Delizia" (Friuli-Venezia Giulia 12° 1992 867 ± 9 ND
Rosato del Salento "Salice" (Puglie rd) 12.5° 1986 943 ± 21 5 ± 0.7 0.5
Rosso del Salento "Merum" (Puglie rd) 15.2° 1971 876 ± 11 93 ± 4 9.6
Rosso Pugliese "Mercurio" (Puglie rd) 12° 1964 664 ± 14 114 ± 4 14.7
Spanna "Sogno di Bacco" (Piedmont rd) 13° 1964 732 ± 28 65 ± 4 8.2
Teroldego Rotaliano "Mezzacorona" (Trentino-Alto Adige rd) 12° 832 ± 25 ND

1992
Teroldego Rotaliano (Trentino -Alto Adige rd) 12° 1996 780 ± 19 22 ± 3 2.7
White Wines
Chardonnay "La Pieve" (Veneto rd) 11° 1993 349 ± 13 ND
Falerno "Cenatiempo" (Latium rd) 12° 1969 275 ± 3 23 ± 0.2 7.7

Picolit "Di Cialla" (Friuli-Venezia Giulia rd) 14° 1992 572 ± 1 ND
Prosecco "Vigneto ai Casali" (Veneto rd) 11° 1993 530 ± 13 ND
Soave "Falconi" (Veneto rd) 11 ° 1993 349 ± 5 ND

Tocai Italico "San Matteo" (Veneto rd) 10° 1993 369 ± 12 ND
Tocai Friuliano "Rubini" (Friuli -Venezia Giulia rd) 12.5° 1992 237 ± 7 ND
*Values are the average of three determinations with the standard deviations; the tradenames of the wines are given in
quotation marks.
** Traces (tr) are <1.0 mg/L.

Page 187
isothiocyanate derivatization [ 219]. Samples were hydrolyzed by adding 4.83 g of barium hydroxide
and 5 mL of boiling water to 500 mg infant formula. The mixture was deaerated by nitrogen flow and
then heated at 120°C for 8 h. After hydrolysis, the pH of the mixture was adjusted to 3-4 with HCl,
filtered, and diluted to 25 mL with water. An aliquot of 163 µL was evaporated to dryness under
vacuum; norleucine internal standard (10 µL of 4-nmol/µL) was added, and the sample vacuum dried
again. A solution of ethanol:water:triethylamine (2:2:1 v/v) was added to the sample (2 µL solution/100
nmol amino acid) and vacuum dried again. The derivatizing reagent
[ethanol:triethylamine:water:phenyl isothiocyanate (7:1:1:1)] was added to the amino acid residues in
the ratio of 5 µL/100 nmol amino acid and allowed to stand for 20 min at room temperature, and then
the excess reagent was removed under vacuum.
Derivatized amino acids can be stored at -20°C for weeks. Before the HPLC analysis, the dry residue
was dissolved in 400 µL of 5 m/M sodium acetate buffer (pH 7.4) containing 5 vol% acetonitrile. The
mixture was sonificated for 1 min and then filtered (0.22-µm membrane filter). Separation was carried
out on an octadecylsilica column (250 × 4.6 mm I.D., 5 µm particle size). The isocratic eluent consisted
of 17 mM sodium acetate containing 325 µL/L triethylamine (pH adjusted to 6.8 by acetic acid) and
acetonitrile (84:16 v/v). A typical chromatogram is shown in Figure 2.97. The limit of detection and
quantification was determined [ 220] and was found to be 3.9 nmol (18 mg/g sample) and 11.3 nmol (51
mg/g sample), respectively. Some validation parameters of the method are presented in Table 2.92. The
data prove that the derivatization step causes the highest increase in the relative standard deviation,
whereas the contribution of other steps to the relative standard deviation is markedly lower. The
relationship between tryptophan quantity and detector response was linear between 20 nmol and 200
nmol of tryptophan (coefficient of correlation = 0.998). The accuracy of the derivatization was 104 ±
6%. The validation parameters indicate that this isocratic HPLC method is sensitive, reproducible, and
suitable for the determination of the tryptophan content in infant formulas. The tryptophan contents
determined are listed in Table 2.93.
Monosodium glutamate (MSG) is extensively used in various foods as a flavor enhancer. Because large
amounts of MSG can cause undesirable physiological effects, its accurate and rapid determination is of
considerable importance. The majority of methods used for the determination of MSG content of foods
use either precolumn or postcolumn derivatization such as dansyl chloride [221], dabsyl chloride [ 222],
and PITC [ 223,224].
PITC was also used in a study dealing with the HPLC determination of free glutamic acid in foods
[225]. Samples were prepared by mixing 1-5 g ground food with 30 mL of 0.02 M potassium
dihydrogen phosphate and allowed to stand for 15 min. Then 30 mL of acetone was added, and the
mixture was left to stand 15 min again, filtered, and diluted to 100 mL with 0.02 M potassium
Page 188
Figure 2.97.
Chromatogram of infant formula. Peaks:
1 = norleucine; 2 = typtophan.
Science—NL, Sara Burgerhartstraat 25,
1055 KV Amsterdam, The Netherlands)
dihydrogen phosphate. An octadecyl- or octyl-bonded silica SPE column was washed with methanol
and then equilibrated with 0.02 M potassium dihydrogen phosphate. Two solutions of 2 mL of amino
acid extract were passed through the column, and the second one was used for derivatization: 30 µL
extract was dried under vacuum and 30 µL of methanol:water:triethylamine (1:1:1 v/v) was added to
the residue and dried again. Derivatization reagent was prepared by mixing 100 µL of PITC, 100 µL of
triethylamine, 100 µL of water, and 700 µL of methanol; then 30 µL of this reagent was added to the
dry residue of amino acids, allowed to stand for 30 min, evaporated to dryness, and redissolved in 300
µL of eluent A. Eluent A was prepared by dissolving 11.45 g of sodium acetate in 900 mL of water and
0.5 mL triethanolamine. Acetic acid was used to adjust the pH to 6.4. Then 60 mL of acetonitrile was
added, and the solution was diluted to 1 L with water.
Eluent B was water:acetonitrile (2:3 v/v). Separation was carried out on an octadecylsilica column
(particle size 5 µm) using UV detection at 254 nm. The gradient is shown in Table 2.94. The content of
free glutamic acid in various foods determined by this HPLC method are compiled in Table 2.95. Since
the
Page 189
TABLE 2.92. Reproducibility Studies; Relative Standard Deviation

for Peak Areas and Retention Times of Tryptophan.
Precision X1 S n–1 RSD (%) X2 Sn–1 RSD (%)
Instrumental* 0.68 0.009 0.88 1.93 0.015 0.7
Derivatization** 0.72 0.06 8.29 1.93 0.03 1.55

Method*** 0.82 **** 0.09 **** 11.5 2.00 **** 0.05 **** 2.33
* Calculated from eight injections of the same standard (100 nmol) on two different days.
** Checked by analysing three standards of the same concentration (100 nmol of tryptophan and 100 nmol of
internal standard) twice.
*** Established by analysis (hydrolysis and derivatization procedure) of three samples (infant formulas) twice.
**** mg per 100 g.
X 1 = Relative area; X2 = Relative retention time; Sn–1 = standard deviation.
derivatization methods are sometimes time-consuming and may increase the relative standard deviation
of the analysis, an indirect conductometric method was developed for the liquid chromatographic
determination of MSG in foods [ 226]. The preparation of nonstarchy food samples was carried out by
adding 100 mL water to 20 g of sample, and the mixture was sonificated for 15 min, and then 6 g
activated charcoal was added. After a half hour (with occasional agitation), it was filtered through a
Whatman No. 542 filter paper and washed several times. The filtrate was diluted to 200 mL with water.
In the case of starchy food samples, the extraction was carried out with an acetone:water (1:1 v/v)
mixture. Before the last steps, two drops of 4 M HCl was added, and the acetone was evaporated. A
Dowex 50W-X8 (H +, 100-200 mesh) column (65 × 9 mm I.D.) was used for the clean-up of the
samples. The column was
TABLE 2.93. Tryptophan Contents of Infant Formulas.

Tryptophan
Protein Content
Infant Formula (g/100 g) g/100 g Sample g/100 g Protein
Adapted starting 12.17 0.108 ± 0.011 0.848 ± 0.089
Follow-up 15.86 0.101 ± 0.008 0.639 ± 0.053
Low-birthmass 15.03 0.099 ± 0.006 0.659 ± 0.042
Without lactose 15.29 0.111 ± 0.021 0.726 ± 0.135
Soy formula 14.42 0.109 ± 0.024 0.754 ± 0.164
Hypoallergenic 14.83 0.079 ± 0.016 0.535 ± 0.109
Page 190
TABLE 2.94. Gradient Used for HPLC Analysis.

Time (min) Solvent A (%) Solvent B (%) Flow (mL/min)
0 100 0 1
0.5 100 0 1
10 54 46 1
10.5 0 100 1
12 0 100 1
14 100 0 1.5
20 100 0 1
Reprinted from Reference [ 225] with permission from Taylor & Francis.
equilibrated by passing 100 mL of 4 M HCl, followed by about 100 mL water until chloride ions were
absent in the eluent (silver nitrate test). Then 10 mL of sample were passed through the column at a
flow rate of 0.5 mL/min and washed with 5 mL water and 10 mL of 0.8 M HCl. MSG was eluted from
the column in the form of glutamic acid with 18 mL of 1 M HCl followed by 5 mL of water. The
volume of eluate was adjusted to 100 mL with water. A 10-mL aliquot was freeze-dried and redissolved
in 10 mL of HPLC eluent. The dansylation method served as a control. An Econosil CN column (250 ×
4.6 mm I.D., particle size 5 µm) was used for the separation of MSG from the other components in the
food samples. The eluents were: (1) water:acetonitrile: tetrahydrofuran (77:20:3 v/v/v) containing 1 mM
perchloric acid and (2) water:acetonitrile:tetrahydrofuran (77:20:3 v/v/v) containing 1 mM
trichloroacetic acid. The flow rate was 1 mL/min, and the column temperature was set to 30°C. The
peaks of MSG were detected as negative changes in conductance by the conductivity detector. The
method successfully separated MSG from the coeluted other amino acids as shown in Figures 2.98 and
2.99. The baseline separation indicates the good separation capacity of this HPLC system for MSG. It
was found that the coefficient of correlation between the peak area and MSG concentration in the
samples was larger than 0.9995. The relative standard deviation of the determinations depended
considerably on its amount varying between 3% and 14%. The recovery was 96.1%. The MSG content
of various food samples are presented in Table 2.96. The data prove that no significant difference can
be found between the MSG contents determined with the indirect conductometric detection and with the
established dansylation method. The difference between the results determined with the two acidic
additives was also not significant, proving that they can be used alternatively for the analysis of MSG in
food samples.
The results in the analysis of amino acids by various HPLC methods have been critically reviewed
[227].
Page 191
TABLE 2.95. Free Glutamic Acid in Various Foods.

Free Glutamic Acid (ppm)
Found in
Food Unfortified Food Fortification Level Recovery (%)
Soups
Cream of mushroom * 3210 2310 109
Turtle **,***,**** 450 1320 109
Red consommè 720 360 121
Lobster bisque **,*** 2880 3240 66
Vegetable beef***, * 310 1060 105
Vegetable* 2920
Chicken broth* 1270
Shrimp bisque*, ***,§ 11,700
Sauces
Soy
Brand 1 **,**** 490 340 125
430 105
850 111
Brand 2 9170 32,200 107
Brand 3 9350 11,255 112
Brand 4 10,200
Brand 5 6940
Duck** ND §§ 280 105
520 91
1000 94
Mustard ** ND 220 99
510 114
870 110
Spices
Brand 1 * 61,000
Brand 2 * 88,000
Brand 3 * 136,000
Brand 4 * 886,000
Brand 5 ND 259,000 85
Brand 6 ** ND 1030 90
Processed food flavors
Model process 66,000 32,000 72
Grilled beef 96,000 220,000 107
Natural prime rib 73,000
Parmesan cheese " 3640 4439 241
12,700 5800 188
5000 1800 172

Vegetables
Green beans
Brand 1 26 1100 66
Brand 2 31
Peas
Brand 1 170
Brand 2 340

Page 192
TABLE 2.95.
Free Glutamic Acid (ppm)
Found in
Food Unfortified Food Fortification Level Recovery (%)
Vegetables—(continued)
Corn
Brand 1 240 360 121
Brand 2 500
Isolated soy protein ND
Chinese rice* 3560 2587 93

Chocolate 120 1800 98
Potato chips* 1570 5400 84

Rice cakes 750 3552 71
Autolyzed yeast powders
Type 1 " " 49,000 123,000 66
55,000 132,000 105
60,000
62,000
Type 2 49,000 123,000 66
Type 3 85,000 29,000 84
Type 4 77,000
Tomato products
Paste
Brand 1 *** 6360 3350 99
4690 69
9830 82
Brand 2 6240 3540 99
5130 113
9760 89
Sauce 1840 930 68
2260 127
4230 96
Soup *** ," 1390 788 98
1660
1550
Fresh tomato# 2270 425 197
* Label declared MSG.

** Label declared no MSG.
*** Label declared natural flavors.
**** Label declared hydrolyzed vegetable protein.
§ The shrimp bisque was a dried product. All other soups were reconstituted.
§§ND = Not detected at <20 ppm, according to the detection limit, which is the mean of baseline
amplitude plus 3 × SD of background.
" Triplicate analysis.
"" Quadruplicate analysis.
# Recovery is high possibly because spiking level is low compared with the level in the tomato.
Repreinted from Reference [ 225] with permission from Taylor & Francis.
Page 193
Figure 2.98.
Chromatogram of MSG from a soup base sample with indirect conductometric
detection. Mobile phase, water:acetonitrile:tetrahydrofuran (77:20:3),
1 mM HClO 4 MSG peak at 7.8 min.
Page 194
Figure 2.99.
Chromatogram of MSG (approximately 0.12 µg) from a baby food sample with
indirect conductometric detection. Mobile phase, water:acetonitrile:
tetrahydrofuran (77:20:3), 1 mM HClO4 MSG peak at 7.8 min.
Page 195
TABLE 2.96. Determination of MSG in Food Products with Mobile Phases:

(i) Water:Acetonitrile:tetrahydrofuran (77:20:3) in 1 mM HClO4 and
(ii) Water:Acetonitrile:tetrahydrofuran (77:20:3) in 1 mM CCl3COOH.
Proposed Method (%, m/m)

Mobile Phase
(i) (ii)
LC Method with
Sample Description Found Mean * Found Mean* Dansylation (%)
1 Soup base (Brand1) 0.71 0.71 0.73 0.74 0.74
0.72 (0.8) 0.73 (1.6)
0.71 0.75
2 Soup base (Brand 2) 16.3 16.3 16.1 16.5 17.3
16.3 (0.4) 16.8 (2.3)
16.4 16.7
3 Soup base (Brand 3) 17.5 17.2 16.9 16.9 18.0
16.9 (1.8) 17.1 (0.9)
17.1 16.8
4 Baby food 0.007 0.007 0.006 0.007 0.007
0.007 (7.9) 0.007 (14.3)
0.008 0.008
5 Oyster extractives 0.41 0.41 0.41 0.42 0.40
0.40 (1.4) 0.41 (2.8)
0.41 0.43
6 Oyster-flavored suace 0.32 0.31 0.32 0.33 0.32
(Brand 1)
0.30 (3.2) 0.34 (3.5)
0.31 0.32
7 Oyster-flavored sauce 3.01 3.03 2.95 3.00 2.89
(Brand 2)
3.08 (1.4) 2.93 (3.3)
3.00 3.11
8 Spice sauce 1.77 1.76 1.75 1.71 1.84
1.74 (0.9) 1.74 (3.6)
1.76 1.64
9 Pistachios 0.068 0.069 0.069 0.067 0.068
0.071 (3.0) 0.065 (3.1)

0.067 0.066
*Mean of triplicate measurements in percentage by mass and relative standard deviation of the results (%) in
parentheses.
Page 196
2.3.1.2—
Other Chromatographic and Electromigration Methods
Some decades ago various TLC methods were extensively used for the separation and semiquantitative
determination of amino acids both in derivatized and underivatized forms. However, the widespread use
of the more precise and reliable HPLC and GLC methods considerably decreased the application of
TLC for the analysis of amino acids. The newest results in this field have been recently reviewed [ 228].
Although it has been proven many times that the various electromigration methods such as capillary
zone electrophoresis [229] and micellar electrokinetic chromatography [ 230] are suitable not only for the
separation of Dns amino acids, but also for their enantiomeric separation [ 231], these methods have not
been frequently used for the determination of the amino acid composition of foods. The micellar
electrokinetic capillary chromatography (MECC) of dansylated amino acid derivatives from feedstuffs
and skin was reported [232]. The hydrolysis of the protein samples and the derivatization of the amino
acids by dansyl chloride was carried out using traditional techniques. The effect of MECC conditions
such as voltage, temperature, pH, and buffer composition on the separation efficacy of dansyl amino
acids was studied in detail. It was established that the concentration of SDS in the buffer has a marked
impact on the separation of DNS amino acids, the optimum being 150 mM. The concentration of boric
acid influenced the separation of Asn and Ser, and good separation was achieved at 100 mM
concentration. The pH also influenced the separation, with pH 7.5 and 8.3 found to be the best values.
The optimal temperature and voltage were 27°C and 15 kV, respectively. A wavelength of 216 nm was
used for detection. The best eluent also contained 20% methanol. The separation of 21 amino acids in
the optimized method is shown in Figure 2.100. The migration order and relative migration times
(RMT) are given in Table 2.97. The detection limit of the method varied from 0.7-1.3 µm, and the
relative standard deviation of the RMT values was 0.09-0.70%, proving that the MECC method can be
successfully used for the determination of the amino acid profile of foodstuffs.
Since the volatility of amino acids is negligible, they cannot be separated by gas-liquid
chromatographic methods. Appropriate derivatization agents and processes make the amino acids
volatile, and these derivatives are suitable for GLC analysis. The GLC method for the analysis of amino
acids was evaluated earlier [ 233]. Thus, amino acids can be N(O, S)-isobutyloxycarbonylated (isoBOC),
then recovered by two -step solid-phase extraction and tertbutylmethylsilylated (TBDMS). The resulting
derivatives are volatile and suitable for GLC analysis [ 234,235]. The edible seeds, nuts, and beans
screened for the free nonprotein and protein amino acids are listed in Table 2.98. Food samples were
ground (80-100 mesh), and 500 mg were extracted
Page 197
Figure 2.100.
Separation of 21 amino acids in the optimized
method. Conditions: 100 mM boric acid,
pH 8.3, 27°C, 15 kV, 150 mM SDS, 216 nm,
vacuum injection 1 s.
The Netherlands.)
with 4 × 1 mL water in a vortex mixer. A saturated solution of picric acid was added to the extract (200
µL to 1 mL), and the mixture was shaken and centrifuged (300 g, 2 min). The supernatant was acidified
with 10% sulfuric acid to pH < 2 and then washed with 3 × 2 mL ethyl acetate and 2 × 2 mL diethyl
ether. The pH of the aqueous phase was adjusted to pH 11 with sodium carbonate. Then isobutyl
chlorformate was added and mixed in a vortex mixer and then washed with diethyl ether. The aqueous
phase was acidified again with 10% sulfuric acid to pH 1-2 and saturated with NaCl. The mixture was
Page 198
TABLE 2.97. Migration Order and RMT Values (relative

to mesityl oxide) for Amino Acids.
Amino Acid RMT Amino Acid RMT
T4HyPro 0.824 Nor 1.206
Thr 0.900 Pro 1.215
Asn 0.909 Met 1.236
Ser 0.913 Ile 1.362
Gln 0.930 Leu 1.416
Ala 0.960 Phe 1.571
Glu 0.982 Trp 1.604
α-ABA 0.989 Lys 2.055
Mesityl oxide 1.000 Arg 2.076
Asp 1.015 DiLys 2.138
Gly 1.027 Tyr 2.145
Cysteic acid 1.055 Trypt 2.156
Val 1.104 DiHyLys 2.160
τ-ABA 1.105 Cad 2.176
C4Hy-D-Pro 1.142 DiTyr 2.191
C4Hy-L-Pro 1.144
Conditions: 100 mM boric acid, pH 8.3, 25°C, 15 kV, 150 mM SDS, 216 nm, vacuum
injection 1 s.
loaded onto a Chromosorb P column and the isoBOC amino acids were eluted with diethyl ether. The
eluate was dried under nitrogen, and the residue was reacted with N-methyl-(trimethylsilyl)-
trifluoroacetamide in tetrahydrofuran. Both DB -5 (SE-54 bonded phase) and DB-17 (OV-17 bonded
phase) capillary columns were used for the separation of the amino acid derivatives (30 m × 0.25 I.D.,
0.241 µm film thickness). Column temperature was initially 150°C for 2 min and then increased to 280°
C at 3°C/min rate for split injection (30:1). In the case of splitless injection, the initial column
temperature was 80°C for 2 min and then increased to 150°C at 30°C/min, held for 1 min, and then
raised to 280°C at 3°C/min rate. The injector temperatures were 280°C and 260°C for split and splitless
injection mode, respectively; the temperature of the flame ionization detector was 300°C. Amino acids
were identified by GLC-mass spectrometry. Chromatograms of nonprotein and protein amino acids in
kidney bean (Phaseolus vulgaris L.) separated on DB-17 and DB -5 columns are shown in Figure 2.101.
The good separation capacity of the method allowed the quantitative determination of nonprotein and
protein amino acids in each food sample. The results of analyses are shown in Tables 2.99-2.101. The
data prove that this GLC method is suitable for the separation and quantitative determination of not
only the protein amino acids, but also the nonprotein ones, which may have an adversary health effect
in foods.
Page 199
TABLE 2.98. Edible Seeds, Nuts and Beans Screened

for the Free Nonprotein and Protein Amino Acids.
Common Name Scientific Name Family
Adzuki bean Phaseolus angularis W.F. Wight Fabaceae
Almond Pinus dulcis Mill. Rosaceae
Cacao Theobroma cacao L. Sterculiaceae
Capsici seed Capsium annuum L. Solanaceae
Chestnut Castanea deritata Marsh. Fagaceae
Coffee bean Coffea arabica L. Rubiaceae
Ginkgo nut Ginkgo biloba L. Ginkgoaceae
Green pea Pisum sativum L. Fabaceae
Hazelnut Corylus avellana L. Corylaceae
Kidney bean Phaseolus vulgaris L. Fabaceae
Mung bean Phaseolus radiatus L. Fabaceae
Peanut Arachis hypogaea L. Fabaceae
Perilla seed Perilla frutescens Britton Laminaceae
Pine nut Pinus koreaiensis Sieb. et Zucc Pinaceae
Sesame seed Sesamium indicum L. Pedaliceae
Sunflower seed Helianthus annuus L. Asteraceae
Walnut Juglnas regia L. Juglandaceae
Watermelon seed Citrullus vulgaris Schrad Cucurbitaceae
Wultari bean Phaseolus vulgaris L. Fabaceae
A similar method was used for the determination of amino acid profile of wines [ 236]. The study was
motivated by the fact that free amino acids contribute to the taste of wine. The results are compiled in
Table 2.102. The data indicate that both the total amino acid content and the amino acid composition of
wines show considerable differences. Stepwise discriminant analysis suggested that six amino acids
(Ala, Asp, Glu, Gly, On, and Val) are the most characteristic of the wines; therefore, they can be used
for identification of wine types.
The determination of the quality and quantity of D-amino acids in food is of considerable importance
because they may decrease the nutritional value and may overload the D-amino oxidase system [237 ].
D-amino acids mainly occur in fermented food products as a consequence of microbial modifications
[238, 239]. The D-amino acid content of Portuguese wines was determined by using GLC [ 240]. Wine
samples (20 mL) were mixed with 80 mL of 95% ethanol and thermostatted at -10°C for 10 min. The
mixture was centrifuged at 3.500 rpm for 10 min. The supernatant was concentrated to 10 mL in a
rotary evaporator. Amino acids were isolated by passing through the solution on an Dowex 50W-X8
column with 4 M NH4OH. The eluate was concentrated to 2 mL, and an aliquot of 250 µL was dried in
a derivatization vial and left
Page 200
Figure 2.101.
Chromatograms of nonprotein and protein amino acids in kidney
bean (Phaseolus vulgaris L.) separated on DB-17 (30 m × 0.25
mm I.D.) and DB -5 (30 m × 0.25 mm I.D.) dual capillary column
system. 3 = alanine; 4 = glycine; 6 = valine; 7 = β-alanine;
10 = leucine; 12 = isoleucine; 13 = threonine; 17 = τ-aminobutiric acid;
19 = pipecolic acid; 20 = S-methylcysteine; 21 = pyroglutamic acid;
31 = phenylalanine; 33 = aspartic acid; 38 = glutamic acid;
39 = asparagine; 42 = α-aminoadipic acid; 43 = glutamine;
49 = lysine; 50 = histidine; 51 = tryptophan;
54 = unidentified; 55 = unidentified.
kind permission from Elsevier Science—NL, Sara Burgerhartstraat
25, 1055 KV Amsterdam, The Netherlands.)
overnight over P 2O5 in vacuum. The residue was dissolved in 100 µL of 3 M/L HCl/isopropanol and
heated at 110°C for 30 min. The sample was cooled at room temperature, evaporated to dryness in
nitrogen, and 50 µL of pentafluoropropionic anhydride and 100 µL of CH 2Cl2 were added. The mixture
was heated at 150°C for 15 min. After cooling, it was injected onto a Chirasil-L-Val capillary column
(15 m × 0.25 mm I.D.). The analytical conditions were: split ratio 1:25; injector and detector
temperatures 250°C; initial column temperature 70°C for 3 min, heating to 200°C at 3°C/min; carrier
gas hydrogen; flame ionization detector.
TABLE 2.99. Nonprotein and Protein Amino Acids Found in Beans.
Amount (% normalized area)*
Kidney Mung Green Wultari Adzuki
No. Amino Acid Bean Bean Pea Bean Bean Cacao
3 Alanine 11.1 25.7 8.3 11.2 20.2 46.8
4 Glycine 4.0 17.0 10.5 10.5 12.6 12.2
6 Valine 7.3 15.3 5.5 8.2 12.5 61.6
7 β -Alanine 0.7 3.8 2.8 3.6 4.4
10 Leucine 3.1 4.8 2.5 1.9 5.2 35.1
12 Isoleucine 2.5 3.7 2.4 1.7 3.6
13 Threonine 1.9 8.3 21.1 7.8 17.4 29.5
17 τ-Aminobutiric acid 6.9 32.7 19.9 59.2 68.8 31.7
19 Pipecolic acid 100.0 12.8 2.2 100.0 69.9
20 S-Methylcysteine 4.4 14.9 3.9
21 Pyroglutamic acid 6.4 10.1 8.0 1.4 7.5 2.3
31 Phenylalanine 1.9 1.9 1.9 1.3
33 Aspartic acid 69.5 53.4 4.2 10.2 65.7 100.0
38 Glutamic acid 56.5 100.0 100.0 24.7 100.0 93.0
39 Asparagine 36.3 32.0 58.8 46.3 44.7 7.7
42 α -Aminoadipic acid 1.4 5.8 1.9 9.3
43 Glutamine 3.0 3.6 3.4 2.2 3.3 6.7
49 Lysine 1.3
50 Histidine 1.1 trace 0.6
51 Tryptophan 1.2 1.4 0.6
54 Unidentified 10.1 7.6
55 Unidentified 17.0 10.7

* Percentage of peak area normalized to the largest peak.
Reprinted from Reference [ 235], copyright © 1995. With kind permission from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055
KV Amdsterdam, The Netherlands.
A typical chromatogram is shown in Figure 2.102. The presence of Denantiomer was proven for almost every
amino acid. Especially high quantities of D-Ala, D-Asx, D-Glx, and D-Leu were observed. The average relative
concentration of free D-amino acids in Roupeiro wines (vintages from 1978 to 1989) are shown in Figure 2.103.
It has been stated that the high concentration of D-Ala in vintages 1978-1981 excludes the probability of natural
isomerization, and the ratio of D-enantiomers is not suitable for the determination of the age of wines. However,
cluster analysis suggested that the concentration of D-amino acids is a valuable indicator of the vinification
technology. Wines form two distinct groups on the cluster dendogram: wines prepared with endogenous yeast, no
inoculation, and no temperature control and wines produced with inoculation of special yeasts and temperature
control of the fermentation
Page 202
TABLE 2.100. Nonprotein and Protein Amino Acids Found in Seeds.

Sesame Sunflower Capsici Watermelon Perilla
No. Amino Acid Seed Seed Seed Seed Seed
3 Alanine 43.2 37.3 0.8 23.0 33.1
4 Glycine 30.6 26.1 0.8 19.6 15.6
6 Valine 32.4 21.1 15.6 5.8
7 β -Alanine 6.7
10 Leucine 32.1 12.2
11 allo-Isoleucine 1.8
12 Isoleucine 21.9 16.7
13 Threonine 19.8 7.1 18.2 9.5 6.0
14 Serine 32.9 20.6 11.2 30.7
16 Proline 17.7 22.5 1.0 16.9 5.7
17 τ-Aminobutiric acid 16.5 8.7 0.8 45.8 11.3
21 Pyroglutamic acid 2.5 4.6 1.2 2.8 7.7
23 Methionine 2.1
31 Phenylalanine 16.7 7.1
33 Aspartic acid 76.7 100.0 84.2 57.0 65.1
38 Glutamic acid 100.0 82.5 10.1 90.8 100.0
39 Asparagine 55.3 28.9 100.0 21.2 42.8
42 α -Aminoadipic acid 2.0 1.6
43 Glutamine 11.2 4.2 2.6 21.4 1.6
49 Lysine 5.6 3.8
50 Histidine 11.1 trace
51 Tryptophan 2.6 5.0
52 Unidentified 10.1 NC
53 Unidentified NC
NC = not calculable because of incomplete separation between peaks 52 and 53.
process. This result suggests that the enantiomeric ratio of free amino acids in wines can be used as a
marker of the fermentation technology applied. It has been further concluded that D-amino acids in
fermented products originate mainly from cell autolysis.
The majority of studies dealing with the determination of free and proteinbonded amino acids in foods
use either HPLC or GLC because it has been proven many times that both methods are suitable for such
investigations. However, the number of studies simultaneously using both analytical procedures is
surprisingly low. The D-amino acid content of vegetables and fruits was determined by both GLC and
HPLC. For GLC analysis, the amino acids were isolated on an cation-exchange column and derivatized
as previously described, and the pentafluoropropionyl amino acid 2-propyl esters were analyzed on a
TABLE 2.101. Nonprotein and Protein Amino Acids Found in Nuts.
No. Amino Acid Chestnut Ginkgo Nut Pine Nut Walnut Hazel Nut Peanut
3 Alanine 53.3 3.5 13.5 39.0 17.5
4 Glycine 13.3 3.9 3.6 5.0 22.7 5.8
5 α-Aminobutiric acid 0.9 trace
6 Valine 28.4 9.1 58.7 4.1
7 β-Alanine 0.7 1.3
8 β-Aminoisobutyric acid 0.7
10 Leucine 11.5 5.4 7.1 0.8
11 Allo-Isoleucine 5.7
12 Isoleucine 13.2
13 Threonine 12.5 trace 1.4 1.3 18.3 2.1
14 Serine 38.8 17.4 4.4 5.5 39.6 5.3
16 Proline 34.3 trace 49.3 11.1 7.4 12.3
17 τ-Aminobutiric acid 24.1 12.7 10.3 6.4 5.8
19 Pipecolic acid 5.5 35.0
21 Pyroglutamic acid 7.1 8.3 trace 15.6 5.0
22 4-Hydroxyproline 2.0
23 Methionine 1.4
31 Phenylalanine 10.0 5.9
33 Aspartic acid 10.1 100.0 25.8 25.7 71.3 40.0
38 Glutamic acid 100.0 56.0 13.9 100.0 100.0 100.0
39 Asparagine 72.8 17.2 100.0 4.8 40.2 28.6
42 α -Aminoadipic acid 0.9 1.0
43 Glutamine 2.7 38.1 1.3 5.7 15.4
46 Ornithine 1.5
49 Lysine trace 3.8
50 Histidine trace trace
54 Unidentified trace
Chirasil-L-Val capillary column [ 241]. For HPLC analysis, the amino acids were derivatized as follows: 5
0.4 < sodium borate buffer (pH 10.4), 1 µL derivatizing reagent (260 mM N-isobutyryl-L-cysteine or N-
isobutyryl-D-cysteine and 170 mM o-phthaldialdehyde in 1 M potassium borate buffer, pH 10.4) and 2 µ
amino acid sample were mixed and reacted under controlled conditions. Octadecylsilica column (250 × 4 mm
I.D., particle size 5 µm) and guard column (20 × 2.1 mm I.D.) were used for the separation. Eluent A: 23
Page 204
TABLE 2.102. Free Amino Acids Found in Four Brands of White Wine.
Mean Peak Area Ratio**
Wine * Ala Gly Val Leu lle Ser Pro Gaba Pyg
C1 0.94 1.06 0.13 1.99 0.87 0.21 128.80 NC ***
C2 0.95 1.01 0.07 1.75 0.56 0.14 115.63 NC

C3 0.90 1.07 0.12 1.77 0.65 0.18 120.18 NC
C4 0.32 0.50 ND 1.48 0.34 0.06 40.22 NC
C5 0.45 1.29 ND 2.16 0.94 ND 53.31 NC
Mean 0.71 0.99 0.06 1.83 0.67 0.12 91.63
RSD(%) 42.50 29.64 97.95 14.12 36.01 73.49 45.28 29.84
B1 0.29 1.21 0.34 ND 0.64 ND 7.40 0.39
B2 0.05 0.67 0.16 ND 0.98 ND 2.18 0.44
B3 0.03 0.38 0.06 ND 0.35 ND 0.80 0.30
B4 0.01 0.15 1.06 ND 0.60 ND 0.28 0.11
B5 0.04 0.18 0.07 ND 1.23 ND 0.09 0.09
Mean 0.08 0.52 0.14 0.76 2.15 0.27
RSD(%) 138.22 84.74 87.32 45.46 141.70 60.07 103.22
S1 2.75 1.08 0.29 ND 1.08 ND 26.12 0.19
S2 1.71 1.02 0.18 1.66 0.80 ND 25.98 0.30
S3 1.12 1.10 0.16 1.67 0.60 ND 19.83 0.27
S4 1.62 1.47 0.29 1.33 0.69 ND 36.81 0.73
S5 1.29 1.30 0.15 1.38 0.75 ND 28.01 0.20
Mean 1.70 1.19 0.21 1.21 0.78 27.35 0.28
RSD(%) 37.40 15.64 32.81 57.37 23.15 22.38 34.82 30.45
M1 1.64 1.65 ND 1.94 0.57 ND 32.70 0.28
M2 2.32 2.43 0.20 2.38 1.65 ND 50.23 ND
M3 1.45 1.84 0.13 1.75 0.45 ND 25.41 0.21
M4 3.37 3.11 0.67 ND 2.52 ND 71.62 0.99
M5 4.14 3.71 ND 2.85 0.66 ND 65.71 0.35
Mean 2.58 2.55 0.20 1.78 1.17 49.13 0.37
RSD(%) 44.49 33.92 138.25 60.76 76.34 40.89 101.80 57.74
Mean Peak Area Ratio
Wine * Thr Hsr Asp Cys Glu Asn Lys Orn

C1 1.11 0.06 0.67 tr **** 0.55 0.35 0.73
C2 0.84 0.02 0.61 tr 0.57 0.40 0.64

C3 1.06 0.01 0.72 tr 0.61 0.44 0.66
C4 0.46 ND 0.31 tr 0.26 0.23 0.49
C5 1.42 ND 0.59 tr 0.49 0.37 0.91
Mean 0.98 0.02 0.58 0.50 0.36 0.69
RSD(%) 36.40 138.33 27.48 27.99 22.12 22.27 13.25
B1 1.94 0.38 3.08 tr 2.77 0.77 0.68
B2 2.68 0.25 5.26 tr 4.77 0.57 1.02
B3 0.92 0.07 1.84 tr 2.34 0.24 0.41
B4 0.60 0.06 1.27 tr 1.18 0.16 0.42
B5 1.10 0.13 4.13 tr 3.83 0.41 0.43

Page 205
TABLE 2.102.
Mean Peak Area Ratio
Wine * Thr Hsr Asp Cys Glu Asn Lys Orn
Mean 1.45 0.18 3.12 2.98 0.43 0.59
RSD(%) 58.59 76.35 52.40 46.35 57.51 44.68 47.08
S1 1.04 ND 0.50 tr 0.64 0.20 0.55
S2 0.72 0.30 0.52 tr 0.63 0.26 0.22
S3 0.49 0.21 0.52 tr 1.38 0.26 0.35
S4 0.59 0.21 0.66 tr 0.83 0.33 0.25
S5 0.60 0.13 0.56 tr 0.65 0.19 0.24
Mean 0.69 0.17 0.55 0.83 0.25 0.32
RSD(%) 30.96 66.16 11.63 38.80 22.70 42.55 70.80
M1 1.15 0.22 1.03 tr 0.82 0.35 0.28
M2 1.50 0.57 1.06 tr 0.76 0.31 0.31
M3 0.87 0.47 0.98 tr 0.79 0.31 0.20
M4 3.11 NC 1.88 tr 1.81 0.59 0.44
M5 1.46 0.51 2.16 tr 1.64 0.48 0.26
Mean 1.62 0.35 1.42 1.16 0.41 0.30
RSD(%) 53.91 67.40 39.07 44.34 30.24 29.86 24.98
* Wines: C = California Chardonnay; B = Blue Nun; S = Majuand Special; M = Majuang.
**Mean peak area ratio with respect to cycloleucine (IS) from three replicate runs on DB-5 (30m × 0.25 mm I.D., 0.25
µm film thickness) capillary column programmed from 60°C (held for 2 min) to 150°C at 30 °C/min, and then to 280 °
C at 3°C/min.
*** Not calculable owing to incomplete resolution from proline.
**** Detected at trace level.
mM sodium acetate trihydrate in 990 mL doubly distilled water (pH adjusted to 5.95 by 10% v/v acetic acid),
eluent B: 474 g methanol and 39 g acetonitrile. Gradient elution: from 0-53.5% B in 75 min. Flow rate was 1
mL/min, and excitation and emission wavelengths were 230 and 445 nm, respectively. Similar HPLC
methods have been previously used for the determination of D-amino acids in fruit juice [ 242,243].
Chromatograms showing the successful separation of D- and L-amino acids are shown in Figure 2.104. It
was stated that the HPLC method offers some advantages compared with GLC: it distinguishes the
enantiomers of Asp, Asn, Glu, Gln, Arg, and His while routine GLC does not separate the enantiomers of
Asn, Gln, Arg, and His. The results of amino acid analyses are presented in Tables 2.103 and 2.104. It has
been concluded from the data that D-amino acids occur in various quantities in foods of plant origin and they
are common constituents of foods of plant origin. This finding makes questionable the use of D-amino acid
content as a marker for the microbial status of food products.
Page 206
Figure 2.102.
Chiral capillary gas chromatography of free amino acids
in an 11-year elementary wine of the Vitis vinifera
variety Roupeiro.
the publisher and the corresponding author.)
2.3.2—
Amines
Aliphatic, alicyclic, and heterocyclic amines are frequently present in various foods. They can be found
in many biological materials because of microbial activity [ 244,245]. It is assumed that they are
produced by the activity of bacterial decarboxylases from amino acids [ 246]. Amines influence the
organoleptic characteristics of foods [ 247], and they are potentially toxic; therefore, their separation,
identification, and quantitative determination is of considerable importance. As in the case of amino
acids both HPLC and GLC methods have been extensively used for the analysis of amines in foods.
2.3.2.1—
The suitability of various HPLC methods for the separation and quantitative determination of biogenic
amines has been proven for a wide variety of food products. Thus, HPLC was used for the analysis of
amines in fish and fish
Page 207
Figure 2.103.
Relative enantiomeric concentrations of free D-amino acids in elementary wines of
the variety Roupeiro corresponding to the vintages from 1978 to 1989.
(Repinted from Reference [ 240] with permission from the publisher and the corresponding author.)
Page 208
Figure 2.104.
Elution profile of amino acids (''aminogram") from freshly pressed
orange juice, derivatized directly with (a) N-isobutyryl-L-cysteine and
(b) N-isobutyryl-D-cysteine, and (c) section of aminogram of processed
orange juice with unpleasant taste and brownish color,
derivatization as in (b).
TABLE 2.103. Relative Amounts * (% D) of Most Abundant Native D -Amino Acids (D-AA) in Cabbage and Garlic and Rela
and Absolute** Amounts of D-AA in Pickled Cabbage.
Red Green Green Green White White
Cabbage **** Cabbage § Cabbage § Cabbage §§ Cabbage " Cabbage ""
Pickled Cabbage
D-AA *** (%D) (%D) (%D) (stem) (%D) (% D) (% D) (% D)
D-Ala 2.5 3.8 0.8 2.6 0.8 1.7

D-Asx 1.5 0.9 0.9 0.4 1.6 1.3
D-Glx 0.3 — 0.5 0.8 0.6 0.4
D-Leu — 1.0 — — — —
D-Lys — — — — — —
D-Val 1.0 — 0.9 — — — —
* Determined by GC on Chirasil-Val, % D = 100.D/(D ± L).
** Determined using automated amino acid analyzer and ninhydrin derivatization.
*** Asx = Asp + Asn; Glx = Glu + Gln.
**** Cultivar (cv) `Wanner'.
§ cv. `Winterboor'.
§§ cv. not known.
7 cv. `Quisto'.
"" cv. `Filder Sitzkraut'.
# D-AA 0.11 g/L; AA totally 3.43 g/L; (—) = not detected (<0.1% or not determinable).
Reprinted from Reference [ 241] with permission from Chromatograhia.

TABLE 2.104. Relative Amounts [% D = 100D/(D + L) and Absolute Amounts (µ mol/L) of Native D -Amino Acids (D-AA) in Fruits an
Determined by Derivatization with OPA-IBLC and HPLC.
Apple2 Apple3 Apple 4 Apple5
D-AA 1 %D µ mol/L %D µ mol/L %D µ mol/L %D µ mol/L
D-Asp 0.4 3.2 0.5 21.2 0.3 6.9 0.4 4.6

D-Glu 0.5 3.4 0.5 1.9 0.4 3.0 0.2 1.0
D-Asn 0.7 14.6 0.2 18.3 0.3 27.3 0.4 29.6
D-Ser 1.7 3.4 0.6 2.1 1.1 2.6 1.1 2.2
D-Gln — — — — — — —
D-Ala 2.7 2.9 2.1 3.2 1.8 3.8 2.1 2.9
D-Arg — — — — — — —
Pear7 Grape8 Grape9 Tomato 10
D-AA 1 %D µ mol/L %D µ mol/L %D µ mol/L %D µ mol/L
D-Asp 0.5 4.7 1.9 7.6 0.9 1.6 0.2 1.7
D-Glu 0.9 3.6 0.8 8.9 0.7 3.9 0.1 1.9
D-Asn 0.4 9.1 — — — — 0.2 1.8
D-Ser 1.1 5.4 0.6 4.3 1.2 1.9 0.1 0.7
D-Gln — — — — — — 0.2 6.3
D-Ala 2.1 3.5 0.6 10.5 1.0 5.4 0.7 2.1
D-Arg — — 0.6 13.9 1.2 8.2 — —
Orange11 Clementine 10,12 Grapefruit 10, 12
D-AA 1 %D µ mol/L (%D) (%D)
D-Asp 0.4 0.4 1.0 0.7

D-Glu 1.2 13.7 1.3 1.1
D-Asn 0.5 9.6 0.5 3.4
D-Ser 0.2 4.3 0.3 0.3
D-Gln — — — —
D-Ala 1.3 14.3 0.8 0.9
D-Arg 0.4 7.1 0.8 1.0
1 The LC method distinguishes between Asp/Asn and Glu/Gln.
2 Cultivar (cv.) `Golden Delicious.'
3 cv. `Goldparmäne.'
4 cv. `Jonathan'.
5 cv. `Jonagold'.
6 cv. `Granny Smith'.
7 cv. `Alexander Lukas'.
8 cv. `Kerner'.
9 cv. `Trollinger'.
10 cv. not known.
11 cv. `navel orange'
12 Absolute amounts not determined; (—) = not detectable (<0.1%).
IBLC = N-isobutyryl-L-cysteine.
Page 211
products [ 248]. Sample preparation was carried out as follows: Bones and skin were eliminated from
fresh tuna and hake, while head, bones, and gut were eliminated from fresh anchovies, herring, and
sardines. Then the fishes were triturated and homogenized. In the case of fish products, the covering
liquid was removed, and the sample was dried with absorbent paper and treated as fresh fish samples:
10 g of homogenized samples were mixed with 15 mL 0.6 N HClO4 for 10 min and centrifuged at 3000
rpm for 10 min. The supernatant was decanted and the procedure repeated with 10 mL 0.6 N HClO 4.
The two supernatants were combined and filled to 25 mL with 0.6 N HClO4. When the content of total
amines is higher than 50 mg/kg, the sample weight can be reduced accordingly.
Separation was carried out on an octadecylsilica column of 150 × 3.9 mm I.D. (particle size 4 µm).
Mobile phase A: 0.1 M sodium acetate and 10 mM sodiumoctanesulfonate, pH adjusted to 5.20 with
acetic acid; mobile phase B: 0.2 M sodium acetate and 10 mM sodium octanesulfonate (pH adjusted to
4.50 with acetic acid) + acetonitrile (6.6 + 3.4). The gradient elution program is shown in Table 2.105.
For the analysis of biogenic amines in ripened fish products, gradient elution was slightly modified to
avoid the interference of other components present in the samples (Table 2.106). A postcolumn
derivatizing agent was prepared by dissolving 15.5 g boric acid and 13.1 g potassium hydroxide in 500
mL of water and adjusting the pH to 10.5-11.0 with 30% KOH. Then 1.5 mL of 30% Brij-35, 1.5 mL of
mercaptoethanol, and 0.1 g ortho-phthaldehyde dissolved in 2.5 mL of methanol was added. Excitation
and emission wavelengths were 340 nm and 445 nm, respectively. Flow rate was 1 mL/min. It was
established that perchloric acid is a more selective extraction agent for biogenic amines than
trichloroacetic acid or methanol. Biogenic amines histamine (HI), tyramine (TY), β-phenylethylamine
(PHE), serotonin (SE), tryptamine (TR), cadaverine (CA), putrescine (PU), agmatine (AG), spermine
(SM) and spermidine (SP) were well separated with this HPLC method (Figure 2.105). The method
showed excellent validation parameters; it was
TABLE 2.105. Gradient Elution Program.

Solvent*
Operation Elution Time (min) A (%) B (%)
Elution period 0 80 20
50 20 80
52 20 80
Return and equilibration 54 80 20
64 80 20
* Changes of A% and B% following an exponential function of second order.

International.
Page 212
TABLE 2.106. Table Gradient Elution Program for the Determination of Biogenic Amines
in Ripened Anchovy Samples (pH of solvent A 4.90).
Solvent
Operation Elution Time (min) A (%) B (%)
Elution period 0 90 10
70 0 100
72 0 100
Return and equilibration 74 90 10
74 90 10

International.
linear for each amine between 0.25 mg/L and 8.00 mg/L, the regression coefficients being higher than
0.9950. Precision was from 0.70-9.75%, and detection limits were less than 0.25 mg/kg for PU, CA, HI,
PHE, and AG and less than 1 mg/kg for SE, TY, TR, SM, and SP. The effect of frozen storage on the
content of biogenic amines in fresh anchovy and fresh hake is shown in Tables 2.107 and 2.108. The
data prove that, except for agmantine, frozen storage did not increase the amount of biogenic amines in
fish samples. The authors stated that the method is suitable for the determination of, not only histamine
according to EC regulations, but also for the determination of nine other biogenic amines in fish and
fish products.
Because of the marked biological activity of histamine in humans, many HPLC methods have been
developed for its determination in various matrices [ 249,250]. The efficiency of TLC and HPLC for the
quantitative determination of histamine in fish tissues has been recently compared [ 251]. Samples were
prepared by mixing 5 g of tissue with 10 mL of 5% trichloroacetic acid solution and then purified with
a mixture of n-butanol, n-heptane, and chloroform. Histamine was extracted with 3 mL 0.2 N HCl.
Aliquots of this solution (1.3 µL and 5 µL) were spotted onto TLC plates and developed with
ethanol:NH4OH (80:20 v/v). Histamine was detected by iodoplatinate (0.25 g PtCl6, 5 g KI and 2 mL of
cc.HCl diluted to 100 mL with deionized water), by ninhydrine (0.5 g ninhydrine/100 mL acetone), and
by fluorescamine (0.0125 g fluorescamine/100 mL acetone). In the case of the ninhydrine reagent, the
plates were heated at 60°C. Two different methods were used for the HPLC determination of histamine:
(1) using UV detection at 210 nm, an aqueous solution of 0.4 M KH2PO 4 as eluent (pH = 4.5) and a
cation-exchange column; (2) derivatizing histamine with OPA, using fluorescence detection (excitation
305-395 nm, emission 430-470 nm), 0.05 M NaH 2 PO4: acetonitrile (70:30 v/v) eluent and
octadecylsilica column. The Rf value of histamine was 0.61 in this TLC system. It was established that
each reagent is suitable for the detection of histamine at 50 ng/spot quantity; however, the
fluorescamine
Page 213
Figure 2.105.
Liquid chromatograms of biogenic amines.
(A) standard solution; (B) fresh tuna sample;
(C) canned tuna sample. Peak identities: 1 = putrescine,
2 = tyramine, 3 = cadaverine, 4 = serotonin, 5 = histamine,
6 = agmatine, 7 = β-phenylethylamine, 8 = spermidine,
9 = tryptamine, 10 = spermine.
(Reprinted from Reference [ 248 ] with permission,
copyright © 1995, by AOAC International.)
Page 214
TABLE 2.107. Biogenic Amine Contents of Fresh Anchovy* Before and After
2 and 4 days of Frozen Storage.
X ± SD Analysis
Initial Content Content After Storage of Variance
Amine X ± SD ** 2 Days 4 Days F***exp p
PU 0.15±0.11 0.25±0.24 0.40±0.35 1.87 0.19

TY 0.55±0.63 0.22±0.35 0.75±0.64 0.74 0.50
CA 0.11±0.07 0.15±0.15 0.25±0.18 1.67 0.23
SE ND ND ND — —
HI 0.50±0.55 1.00±0.55 1.00±0.69 1.88 0.19
AG 0.65±0.30 0.25±0.25 1.24±0.71 6.22 >0.05
PHE 1.50±0.45 1.75±0.60 1.40±0.20 0.97 0.40
SD 2.95±1.04 2.45±0.60 2.22±0.55 1.46 0.26
TR 0.10±0.15 ND 0.10±0.22 0.72 0.50
SM 6.35±2.18 6.55±1.33 5.75±1.75 0.37 0.70
* Results obtained for fish muscle after head, bones, and gut are eliminated.
** Mean ± standard deviation in mg/kg.
*** F tab(2,15,0.05) = 3.68.
TABLE 2.108. Biogenic Amine Contents of Hake* Before and After 2 and 4
Days of Frozen Storage.
X ± SD Analysis
Initial Content Content After Storage of Variance
Amine X ± SD ** 2 Days 4 Days F***exp p
PU 0.75±0.80 0.35±0.90 0.30±0.80 0.48 0.63

TY ND ND ND — —
CA 3.10±4.15 5.70±4.35 3.05±2.75 0.05 0.95
SE ND ND ND — —
HI ND ND ND — —
AG 1.85±1.10 5.90±2.95 4.95±2.05 5.70 >0.05
PHE ND ND ND — —
SD 2.40±0.65 2.40±1.15 2.75±0.60 0.35 0.70
TR ND ND ND — —
SM 7.45±1.75 7.70±0.65 8.70±1.90 1.17 0.34
* Results obtained for fish muscle after bones and skin are eliminated.
** Mean ± standard deviation in mg/kg.
*** F tab(2,15,0.05) = 3.68.
Page 215
TABLE 2.109. Comparison of Sensitivity of the Two Techniques for the Determination of
Histamine in Standard Solutions After High Performance Liquid Chromatographic Elution.
Regression Data
Parameter Absorbance Fluorescence

Concentration range 0–10 ppm 0–2 ppm
Intercept value (a) 0.42 –2.31
Confidence interval of the intercept (95%) (–5, 6) (–4.9, 0.25)
Standard deviation of the intercept 2.19 1.26
Slope value (b) 11.756 56.083
Confidence interval of the slope (95%) (10.8, 12.7) (53.5, 58.7)
Standard deviation of the slope 0.38 1.29
Correlation coefficient (r) 0.9974 0.9901
Detection limit 10 ng 1 ng
reagent gave a clearer spot. The regression data of the HPLC methods are given in Table 2.109. Not
only the sensitivity, but also the selectivity, of the flourescence method was higher than that of the
absorption method, advocating the use of derivatization and fluorescence detection for the selective and
sensitive determination of histamine in fish tissue. The histamine content of 10 anchovy samples
determined with both HPLC methods is listed in Table 2.110. The mathematical statistical evaluation of
the data indicated that no significant difference can be found between the results of the methods; that is,
they can be alternatively used for the determination of histamine in fish tissue.
TABLE 2.110. Concentration of Histamine in Anchovy Samples Analyzed by

HPLC and UV-Vis or Fluorescence Detectors.
Concentration (ppm) from Concentration (ppm) from
Sample No. Absorbance Fluorescence
1 5.50 5.60
2 7.50 7.35
3 4.50 4.87
4 6.00 5.61
5 3.00 3.36
6 2.25 2.18
7 3.34 3.35
8 3.50 3.55
9 3.75 3.60
10 3.54 3.50
Page 216
It is well known that various heterocyclic amines are formed during the cooking of foods with a high
protein content. Since these compounds show potent mutagenic activity, their isolation and quantitative
determination is of considerable importance. HPLC is a method of preference for the analysis of this
type of compounds [ 252]. Because of the low quantity of heterocyclic amines, not only the traditional
UV detection, but also electrochemical [ 253,254], fluorescence [255] and mass spectrometric detection
modes [ 256] were used to decrease the detection limit. Because the recovery of heterocyclic
Figure 2.106.
Clean-up procedure scheme.
Page 217
mutagenic amines considerably depends on the conditions of extraction, a solid-phase extraction

method was optimized for this purpose [ 257]. The best extraction procedure is shown in Figure 2.106. It
has been stated that extract A contains IQ (2-amino-3-methylimidazo[4,5-f]quinoline), MeIQ (2-amino-
3, 4-dimethylimidazo[4,5-f]quinoline), MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), and
4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline), while extract B contains Trp-P-1 (3-
amino-1,4-dimethyl-5H-pyrido[3,4-b]indole), Trp-P-2 (3-amino-1-methyl-5H-pyrido[3,4-b]indole),
harman (1-methyl-9H-pyrido[4,3-b]indole), norharman (9H-pyrido[4,3-b]indole), PhIP (2-amino-1-
methyl-6-phenylimidazo [4,5-b]pyridine), AαC (2-amino-9H-pyrido[2,3-b]indole), MeAαC (2-amino-
3-methyl-9H-pyrido[2,3-b]indole), and Glu-P-1 (2-amino-6-methyldipyrido [1,2-a:3' 2'-d]imidazole).
The percentages of recovery in the global clean-up procedure are shown in Table 2.111. The data
indicate that this combined extraction method coupling diatomaceous earth, propylsulphonyl silica gel,
and octadecylsilica is suitable for the determination of imidazopyridine and indolpyridine derivatives in
meat samples, and it successfully separates imidazopyridine and indolpyridine derivatives from those of
quinoxaline and quinoline. The recovery of Glu-P-1 was 81% in extract B. Separation was carried out
on an octadecylsilica column (250 × 4.6 I.D., particle size 5 µm). Extract A was analyzed with eluent
50 mM ammonium acetate (pH 4.0):acetonitrile (90:10) using aniline as the internal standard. Extract B
was analyzed with eluent 50 mM ammonium acetate (pH 5.7):acetonitrile (70:30) using 1-
naphthylamine as the internal standard. Both electrochemical (optimal working potential + 1000 mV)
and fluorescence detection were used (excitation 300 nm, emission 425 nm, except for Glu-P-1 where
excitation and emission were
TABLE 2.111. Percentages of Recovery in the Global Clean-Up Procedure (n = 5).

Percent Recovery of the Total Process
20 ng 100 ng 20 ng
Analyte Mean RSD (%) Mean RSD (%) Mean RSD (%)
Trp-P-2 101 6 103 4 104 5
Trp-P-1 101 7 96 5 98 5
PhIP 86 10 90 6 94 9
Harman 77 7 102 6 94 9
Norharman 60 12 98 5 91 8
AαC 59 13 56 13 68 13
MeAαC 62 8 71 7 89 5
Page 218
360 nm and 450 nm, respectively). Chromatograms of beef extract and spiked beef extract show the
good separation capacity of the HPLC system (Figures 2.107 and 2.108). The results of the analysis of
beef extract are listed in Table 2.112.
The quantity and composition of biogenic amines in wine and other beverages have also been
vigorously studied, mainly by various HPLC methods. A two -step SPE method was developed for the
removal of polyphenolic compounds from wine and for the concentration of biogenic amines [ 258]. In
the first step, the wine sample was passed through on an strong anionic exchanger and then eluted with
methanol. In the second step, an octadecylsilic cartridge
Figure 2.107.
HPLC-electrochemical detection (ED)
chromatogram of extract B of a beef extract
sample. Mobile phase 50 mM ammonium acetate
(pH 5.7):acetonitrile (70:30), at 0.8 mL/min.
(A) Extract B in 50 µL IS solution; (B) extract B
spiked before the clean-up (250 ng) dissolved in
100 µL of IS solution. Peaks: 1 = Trp-P-2; 2 = Trp-P-1;
3 = harman; 4 = PhiP; 5 = norharman; 6 = A αC;
IS = 1-naphthylamine; 7 = MeAαC.
The Netherlands.)
Page 219
was used. The retention of amines was enhanced by adding a decanesulfonate ion-pair forming agent to
the solution. After SPE, the biogenic amines were derivatized with OPA. Separation was carried out on
an octadecylsilica column (250 × 4.6 I.D., particle size 5 µm) using gradient elution. Solvent A was 1%
tetrahydrofuran in 0.05 M aqueous sodium acetate, and solvent B was methanol. Gradient elution began
with 55% methanol and finished after 25 min with 80% methanol. The column was thermostatted at 60°
C. The flow rate was 1 mL/min, and excitation and emission wavelengths were 330 nm and 445 nm,
Figure 2.108.
HPLC-fluorescence chromatogram of extract
B of a beef extract sample. Mobile phase 50 mM
ammonium acetate (pH 5.7):acetonitrile (70:30), at 0.8
mL/min. (A) Extract B in 50 µL IS solution; (B) extract
B spiked before the cleanup (250 ng) dissolved in
100 µL of IS solution. Peaks: 1 = Trp-P-2; 2 = Trp-P-1;
3 = harman; 4 = PhiP; 5 = norharman; 6 = A αC;
IS = 1-naphtylamine; 7 = MeAαC.
Page 220
TABLE 2.112. Analysis of Beef Extract.

Analyte Recovery (%) Concentration * (ng/g)
IQ 82±7 ND
MeIQ 99±4 ND
MeIQx 87±12 ND
4,8-DiMeIQx 78±7 ND
Glu-P-1 81±4 ND
Trp-P-1 91±9 ND
Trp-P-2 74±8 14±5 **
PhIP 55±3 ND
Harman 70±7 110±20
Norharman 73±9 53 ±17
A αC 68±4 2±1 **
MeAα C 70±7 ND
* Value corrected by percent recovey.

** Identity not confirmed by DAD.
Confidence intervals expressed as standard deviations.
Netherlands.
respectively. The method was found to be suitable for the separation of 15 biogenic amines, which
eluted in the following order: ethanolamine, histamine, methylamine, ethylamine, tyramine,
isopropylamine, propylamine, tryptamine, butylamine, phenethylamine, putrescine, 3-
methylbutylamine, amylamine, cadaverine, and hexylamine. A chromatogram of red wine is shown in
Figure 2.109. The detection and quantification limit for the biogenic amines were found to be 20-90
µg/L and 40-200 µg/L, respectively, proving the good sensitivity of the method.
A slightly different method was used for the determination of seven biogenic amines in young Rioja
wines (Spain) [ 259]. The wine samples were derivatized with OPA without any prepurification step. The
octadecylsilica column was of 150 × 4.6 mm I.D (particle size 3 µm). The eluent was similar, but the
gradient elution was different (Table 2.113). The excitation and emission wavelengths were 340 nm and
420 nm, respectively, and the column temperature was 45°C. A typical chromatogram of a rose wine is
shown in Figure 2.110. The amine content of yound Rioja wines is compiled in Table 2.114. The data
revealed that the content of various amines in the young Rioja wines is relatively low. It was further
stated that this method, using standard addition, is reliable and rapid and does not need complicated
sample preparation.
Derivatization with OPA has also been used for the determination of histamine in wine and champagne
[260]. Histamine was separated on a cation exchanger column containing 2 g of Amberlite CG -50
equilibrated with sodium acetate buffer. The buffer was prepared by dissolving 16.1 g sodium
Page 221
Figure 2.109.
Example of red wine analyzed using the proposed
procedure. Peaks: 1 = ethanolamine; 2 = histamine;
4 = ethylamine; 5 = tyramine; 6 = isopropylamine;
8 = tryptamine; 10 = phenethylamine; 11 = putrescine;
14 = cadaverine, ◊ = peak corresponding to the
excess of OPA; * = unknown.
The Netherlands.)
acetate in water and the pH adjusted to 4.8 with acetic acid and then diluted to 1 L. Mixtures of 20 mL
wine or champagne and 50 mL sodium acetate buffer were passed through the column and washed with
200 mL buffer. Histamine was eluted from the column with 50 mL 0.2 N HCl and diluted to 50 mL.
The derivatizing reagent was prepared by dissolving 100 mg OPA in 10 mL of methanol.
Mercaptoethanol was omitted because it was established that OPA selectively derivatizes histamine
when the reagent does not contain mercaptoethanol. Derivatization was carried out by mixing 2 mL of
histamine solution with 1 mL of 1 N NaOH and 0.1 mL of OPA reagent. After 3 min, 2 mL of 0.7 N
HCl were added, and the sample was immediately injected. Separation was carried out on an octadecyl
column (250 × 4 mm I.D., particle size 4 µm) using fluorescence detection (excitation and emission
wavelengths being 350 nm and 450 nm, respectively). Isocratic eluent was 30% acetonitrile and 70%
mixture of sodium acetate (4.1 g/L, pH 6.9) + acetonitrile + tetrahydrofuran (100 + 10 + 1).
Prepurification of histamine on the cation exchanger column is necessary because the determination of
histamine was not possible
Page 222
TABLE 2.113. Chromatographic Conditions.

Time Flow Rate %A %B Curve
0 0.6 60 40
5 0.6 60 40
15 0.6 40 60 3
30 0.6 0 100 2
15 0.6 60 40 1
10 0.6 60 40
A = 0.05 M acetate buffer (pH 6.6):tetrahydrofuran 99:1.
B = methanol.
by the direct derivatization of wine or champagne [Figure 2.111]. The detection limit of the
determination of histamine was 0.05 mg/L. When the OPA reagent contained mercaptoethanol and a
gradient elution was used, tyramine, putrescine, and cadaverine can also be determined in one run.
Since the same derivatization procedure can be used for amino acids and biogenic amines, their
simultaneous determination can also be carried out. The
Figure 2.110.
Typical chromatogram of a rose wine without addition.
1 = histamine; 2 = methylamine; 3 = ethylamine; 4 = tyramine;
5 = 2 -phenylethylamine; 6 = putrescine; 7 = cadaverine.
Page 223
TABLE 2.114. Quantitative Analysis of the Amines (mg/L)

in Samples of Young Rioja Wines.
Sample H M E T Ph P C
Red wine 4.67 <0.4 1.16 3.39 0.59 7.80 0.54
White wine 0.30 0.80 0.76 <0.4 0.79 1.96 <0.2
Rose wine 0.58 <0.4 0.31 <0.4 0.55 4.30 <0.2
H = histamine; M = methylamine; E = ethylamine; T = tyramine; Ph = 2-phenylethylamine; P =
putrescine; C = cadaverine.
derivatization agent 9-fluoromethyl chloroformate (FMOC) has been successfully used in the reversed-
phase HPLC determination of free amino acids [ 261], protein-bonded amino acids [262], and biogenic
amines in meat [ 263]. FMOC derivatization was used for the simultaneous determination of biogenic
amines and their precursor amino acids in wines [ 264]. Derivatization was carried out by mixing 20 µL
of the wine sample with 50 µL of borate buffer (0.2 M boric acid, pH adjusted to 8.5 with 5 M NaOH)
and with 100 µL of derivatizing reagent (8 mg FMOC/mL acetonitrile). After 3 min, 50 µL of 0.5 M
NH3 solution is added. After other 3 min, 300 µL of a quenching solution [acetonitrile:acetic acid:water
(20:2:3 v/v)] is added, and the sample is injected into the HPLC system. An octadecylsilica column
(200 × 2.1 mm I.D., particle size 5 µm), 0.3 mL/min flow rate, and fluorescence detection (excitation
and emission wavelengths 263 nm and 313 nm, respectively) were used. Solvent gradient is shown in
Table 2.115. The mean concentration and standard deviation of biogenic amino acids and their
precursor amino acids are given in Table 2.116. Mathematical-statistical calculations found significant
differences between content of arginine, ornithine, putrescine, histamine, agmatine, spermine,
spermidine, and tyramine in red, rosè, and white wines.
HPLC methods have been used for the determination of histamine in wine as a phenylisothiocyanate
derivative [ 265], for the analysis of biogenic amines in wine after derivatization with o-phthalaldehyde
[266], with dansyl chloride [ 267], or with both of them [ 268]. Similar HPLC methods were used for the
analysis of biogenic amines in cheese [ 269], in chocolate and cheese [ 270], and in other food products
[271].
2.3.2.2—
Gas-Liquid Chromatography
Gas-liquid chromatographic methods have also been extensively used for the separation and
quantitative determination of volatile amines in various matrices. Earlier studies used underivatized
amines as solutes [272,273]; however, polar amine gave tailed peaks that deteriorate the efficacy of the
analysis [ 274]. To overcome this difficulty, derivatization has also been used in the GLC
Page 224
Figure 2.111.
Chromatogram of white wine (A) without prepurification
and (B) after prepurification on a cation exchanger.
(Reprinted from Reference [ 260], copyright © 1995,
Wissentschaftliche Verlagsgesellschaft mbH,
Stuttgart, Germany.)
analysis of amines [ 275]. It was proven that secondary amines can be selectively derivatized with
diethylchlorothiophosphate (DECTP) and then determined with GLC [ 276]. This method was used for
the determination of secondary amines in a wide variety of liquid and solid foodstuffs.
Liquid samples were directly used for derivatization (0.05-0.2 mL) while solid samples (0.2-1.6 g) were
homogenized in 4 mL of 0.05 M HCl and then centrifuged at 2000 g for 10 min. The supernatant was
decanted and the extract repeated once again. The combined supernatant was used for derivatization
TABLE 2.115. Solvent Gradient. Flow Rate 0.3 mL/min.
0 15 85
18 15 85
18.1 38 62
25 40 60
30 40 60
67 42 58
103 85 15
110 85 15
(0.1-0.4 mL). N-methylcyclohexylamine internal standard (0.1 mL of 10 µm) was added to the sample,
neutralized with 2 M NaOH, and diluted with water to 1.6 mL. Then 0.2 mL of 0.5 M phosphate buffer (pH 8)
and 0.2 mL of 0.2 M OPA dissolved in acetonitile were added to the sample and allowed to stay for 2 min at
room temperature. After 2 min, 0.2 mL of 10% sodium carbonate and
TABLE 2.116. Mean Concentration and Standard Deviation for the Different
Compounds Found in Wines of the Vallèe du Rhone (in mg/L).
All Samples Red Wines Rosè Wines White Wines

Biogenic Amines and
Precursor Amino Acids Middle SD Middle SD Middle SD Middle
AR 22.4 14.8 27.5 14.7 10.5 5.6 16.9
OR 3.7 4.4 5.0 4.8 1.0 1.2 1.9
AG 17.3 9.5 21.6 8.4 9.0 3.7 10.3
PU 7.7 6.8 10.8 6.7 2.5 0.9 1.9
CA 0.2 0.4 0.2 0.2 0.4 0.9 0.1
SP 0.5 0.6 0.6 0.6 0.4 0.5 0.3
SM 0.1 0.2 0.1 0.1 0.2 0.4 0.1
HI 8.9 10.9 8.2 6.7 13.5 22.0 7.3
PA 13.0 5.8 13.7 5.9 11.3 4.8 12.6
TS 3.5 2.3 3.3 2.3 3.3 1.5 4.4
HA 2.5 2.6 3.7 2.5 0.4 0.6 0.1
PE 1.7 1.5 1.9 1.6 1.5 1.4 0.9
TA 3.1 2.1 3.7 2.3 2.3 1.7 2.2
n = 84 samples; red wines = 54; rosè wines = 15; white wines = 15; PU = putrescine; HI = histidine; HA = histamine; OR =
ornithine; Ar = arginine; AG = agmatine; PA = phenylalanine; PE = phenylethylamine; CA = cadaverine; TS = tyrosine; TA =
tyramine; SP = spermidine; SM = spermine.
Page 226
0.2 mL of 1% DECTP disolved in acetonitrile were added to the mixture and incubated at 60°C for 10
min. The excess of reagent was removed by adding 0.2 mL of 50 mM cysteic acid and incubating again
at 60°C for 5 min. After the second incubation, the reaction mixture was extracted with 0.2-0.4 mL of
n-hexane. Hexane solution was injected into the GC-flame photometric detection (FPD) system. A
fused-silica capillary column (15 m × 0.53 mm I.D., 1.0 µm film thickness) of cross-linked DB-1701
was used for the analyses. Column temperature changed from 100°C to 260°C at 10°C/min; injection
and detection temperatures were 280°C. Nitrogen flow rate was 10 mL/min. A flame photometric
detector was used (P-filter) with a 526-nm interference filter. Since OPA reacted with the primary
amines, they do not interfere with the derivatization of the secondary amines with DECTP. Some
chromatograms are shown in Figure 2.112. The detection limit depended on the structure of solutes and
Figure 2.112.
Typical gas chromatograms obtained from (A) standard (containing
10 nmol of dimethylamine and 1 nmol of other secondary amines); (B) milk
(0.1 mL); (C) red pepper (6.4 mg); and (D) oyster (52 mg). The arrows
show that the recorder response was raised up to eight-fold after ca.
4 min from sample injection. Peaks: 1 = dimethylamine;
2 = N-methylethylamine; 3 = diethylamine; 4 = di-n-propylamine;
5 = pyrrolidine; 6 = piperidine; 7 = morphorine; 8 = di -n-butylamine;
9 = hexaethyleneimine; 10 = N-methylcyclohexylamine (I.S.);
11 = N-methylbenzylamine.
Page 227
varied from 0.05-0.2 pmol injected; the coefficient of correlation of the linear relationship between
detector signal and the amount of solutes was about 0.991. The average recovery varied between 91%
and 113%, with a relative standard deviation of 0.4-8.2%. The second amine contents of various
foodstuffs are listed in Table 2.117. The data indicate that fish and fish products are rich in amine
(DMA), while spices contain a considerable quantity of pyrrolidine (PYR). The secondary amine
content of vegetables, grains, and meats was markedly low.
The carcinogenic N-nitrosodimethylamine (NDMA) in beer was also determined with GLC coupled
with stable isotope dilution chemical ionization mass
TABLE 2.117. Secondary Amine Contents in Various Foods Obtained

from Commercial Sources.
Content (nmol/g or nmol/mL)*
Sample DMA MEA EA DPA PYR
Rice ND ND ND ND ND
Bread 10.3±0.4 ND ND ND 3.4±0.3
Red beans ND ND ND ND ND
Soy beans ND ND ND ND ND
Almond 38.1±1.0 ND ND ND ND
Black pepper 165±12 ND ND ND 453±38
Red pepper 42.9±2.5 ND ND 120±3 661±36
Wasabi ND ND ND ND 3.0±0.03
Garlic 96.1±2.9 19.4±0.01 ND 110±0.4 277±0.3
Onion 10.2±0.1 ND ND ND ND
Cabbage 14.3±0.8 ND ND ND ND
Banana 4.8±0.2 ND ND ND 1.6±0.1
Dried fungi 212±10 ND ND ND 45.9±5.3
Salted algae ND ND ND ND ND
Salted pollack roe 2086±73 ND ND 2.5±0.2 15.0±1.2
Cod (dried) 1043±38 ND ND ND ND
Mackerel 2129±390 ND ND ND 16.4±1.9
Oyster 39.3±2.0 1.6±0.1 ND ND ND
Beef 26.4±0.4 ND ND ND ND
Pork ND ND ND ND ND
Chicken 18.9±0.6 ND ND ND ND
Egg white ND ND ND ND ND
Egg yolk 109±3 ND ND ND ND
Yogurt 483±50 ND ND ND 19.2±1.9
Cheese 29.8±2.9 ND ND ND 17.4±1.2
Cow's milk 147±7 ND ND ND 50.1±0.4
Wine 26.4±0.2 ND 352±25 52.9±6.1 7.5±0.6
Beer 64.5±5.7 2.5±0.1 ND ND 7.9±1.1
Sake ND ND ND ND 10.3±0.1
Bean jum ND ND ND ND ND

Page 228
TABLE 2.117.
Content (nmol/g or nmol/mL) *

Sample PIP MOR DBA HMI NMBzA
Rice ND ND ND ND ND
Bread 3.5±0.3 ND 4.2±0.1 ND ND
Red beans ND ND ND ND ND
Soy beans ND ND ND ND ND
Almond ND ND 1.1±0.05 ND ND
Black pepper 6351±368 ND 9.0±0.5 ND ND
Red pepper ND 20.4±0.1 21.6±1.3 ND ND
Wasabi ND ND 4.2±0.3 ND 2.0±0.1
Garlic ND ND ND ND ND
Onion ND ND ND ND ND
Cabbage ND ND ND ND ND
Banana 2.1±0.05 ND ND ND ND
Dried fungi ND ND ND ND ND
Salted algae ND ND ND ND ND
Salted pollack roe 6.4±0.3 4.9±0.4 ND ND 6.7±0.5
Cod (dried) ND ND ND ND ND
Mackerel 15.9 ±2.3 ND ND ND ND
Oyster ND ND ND ND ND
Beef ND ND ND ND ND
Pork ND ND ND ND ND
Chicken ND ND ND ND ND
Egg white ND ND ND ND ND
Egg yolk ND ND ND ND ND
Yogurt 18.0 ±1.5 ND ND ND ND
Cheese 29.3 ±3.1 ND ND ND ND
Cow's milk 62.4 ±3.1 ND ND ND ND
Wine 7.9±0.5 8.9±0.04 ND ND 29.8 ±0.9
Beer 22.2 ±1.2 ND 31.2±2.1 ND ND
Sake ND ND 14.5±1.3 ND ND
Bean jum ND ND ND ND ND
* Mean ± SD (n = 4).
Abbreviations: DMA = dimethylamine; MEA = N-methylethylamine; DEA = diethylamine; DPA = di-n-
propylamine; PYR = pyrrolidine; PIP = pipeidine; MOR = morpholine; DBA = di-n-butylamine;
HMI = hexamethyleneimine; NMBzA = N-methylbenzylamine.
spectrometry [ 277]. Two hundres grams of beer were weighted in a 500-mL distillation flask containing
40 g of NaCl, and then 1 mL of 10% aqueous sulfamic acid solution and 50 µL of [2H6]NDMA (10 µg
[2H6] NDMA/mL isoamyl alcohol) were added. The sample was distilled, and about 75 mL distillate
was collected. The receiver was cooled in an icebath. The distillate was extracted three times with 35-
35 mL of dichloromethane and then dried by
Page 229
Figure 2.113.
Total ion chromatogram (TIC) and SIM traces relative to protonated
molecular ion of NDMA (m/z 75.0) and [2H 6]NDMA (m/z 81.0) from a
beer sample containing 1.36 µg/kg NDMA (peak indicated
by the arrow in the TIC).
passing through a column containing 30 g of anhydrous sodium sulfate. The dried extract was
evaporated to 1.0-1.3 mL at 60°C and injected in the GLC system. A fused-silica capillary column
(CPWax 52CB; 25 m × 0.25 mm I.D., 0.2 µm film thickness) and a dectivated fused-silica tube (1.5 m
× 0.32 mm I.D.) precolumn were used for the analysis. The carrier gas was helium, and the column
head pressure was 50 kPa. Column temperature was initially 35° C for 1 min, then heated to 55°C at a
rate of 70°C/min followed by a 7-min isothermal step, heating again to 70°C at a rate of 3°C/min, and
finally heating to 180°C at a rate of 20°C/min. The source temperature was 200°C and the filament
emission current and electron energy were 300 µA and 150 eV, respectively. Methane was the reagent
gas for chemical ionization (1.0 Torr source pressure), and perfluorotributylamine was the calibration
gas. Quantitative determination was carried out by the selected-ion monitoring (SIM) of [M + H]+ ion
of NDMA (m/z = 75.0) and [ 2H6]NDMA (m/z = 81.0). The dwell time was 0.100 s per ion. A typical
chrmatogram is shown in Figure 2.113. The detection limit of the method defined as the lowest amount
of NDMA showing a signal equal to the blank + 3. SD was found to be 0.04 µg/kg. The relative
standard deviation of the method was 0.73% and the recovery 78 ± 2.1% at 95% significance level. The
validation parameters indicate that the method is sensitive, reliable, and can be successfully used as an
alternative to GLC coupled to thermal energy analysis.
Page 230
GLC methods combined with mass spectrometry using trifluoroacetonitrile as a derivatization agent
were used for the identification of new volatile amines in grapes and wines [ 278,279] and in other
biological samples [ 280].
2.3.3—
Peptides
Peptides play a considerable role in foodstuffs. They show considerable biological activity, as well as
sensory and technological properties [ 281]. Thus, peptides without aromatic amino acids can be used in
the treatment of amino acid disorders such as phenylketourea; can increase the bioavailability of
calcium [ 282,283], preventing tooth enamel demineralization; have an emulsifying capacity; and
contribute to the taste, exhibiting a bitter, salty, and metallic taste according to their chemical structure.
Because peptides show negligible volatility, GLC methods cannot be used for the analysis of peptide
mixtures. Because of its relatively low separation capacity, TLC has found only limited application in
the separation of peptide mixtures. HPLC has been the method of preference for peptide analysis. The
various HPLC methods used for the separation and quantitative determination of peptides have been
discussed in detail elsewhere [ 284]. Many efforts have been devoted for the study of the peptide
composition of various dairy products such as milk [ 285], cheese, milk protein hydrolysates, and so
forth. Since peptides are composed of amino acids, many methods used for the HPLC separation of
amino acids can also be used for the analysis of peptides after slight modification. Peptides from skim
milk were isolated by adding 100 mL of 24% w/v aqueous trichloroacetic acid (TCA) solution to 100 g
of skim milk and mixed for 30 min at 4°C [ 286]. Afterward, the volume was adjusted to 250 mL with
distilled water and centrifuged at 4°C. A 125-mL aliquot of supernatant was passed through an
octadecylsilica cartridge preconditioned with 10 mL of methanol, 10 mL of water, and 10 mL of 0.1%
aqueous solution of trifluoroacetic acid (TFA). Peptides were removed from the cartridge using a
mixture of acetonitrile:0.1% TFA in water (9:1 v/v). Injection volume was 50 µL. Peptides were
separated on an octadecylsilica column using gradient elution. Eluent A and B were 0.1% TFA in water
and acetonitrile, respectively. Flow rate was 1 mL/min. Both absorption at 214 nm and postcolumn
derivatization with OPA and subsequent fluorescence detection were used for the determination of
peptides. Peptide chromatograms from skim milk and from a tryptic hydrolysate of β-A2-casein are
shown in Figures 2.114 and 2.115.
It was established that the method effectively separates the peptides from free amino acids; however,
the interference of aromatic amino acids with the retention of small and hydrophobic peptides has to be
taken into consideration. Postcolumn derivatization with OPA increases detection sensitivity and
selectivity and provides additional information on peptide composition.
Page 231
Figure 2.114.
RP-HPLC chromatogram of peptides isolated from commercial
skim milk and detected by on-line absorbance (214 nm) and
OPA fluorescence. Gradient elution was 0-40% B in 65 min
and then 90% B in 10 min. Detection by fluorescence was
obtained with the OPA reagent containing 28 mM
mercaptoethanol at 50 °C and using a flow rate of 0.5 mL/min.
Figure 2.115.
RP-HPLC chromatogram of peptides from a tryptic
hydrolysate of β-A 2-casein obtained using on -line
absorbance and fluorescence detection. The
chromatographic conditions are the same
as in Figure 2.114.
Page 232
FMOC derivatization has also been used for the separation and identification of hydrophilic peptides in
dairy products [ 287]. A model system was used (dairy pasteurized slurry treated with various enzymes
such as lipases, peptidases, and proteases). The sample was prepared by homogenizing 16 g model
substance with 30 mL water and then centrifuged at 8650 g for 20 min at 4°C. The fat and the aqueous
phases were collected separately. The pellet was treated twice in the same manner. The three fat
fractions were pooled, homogenized in 20 mL water, and centrifuged again. The four aqueous fractions
were combined, filtered, and passed through on a Sephadex G-25 column (200 cm × 4 cm I.D.) using
water as the eluent at 84 mL/min flow rate and 206 nm detection wavelength. Fractions of 84 mL were
collected and submitted to sensory analysis and further separation. FMOC derivatization was carried
out by drying 0.2 mL of gel filtrate, resuspending in 0.2 mL of sodium borate buffer (0.5 M, pH 7.8),
and adding 0.2 mL of FMOC reagent (5.8 mM in acetone). After 45 s of mixing, the content of the
reaction vial was extracted with 0.4 mL of pentane-ethyl acetate (80:20). Nonderivatized peptides were
separated on an octadecylsilica column (250 mm × 4.6 mm I.D., particle size 5 µm) using a linear
gradient of solvent A, decreasing from 80% to 20% in 30 min at 25°C [solvent A was 0.115% TFA in
water and solvent B was 0.1% TFA in acetonitrile:water (60:40)]. The flow rate was 1 mL/min and the
detection wavelength 214 nm. FMOC derivatives were separated on the same column using different
gradient elution. Solvent A was acetonitrile:100 mM ammonium acetate buffer (pH 3.8) (20:80), and
solvent B consisted of acetonitrile:100 mM ammonium acetate buffer (pH 4.2) (80:20). Gradient started
10 min after injection; the gradient was from 30% to 80% B in 50 min and 100% B for 10 min. The
column was thermostatted at 40°C, flow rate was 1 mL/min, and the detection wavelength was 260 nm.
The main hydrophilic peptide derivatives were collected and then separated again using an isocratic
elution with eluent acetonitrile:100 mM ammonium acetate buffer (pH 3.3) (30:70). Collected peptide
fractions were sequenced. The results of gel filtration and those of subsequent sensory evaluation of the
fractions are shown in Figure 2.116. The sensory characteristics of the fractions differ considerably,
indicating that they depend markedly on the molecular mass of peptides. Fractions 12 and 13, showing
favorable sensory characteristics, were submitted to HPLC analysis. The corresponding chromatograms
are shown in Figure 2.117. After HPLC separation, the amino acid sequence of 15 peptides and their
taste were determined. The results are listed in Table 2.118. Since the taste of the individual synthetic
peptides was not characteristic enough, it was assumed that synergistic effects must occur between the
peptides if they influence the taste of dairy products.
Because of their favorable biological activity, the composition of caseinophosphopeptide (CPP)

mixtures has been vigorously studied, mainly by various HPLC methods [ 288,289] and by immobilized
metal ion affinity
Page 233
Figure 2.116.
Gel filtration chromatogram and sensory characterization of the collected fractions.
Page 234
Figure 2.117.
Separation of peptide derivatives of gel filtration fractions (a) 12 and (b) 13.
Page 235
TABLE 2.118. Amino Acid Sequence of Peptides Isolated from Gel Filtration Fractions 12
and 13 and Sensory Evaluation of Some Synthetic Peptides.
Peptide Taste of Synthetic Concentration of Synthetic
Identificaiton Peptides Peptides for Tasting (mg/mL)
Gly-Gly Tasteless 5
Thr-Gln Tasteless 5
Ser-Thr Tasteless 5
Ala-Gln Slightly bitter and umami [sic] 20
Ala-Gln-Thr
Gly-Val-Ser
Val-Gln Slightly sweet 5
Ile-Asn-Asn
Val-Thr
Leu-Gln
Val-Ala Tasteless 5
Ile-Asn
Ala-Pro Slightly bitter 5
Ile-Thr-Pro
Ala-Pro-Phe-Pro
chromatography (IMAC) [ 290,291]. These two methods have been successfully combined for the
analysis of CPPs [ 292]. Casein hydrolysate was prepared by mixing 50 mL of sodium caseinate (2 × 10-2
g/mL in 2 mM Tris-HCl buffer, pH 7.6) and 50 mL of trypsin (2 × 10-4 g/mL in 2 mM Tris-HCl buffer,
pH 7.6). The mixture was incubated at 37°C for 24 h and then heated at 70°C for 20 min. The pH was
adjusted to 4.6, and the mixture was filtered, concentrated in a rotary evaporator at 40°C, and used for
further analysis. Immobilized metal affinity chromatography was carried out on a chelating sepharose
gel column (6.4 × 1 cm I.D.) and washed with 0.1 M sodium acetate buffer (pH 3.0) for 20 min at a
flow rate of 1 mL/min. A 20 mM FeCl3 solution in 0.01 N HCl was passed through the column at the
same flow rate until the effluent turned yellow. The column was washed with 0.1 M sodium acetate
buffer (pH 3.0, 1 mL/min) for 50 min and then equilibrated with sodium acetate buffer (0.1 M; pH 5.0;
flow rate 1 mL/min) for 20 min. One milliliter of hydrolysate was applied to the column, and first, the
nonphosphorylated peptides were eluted with 0.1 M sodium acetate (pH 5.0, flow rate 1 mL/min).
Phosphorylated peptides were removed from the column by washing the column with 0.05 M
triethylammonium hydrogen carbonate (pH 8.0) for 40 min (flow rate 1 mL/min). This fraction was
concentrated to 1 mL at 40°C and used for HPLC analysis. An octadecylsilica column (250 × 4 mm
I.D.; 5 µm particle size) was used for the separation. Solvents A and B were 0.1% dissolved in water
and acetonitrile, respectively. The linear gradient was from 100% A to 30% A-70% B in 45 min
Page 236
or from 100% A to 82.9% A-17.1% B in 9 min, 79.9% A-20.1 % B in 18 min, 74.3% A-25.7% B in 25
min, 65.8% A-34.2% B in 36 min, and 30% A-70% B in 45 min. Peptides were collected, purified again
by the same HPLC procedure, and sequenced.
The IMAC chromatograms of the original sodium caseinate and its tryptic hydrolysate are shown in
Figure 2.118. The chromatograms clearly show that the IMAC supports interact only with the
hydrolysate and not with the original casein. Linear gradient had a low separation capacity for CPPs;
however, a nonlinear gradient resulted in better separation, as shown in Figure 2.119. The cluster of
peptides present in chromatogram A in Figure 2.119 at about 36-40 min was assumed to contain
peptides complexed with Fe 3+. In order to disintegrate these hypothetical metal-peptide complexes, 2.5
mL of 0.1 M ethylenediaminetetraacetic acid (EDTA) was added to the bound fraction before
concentration. Chromatogram B shows that the separation was markedly improved in
Figure 2.118.
Immobilized metal affinity chromatography of the (A)
unhydrolyzed sodium caseinate and the (B) tryptic
hydrolysate on Fe 3+-chelating sepharose. Eluents:
(A) 0.1 M sodium acetate (pH 5.0); (B) 0.05 M
triethylammonium hydrogen carbonate (pH 8.0). Flow rate
was 1 mL/min. The arrow indicates the time at which
eluent A switches to eluent B. UBF indicates the unbound
fraction and BF indicates the bound fraction, which
was the caseinophosphopeptide.
Preston Publications, a Division of Preston Industries,
Inc., and the corresponding author.)
Page 237
Figure 2.119.
High-performance liquid chromatographic elution patterns of the
bound fraction obtained by immobilized metal ion chromatography
of the sodium caseinate hydrolysate: (A) the bound fraction
without EDTA; (B) the bound fraction with EDTA. Proteolysis was
by trypsin (enzyme/substrate = 1/100, 24 h) and was monitored at
220 nm. The peptides were eluted at room temperature using a
stepwise linear gradient from A [0.1% trifluoroacetic acid (TFA) in
water] to 17.1% B (0.1% A in acetonitrile) in 9 min, 20.1%
at 18 min, 25.7% B at 25 min, 34.2% B at 36 min, and 70% B at 45 min.
(Reprinted from Reference [ 292] with permission from Preston
Publications, a Division of Preston Industries, Inc.,
and the corresponding author.)
Page 238
the presence of EDTA. The amino acid sequences of some isolated peptides are presented in Table
2.119. This method provides a useful technique for the separation and identification of CPPs in a
complicated mixture containing proteins and peptides of various molecular mass and polarity.
RP-HPLC has also been used for the separation of peptides from phosphorylated and dephosphorylated
casein hydrolysates [ 293]. Not only RP-HPLC, but also gel permeation chromatography has been used
successfully for the separation of peptides in various food products. Thus, it has been used for the
determination of the molecular mass distribution of hydrolyzed casein [ 294, 295], for the analysis of
casein-derived peptide in cheese [ 296], and for the study of the composition of whey [ 297,298] and soy
protein hydrolysates [ 299]. Recent achievements, such as fast protein liquid chromatography (FPLC),
considerably increase the selectivity of separation and simultaneously decrease the analysis time [ 300].
HPLC gel permeation columns were used to study the proteolytic procedures in cheese [ 301] and for the
investigation of casein hydrolysates [ 302,303]. FPLC has been used for the analysis of casein and whey
protein hydrolysates, and the influence of adsorption side effects of the cross-linked agarose-based
support on the analysis of hydrolysates was studied in detail [ 304]. The fractions of protein hydrolysates
were eluted from the Superose 12 column with a buffer consisting of 0.1 M Tris-HCl, 0.1 M NaCl, and
10% methanol (pH 7.0). The flow rate was 0.5 mL/min, and detection was carried out at 214 and 280
nm. Hydrolysates were diluted in the elution buffer to give a 0.25% protein equivalent (N × 6.38), and
100µL of the
TABLE 2.119. Amino Acid Sequences of Caseinophosphopeptides.

Peak No.* Primary Structure of the CPP
C1 α s2 (126-136) casein:
Glu-Gln-Leu-SerP-Thr-SerP -Glu-Glu-Asn-Ser-Lys
3 β (33-48) casein:
Phe-Gln-SerP-Glu-Glu-Gln-Gln-Gln-Thr-Glu-Asp-Glu-Leu-Gln-Asp-Lys
8 α s1 (43-58) casein:
Asp-Ile-Gly-SerP-Glu-erP-Thr-Glu-Asp -Gln-Ala-Met-Glu-Asp-Ile-Lys
9 α s2 (138-149) casein:
Thr-Val-Asp -Met-Glu-SerP-Thr -Glu-Val-Phe-Thr-Lys
10 α s1 (106-119) casein:
Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn-SerP-Ala-Glu-Glu-Arg
11 α s1 (104-119) casein:
Tyr-Lys-Val-Pro-Gln-Leu-Glu-Ile-Val-Pro -Asn -SerP-Ala-Glu-Glu-Arg
12 β (2 -25) casein:
Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro -Gly-Glu-Ile-Val-Glu-SerP-Leu-SerP-SerP-
SerP-Glu-Glu-Ser-Ile-Thr -Arg
* Peaks as seen in Figure 2.119.
Reprinted from Reference [ 292] with permission from Preston Publications, a Division of Preston
Industries, Inc.
Page 239
sample solution was injected into the column. The chromatograms of casein and whey hydrolysates are
shown in Figures 2.120 and 2.121. It was of interest that some fractions of casein hydrolysate were
eluted after the total column volume when using enzyme preparation that contains pancreatic
exopeptidases [Figure 2.120(a)]. This anomalous retention behavior was tentatively explained by the
hydrophobic and hydrophilic interactions occurring between the peptide fractions and the support
matrix. It has been further established that Trp accounts for the hydophobic interactions with the
stationary phase. The last fraction in the chromatogram of whey protein hydrolysate prepared on
pilotscale was found to be nitrate ion, which adsorbs strongly to the stationary phase from the water
used for the hydrolysis.
Capillary electrophoresis (CE) as a sensitive and fast separation method has also been used for the
analysis of the hydrolysis products of casein. CE, combined with RP-HPLC, proved to be an effective
tool to study the hydrolysis of αs and β-casein by chimosin [ 305]. Caseins were separately dissolved in
Figure 2.120.
Elution profiles of casein hydrolysates from an FPLC Superose 12
column assayed by absorbance at (a)280 and (b)214 nm.
The elution buffer (pH 7.0) was 0.1 M Tris-HCl-0.1 M NaCl-10% methanol;
flow rate, 0.5 mL/min. Sample volume: 100 µL of 0.25% w/v, protein solution;
(a) rennet casein hydrolyzed with a mixed enzyme preparation, BN2001
(DH, 23%); (b) rennet casein hydrolyzed with a specific proteinase
preparation, Profix (Papain, DH, 10%)
Page 240
Figure 2.121.
Elution profiles of whey protein hydrolysates from
an FPLC Superose 12 column assayed by absorbance
at 214 nm. The elution buffer (pH 7.0) was 0.1 M
Tris-HCl 0.1 M NaCl 10% methanol; flow-rate, 0.5 mL/min.
Sample volume: 100 µL of 0.25% w/v, protein solution;
(a) whey protein concentrate: 1 = IgC, 2 = BSA; 3 = b-Lg,
4 = a-La; (b) laboratory-prepared whey protein hydrolysate;
(c) pilot-scale whey protein hydrolysate showing an
additional peak at 22 mL; (d) 100 µL of general water supply
used to prepare pilot-scale hydrolysate, NaNO3 (20 mg/kg)
eluted in exactly the same position. The void volume
(V 0) and total column volume (V t) are indicated by arrows.
0.075 M ammonium acetate buffer (pH 6.2) at a concentration of 1%; 0.3% chymogen was added, and
the mixture was incubated at 30°C for 18 h or 24 h for αs and β-casein, respectively. Samples were
taken during the hydrolytical procedure and analyzed with CE. Hydrolysis was stopped by heating to
85°C for 10 min. The final products were centrifuged at 3000 g for 20 min, and the pH of the
supernatant was adjusted to 4.6 with acetic acid and allowed to stand for 1 h and centrifuged again.
Ethanol was added to the new supernatant to a 70% end concentration, and the mixture was held at 4°C
overnight and centrifuged again. The supernatant was dried at 80°C, redissolved in 2 mL of water, and
used for HPLC analysis. Untreated fused-silica capillaries, externally coated with polyimide (75 µm
I.D., 60 cm total length, 52.5 cm to the detector), were used for the CE separation. Samples were
injected hydrostatically for 10 s. A 0.5 M sodium phosphate stock buffer (pH adjusted to 7.0 with
NaOH) was used.
Page 241
Figure 2.122.
CE of α s-casein sample (0 h) and of αs -casein hydrolyzed
by chymosin at pH 6.2 for 0.5, 1, and 4 h. Samples were
diluted 1:1 with sample buffer. Run buffer: 0.1 M sodium
phosphate, 4 M urea, pH 7.3. Voltage: 12 kV, current 120 µA.
from VV -GmbH Volkswirtschaftlicher Verlag.)
In the case of caseins and less soluble peptides, a 0.1 M run buffer in 4 M urea (pH 7.3) was prepared
daily. A sample buffer was prepared from a stock buffer diluted to 0.1 M with added urea to 7 M and
dithioerythrol to 10 mM. Casein hydrolysate was diluted with the sample buffer (1:1). Soluble peptides
were separated in 0.05 M phosphate buffer (pH 7.3) using both run and sample buffer. The detection
wavelength was 214 nm. RP -HPLC used gradient elution, buffer A being 0.075 M ammonium acetate
(pH 6.2) and buffer B being 5% buffer A + 95% acetonitrile. The gradients were 17-35% B and 22-45%
B in 0-25 min for the fractionation of the hydrolysis products of αs - and β-casein, respectively. The
flow rate was 1 mL/min, and the fractions were detected at 220 nm and 280 nm.
The electropherograms of intact and hydrolyzed αs-caseins are shown in Figure 2.122. The hydrolysis
of αs -casein is relatively rapid in the presence of chymosin, whereas α2s -casein is not hydrolysed. It was
assumed that the highest
Page 242
peak (marked with I) is the αs1-I-casein, the first hydrolysis product of αs-casein under the action of
chymosin. The peak at 14.5 min was tentatively identified as the Arg1 -Phe 23 peptide from αs1-casein
(αs 1-casein is built up of ( αs1-I-casein and the Arg 1-Phe 23 peptide). The hydrolysis of β-casein is slower
than that of ( αs 1-casein, as shown in Figure 2.123. The separation of soluble peptides by HPLC and their
further separation by CE is presented in Figure 2.124. The separation profile indicates that the
hydrolysis products separated by HPLC can be further analyzed by CE. The combination of both
methods can be used not only for the analysis of the hydrolysis products of casein, but also for the
determination of the peptides in cheeses.
Free solution capillary electrophoresis was used for the analysis of caseinomacropeptide (CMP), a
hydrolysis product of bovine κ-casein [ 306]. The sam-
Figure 2.123.
CE of β-casein sample (0 h) and of β-casein hydrolyzed
by chymosin at pH 6.2 for 1, 2, and 7 h. Run buffer: 0.1 M
sodium phosphate, 4 M urea, pH 7.3.
Voltage = 12 kV, current 118 µA.
Page 243
ple of acid casein was prepared from the milk of a cow homozygotic in κ-casein A. The pH of an
aqueous solution of κ-casein was adjusted to 6.7 at 32°C and 35 µL of chymosin were added to 100 mL
of solution. After 30 min of incubation the pH was adjusted to 4.6 with 1 M HCl, and then the sample
was centrifuged at 1900 g for 10 min. The supernatant was used for CE. N-acetyl neuraminic acid
(NANA) was removed from the samples by mixing them with the same volume of 20 mM phosphate
buffer (pH 6.5) and then incubating with neuramidase (1 µL/100 µL substrate solution). Samples were
hydrodynamically injected at the anodic end and detected 7.5 cm from the catodic end at 214 nm. The
electropherogram of isolated CMP is shown in Figure 2.125. The electropherograms indicate that the
buffer concentration exerts
Figure 2.124.
Top: RP-HPLC fractionation of peptides from α s-casein
hydrolysate soluble at pH 4.6 and 70% ethanol. Below:
Capillary electrophoregrams of fractions a, b, c, and d.
Electrophoresis buffer was 0.05 M sodium phosphate,
pH = 7.3. Voltage = 12 kV, current 170-180 µA.
Page 244
Figure 2.125.
Capillary electrophoresis of isolated CMP, prepared
from acid casein homozygotic in κ-casein.
Electrophoretic conditions: untreated capillary,
75 µm I.D., 52.5 cm to detector (60 cm total length) and
phosphate buffers pH 8: (A) 20 mM, 25 kV; (B)
50 mM, 13 kV (current 99-110 µA). Peak identification:
EOF, formamid; 1 = main form of CMP; 2-6 = other CMP
forms and contaminating proteins/peptides.
a considerable influence on the migration velocity, and higher buffer ionic strength causes high
migration time. It has been established that neuraminidase treatment modifies the position of the
impurities. This finding suggests that the impurities are the glycosylated derivatives of CMP. The
method has also been used for the separation of CMP from rennet whey (Figure 2.126). Because the
method efficiently separates CMP from the other pep-
Page 245
Figure 2.126.
Capillary electrophoresis of rennet whey.
Electrophoretic conditions: 40 mM phosphate,
pH 2.5; 28 kV (current 109-125 µA). Peak identification:
β-lg: β-lactoglobulin; α-la: α-lactalbumin, lg:
immunoglobulins; PP: proteose peptones and/or
phosphopeptides; CMP1: main form of CMP; 2-3:
other CMP forms and contaminating proteins/peptides;
4: CMP containing NANA.
tides and proteins in whey, its use was proposed for the study of the kinetics of rennet coagulation.
CMP was also isolated by using a strong basic anion-exchange resin [ 307]. Emmental cheese whey was
microfiltrated on 1.0-µm membranes, and the filtrate was used for the analysis. CMP was isolated on a
column of macroporous strong basic anion-exchange resin treated with three bed volumes of 1.0 M
NaOH and 1.0 M HCL. The pH of the whey filtrate was adjusted to 5.0 with 1 N HCl, and 200 mL were
passed through the column at a flow rate of 100 mL/min at 5°C. The column was washed with 100 mL
of distilled water and then eluted with 90 mL of 2% NaCl and 100 mL of distilled water. The salt
solution and the first 50 mL of wash water were used for the further separation with FPLC. The
fractionation of filtrate on the strong basic anion-exchange resin is shown in Figure 2.127. The
chromatograms show that CMP is eluted with 2% NaCl (large peak at 226 nm); however, orotic acid,
absorbing at 280 nm, coeluted with CMP. FPLC separation profiles are given in Figure 2.128. The
chromatograms show that the method efficiently isolates CMP from whey. The yield of the CMP
isolation was 72%, and purity of the product was 70-80%.
Although the majority of separations were carried out on peptides of dairy products (mainly cheese and
casein hydrolysates), the number of studies
Page 246
Figure 2.127.
Relative absorbance of the eluate at 226 mm and 280 nm as
a function of eluate volume. Clarified whey (200 mL)
was introduced on the Dialon HPA 75 column (resin
volume 30 mL) at pH 5.0, the column was washed with
water (100 mL), and the adsorbed CMP was eluted with
2% NaCl (90 mL). After washing with water, the column was
ready for the next cycle. The flow rate was kept contant
at 100 mL/h. The process was performed at 5°C.
dealing with the assessment of the peptide profile of other food products is continually increasing.
Thus, RP -HPLC was used for the separation of low-molecular mass hydrophobic bitter peptides in
soybean protein hydrolysates [ 308]. The study was motivated by the finding that enzymatic hydrolysis
of proteins frequently resulted in the formation of bitter peptides. Peptides with hydrophobic amino acid
residues and the conformation of peptides may account for the bitter taste. The isoelectric soluble
soybean protein hydrolysates (ISSPHs) were prepared by adding subtilisin (enzyme/substrate ratio E/S
= 12) to a soybean protein isolate (8% protein). Hydrolysis was carried out at a pH of 8 (temperature
50°C), and it was stopped by lowering the pH to 4.0-4.2 for 30 min. Then the sample was centrifuged (1
h at 8000 g) and the supernatant freeze-dried. The hydrophobic bitter peptide fractions (HBPFs) were
extracted with 2-butanol. The Hydrolysates were first separated according to the molecular mass by gel
permeation HPLC in the molecular mass range of 4000-400,000. Eluent was 0.1 M phosphate buffer
(pH 8.0) containing 0.1% sodium dodecyl sulfate (SDS). Flow rate was 1.0 mL/min and the detection
wavelength 215 nm.
Bitter peptides were isolated for sensory evaluation by preparative gel chromatography on Sephadex G-
25 Fine column (78 × 3 cm I.D.): 70 mg of HPF was dissolved in 1.5 mL water, filtered, and applied to
the column. Eluent was distilled water, flow rate was 15 mL/h, and detection was 215 nm. Fractions of
5 mL eluate were collected. The most bitter fractions were further analyzed on an octadecylsilica
column (250 × 4 mm I.D., particle size 5 µm). The gradient
Page 247
Figure 2.128.
Peptide profiles (FPLC, Superdex 75, 214 nm)
of the CMP standard, the whey, CMP-free whey,
and the CMP fraction. RT' 15.8 = CMP. Eluent 0.02
mM Tris -l buffer (pH 7.8) at the flow
rate of 0.75 mL/min.
elution was carried out as follows: (solvent A = 0.1% v/v TFA in water; solvent B = 0.1% v/v TFA in
acetonitrile). 0-2 min, 0% B; 2-5 min, 0-10% B; 5-65 min, 10-40% B; 65-70 min, 40-70% B; 70-71
min, 70-0% B. Detection was carried out at 220 nm. Fractions were collected, concentrated, and tasted
for bitterness. Fractions not well separated were rechromatographed in 25 mM ammonium acetate (pH
6.0, solvent A) and 60 vol% acetonitrile in 50 mM ammonium acetate (pH 6.0, solvent B). The gradient
was 0-2 min, 0% B; 2-62 min, 0-100% B; 62-63 min, 100-0% B. The buffer was removed
Page 248
from the isolated peptides by using the previous solvents containing 0.1% TFA and the following
gradient: 0-6 min 0-15% B; 6-26 min, 15-25% B; 26-36 min, 25-50% B; 36-37 min, 50-80% B; 37-38
min, 80-0% B. The gel chromatographic separation of peptide samples and their molecular mass and
taste are presented in Figure 2.129 and Table 2.120. The tasting of the separated peptides indicated that
the bitter taste increases by increasing the time of hydrolysis, and it is expressed more in the fractions
containing low-molecular mass peptides.
The separation of peptides on a Sephadex G-25 column is shown in Figure 2.130. The data in Figure
2.130 support the previous conclusions that the bitterness of soluble soybean protein hydrolysates
increase with a decreasing molecular mass of the peptides. The further chromatographic purification of
the bitter peptides by RP-HPLC indicated that they contain minimally 18 fractions (Figure 2.131), and
the fractions can be further separated in pure peptides by
Figure 2.129.
Elution profile of (A) five ISSPHs and (B) their HPFs on a Zorbax Bio Series
GF-250 column (250 × 9.4 mm I.D.). The molecular mass fractions are designated I,
II, and III; I = high-molecular-mass fraction; II = medium -molecular-mass
fraction; III = low-molecular-mass fraction.
Page 249
using a second solvent system. The complexity of the peptide profile of the hydrolysates suggests the
necessity of the use of multistep separation procedures to reach chromatographically pure peptides, the
sequence of which can be further elucidated. The physicochemical parameters of bitter peptides are
given in Table 2.121. The data indicate that the bitter peptides mainly contain hydrophobic amino acid
residues and have leucine, valine, or tyrosine at the C-terminal.
A similar combined method (SEC and RP-HPLC) was used for the separation of peptides from
Yellowfin tuna red muscle myoglobin hydrolysate [ 309]. Myoglobin was isolated from red skeletal
muscle by homogeneizing it with distilled water (1:1 w/v) under ice-cooling. The suspension was
centrifuged at 4°C (3000 g, 15 min) and was saturated to 60% with solid ammonium sulfate. It was
centrifuged again under the same conditions, and the pellet was discarded. The supernatant was
saturated to 80% with solid ammonium sulfate and centrifuged again as described above. The pellet
containing myoglobin was dissolved in water and ultrafiltered and diafiltered. The crude preparation
was purified on a D.E.A.E. Sephacel column (320 × 30 mm I.D.) equilibrated with 50 mM Tris-HCl
buffer (pH 8.6). The eluting buffer was the same to 50 min and then changed to the same buffer
containing 0.2 M NaCl. Flow rate was 60 mL/h and detection 280 nm. An aqueous solution of
myoglobin (1 g/100 mL, pH adjusted to 2.0 with HCl), was hydrolyzed with porcine pepsin (40 mg) at
40°C for
TABLE 2.120. Bitterness and Molecular Mass Distribution of ISSPHs and HPFs Obtained by GP-HPLC on a
Zorbax Bio Series GF-250 Column (250 × 9.4 mm. I.D.).
Area (%)
Bitterness Fraction I: Fraction II: Fraction III:

(score) M τ 30,000-3000 Mτ 3000-1000 Mτ <1000
Sample
ISSPH-DH 3% 0 37.3 7.1 55.6
ISSPH-DH 6% 2 31.5 6.2 62.3
ISSPH-DH 9% 3 23.0 5.8 71.2
ISSPH-DH 12% 3 16.3 5.2 78.5
ISSPH-DH 15% 2 13.9 3.9 82.2
HPF-ISSPH-DH 3% 5 17.2 * 10.5 72.3
HPF-ISSPH-DH 6% 5 14.2 * 10.3 75.5
HPF-ISSPH-DH 9% 5 10.7 * 6.9 82.4
HPF-ISSPH-DH 12% 5 8.3* 3.9 87.8
HPF-ISSPH-DH 15% 5 6.7* 4.1 89.2
* Mτ 10,000-3000
Scores: 0 = no bitterness; 1 = weak, bitter after-taste; 2 = weakly bitter; 3 = bitter; 4 = strongly bitter; 5 =
extremely bitter.
Page 250
Figure 2.130.
Chromatogram of the hydrophobic bitter peptide fraction
from isoelectric soluble soybean protein hydrolysate
(HPF-ISSPH -DH 15%) on a column of Sephadex G-25 Fine:
× = Absorbance at 215 nm; o = bitterness. The most bitter
low-molecular-mass peptide fraction from 290 mL to 340 mL
(shown with a brace) was rechromatographed by RP-HPLC.
The numbers denote elution positions of the following
compounds: 1 = cytochrome c; 2 = angiotensin II; 3 = L-leucine.
Figure 2.131.
RP -HPLC of the most bitter low-molecular-mass peptide fraction on a
Spherisorb ODS-2 column (250 × 4 mm I.D.) with (A) 0.1% (v/v) TFA and (B)
0.1% (v/v) TFA in acetonitrile. The coeluted peptide
material is divided into fractions 1-18.
Page 251
TABLE 2.121. Bitter-Tasting, Low-Molecular Mass Peptides Isolated from the Hydrophobic Peptide
Fraction of Isoelectric Soluble Soybean Protein Hydrolysate Degree of Hydrolysis (DH) 15% (HPF-
ISSPH-DH 15%).
RP-HPLC Hydrophobic
Fraction No. Peptide Eluent B (%) * Residue Sequence MT
8 8-I 19.65 2/3 FLS 365.41
8-II 19.96 3/4 LLPH 487.57
10 10-I 20.77 3/4 LVGY 450.52
10-II 20.54 3/4 IYIG 464.54
11 11-I 21.37 3/4 VYDV 494.53
11-II 21.08 3/4 SVIY 480.54
12 12-I 22.67 4/4 VYFV 526.62
12-II 22.84 3/4 ISIY 494.56
13 13-I 24.17 4/4 VVLY 492.60
14 14-I 24.50 2/3 DIF 392.42
14-II 24.57 4/5 GYPVV 533.61
15 15-I 25.75 4/4 YVVL 492.60
15-II 26.00 3/5 SGFTL 509.54
18 18-I 28.64 3/6 SNLNFL 678.72

* Eluent B = 0.1% TFA in acetonitrile.
3 h. The pH was maintained constant by adding HCl during the hydrolysis. The hydrolysis was stopped
by adjusting the pH to 6.5 with ammonium hydroxide. The reaction mixture was centrifuged (10,000 g,
30 min), and the supernatant was used for further peptide analysis. Size exclusion HPLC of peptides
was carried out on a TSK G 2000 SWG column (600 × 21.5 mm I.D.). Equilibration and elution buffer
was 3 mM ammonium acetate-acetic acid buffer (pH 6). The flow rate was 6 mL/min, and the detection
wavelength was 215 nm. Fractions were collected, freeze-dried, and redissolved in eluent A before RP-
HPLC analysis. The octadecylsilica column (300 × 3.9 mm I.D.) was equilibrated with 10 mM
ammonium acetate-acetic acid buffer (pH 6.0, eluent A). Eluent B was acetonitrile. The optimal
gradients for the separation of the individual peptide fractions are presented in Table 2.122. The flow
rate was 1.5 mL/min, and peptides were detected at 215 nm. Peptides were collected and hydrolyzed
with HCl, and the amino acid composition was determined by derivatization with phenyl isothiocyanate
and by subsequent separation by RP -HPLC. Myoglobin was well separated from the other proteins on
the D.E.A.E. Sephacel column (Figure 2.132). It was established that the yield of the method was
approximately 56% pure myoglobin. Myoglobin hydrolysate was separated in seven fractions on the
TSK G2000 SWG column (Figure 2.133). The chromatograms of the fractions on an RP -HPLC column
are shown in Figure 2.134. The fact
Page 252
TABLE 2.122. Acetonitrile/Ammonium Acetate Buffer Gradient Applied on an RP-HPLC Column,

Optimized for Each Fraction Separated on a TSK g2000 SWG Column (Eluent A: 10 mM ammonium
acetate/acetic acid buffer, pH 6.0; eleunt B: acetonitrile; flow rate 1.5 mL/min).
Fraction Time (min) Eluent A % Eluent B %
I, II 0 100 0
6 94 6
56 70 30
60 100 0
III, IV 0 100 0
56 72 28
60 100 0
V 0 100 0
5 92 8
55 74 26
58 100 0
VI 0 100 0
45 70 30
47 50 50
50 100 0
VII 0 100 0
5 90 10
50 65 35
52 50 50
55 100 0
that each fraction contains more than one peptide indicates the advantages and disadvantages of the SE
separation method. The data show that size exclusion HPLC (SE-HPLC) can be successfully used for
the prefractionation of protein hydrolysates, which enhances the efficacy of the subsequent RP -HPLC
separations. However, its separation capacity is limited. The sequence and the position in the
myoglobin chain of the isolated peptides are listed in Table 2.123. The results indicate that the peptic
hydrolysate of yellowfin tuna myoglobin contains mainly small peptides consisting in, generally, less
then seven amino acid residues. It has been stated that this combined separation process is suitable for
the study of similar hydrolytic processes.
Although UV detection is generally used in the analysis of peptide mixtures, chemiluminescent nitrogen
detection (CLND) [ 310] has also been applied for the estimation of the average molecular mass of such
a type of solutes [ 311]. The separation of the components of soy protein, vegetable protein, whey
hydrolysate, and casein hydrolysate was carried out by size exclusion chromatography using a TSK
G2000 SWXL column (300 × 7.8 mm I.D.). The eluent was water:2-propanol:methanol (70:15:15 v/v)
containing
Page 253
Figure 2.132.
Preparative chromatography on a D.E.A.E. Sephacel column
(320 × 30 mm I.D.) of a Yellowfin tuna red muscle extract.
Equilibrating buffer: Tris/HCl 50 mM, pH 8.6. Eluting buffers:
Tris/HCl 50 mM, pH 8.6 and Tris/HCl 50 mM, pH 8.6,
NaCl 0.2 M. Flow rate: 1.6 mL/min.
Figure 2.133.
Elution profile of peptic digest of myoglobin
on a TSK G2000 SWG semipreparative column
(600 × 21.5 mm I.D.) equilibrated and eluted with
3.0 mM ammonium acetate/acetic acid buffer, pH
6.0. Injection volume: 100 µL (20 mg of total
hydrolysate). Flow rate: 6.0 mL/min.
Page 254
Figure 2.134.
Purification by RP-HPLC on a Deltapak C 18 column of the fractions I to VII issued
from SE-HPLC. Injection volume: 100 µL (1 mg peptides). Flow rate: 1.5 mL/min.
Page 255
TABLE 2.123. Identification of Peptides from a Yellowfin Tuna Red Muscle Myoglobin Peptic Hydrolysate
Isolated by SE -HPLC Followed by RP-HPLC.
Peptide No. Corresponding Myo
Fractions from RP-HPLC -globin Fragment Sequence AA
I I-1 112-115 Glu-Lys-Ala-Gly
I-2 1-4 Ala-Asp -Phe-Asp Phe
I-3 1-3 Ala-Asp -Phe Phe
I-4 7-16 Leu-Lys-Cys-Trp-Gly- Trp

Pro -Val-Glu-Ala-Asp
II II-1 133-134 or 1 -2 Ala-Asp
II-2 as I-3 1-3 Ala-Asp -Phe Phe
II-3 14-17 Glu-Ala-Asp -Tyr Tyr
II-4 129-133 Gly-Ile-Ile-Ile-Ala
II-5 43-50 Ala-Gly-Ile-Ala-Gln-

Ala-Asp
II-6 45-52 Ile-Ala-Gln-Ala-Asp
II-7 112-117 Glu-Lys-Ala-Gly-Leu-Asp
II-8 10-16 Trp -Gly-Pro -Val-Glu- Trp

Ala-Asp
II-9 10-13 Trp -Gly-Pro -Val Trp
II-10 136-146 Glu-Ala-Asn -Tyr -Lys Tyr

Glu-Leu-Gly-Phe-Ser-
Gly
II-11 as I-4 7-16 Leu-Lys-Cys-Trp-Gly- Trp

Pro -Val-Glu-Ala-Asp
II-12 7-15 Leu-Lys-Cys-Trp-Gly- Trp

Pro -Val-Glu-Ala
III III-1 14-16 or 47-49 or Glu-Ala-Asp or Gln-

136-138 Ala-Asp or Glu-Ala-
Asn
III-2 133-135 Ala-Asp -Leu
III-3 15-17 or 137-139 Ala-Asp -Tyr or Tyr

Ala-Asn -Tyr
III-4 136-140 Glu-Ala-Asn -Tyr -Lys Tyr
III-5 46-50 Ala-Gln-Ala-Asp -Ile
III-6 5-7 Ala-Val-Leu
III-7 4-7 Asp -Ala-Val-Leu
III-8 135-139 Leu-Glu-Ala-Asn-Tyr Tyr

III-9 130-135 Ile-Ile-Ile-Ala-Asp-Leu
III-10 136-143 Glu-Ala-Asn -Tyr -Lys- Tyr

Glu-Leu-Gly
III-11 102-106 Ile-Ser-Glu-Val-Leu
III-12 44-49 Gly-Ile-Ala-Gln-Ala-Asp
III-13 14-22 Glu-Ala-Asp -Tyr -Thr- Tyr

Thr -Met-Gly-Gly
III-14 130-134 Ile-Ile-Ile-Ala-Asp
III-15 as II-10 136-146 Glu-Ala-Asn -Tyr -Lys- Tyr

Glu-Leu-Gly-Phe-Ser-
Gly
III-16 130-135 Ile-Ile-Ile-Ala-Asp-Leu
III-17 129-135 Gly-Ile-Ile-Ile-Ala-Asp-Leu

Page 256
TABLE 2. 123.
Peptide No. Corresponding Myo-
Fractions from RP-HPLC globin Fragment Sequence AA
III-18 8-16 Lys-Cys-Trp-Gly-Pro - Trp
Val-Glu-Ala-Asp
IV IV-1 112-116 Glu-Lys-Ala-Gly-Leu
IV-2 14-19 Glu-Ala-Asp-Tyr -Thr- Tyr

Thr
IV-3 as III-7 4-7 Asp -Ala-Val-Leu
IV-4 as II-10 136-146 Glu-Ala-Asn-Tyr -Lys-Tyr

Glu-Leu-Gly-Phe-Ser-Gly
IV-5 10-15 Trp -Gly-Pro-Val-Glu- Trp

Ala
IV-6 79-81 Ala-Ile-Leu
IV-7 10-16 Trp -Gly-Pro-Val-Glu- Trp

Ala-Asp
AA = aromatic amino acid detected by second-order derivative spectrum.
0.1% TFA. The flow rate was 400 µL/min, and the column was thermostatted at 60°C. The effluent was
split after the column and was monitored simultaneously by a UV detector at 214 nm and by CLND
(pyrolysis temperature 1050°C; voltage of the photomultiplier tube 700, detector output 1 V). Solutes
containing nitrogen are converted to nitric oxide at the temperature of pyrolysis. The gas is dried and
mixed with ozone in the reaction chamber. The excited nitrogen dioxide molecules emit light when
returned to the stable ground state. This light is measured by the photomultiplier. The chromatogram of
soy protein hydrolysate is shown in Figure 2.135. The results of both detection methods are similar; the
majority of components are peptides with molecular mass over 10 kD. The similarity of the peptide
profile measured by both methods was tentatively explained by the assumption that the chromophor
aromatic amino acids are evenly distributed among the fractions with different molecular mass. The
results are also similar in the analysis of a hydrolyzed whey protein sample (Figure 2.136) and for whey
hydrolysate; however, they differ considerably in the case of an extensively hydrolyzed casein (Figure
2.137). This discrepancy was attributed to the uneven distribution of chromophores in the low-
molecular mass fractions of the casein hydrolysate. The technique was proposed for the selective
detection of nitrogen-containing peptides and proteins in complex food matrices and as a new tool for
the purity control and molecular mass determination of peptides and various protein hydrolysates.
Page 257
Figure 2.135.
Size exclusion chromatography with a TSK-G2000SWXL
column of a lightly hydrolyzed soy protein with simultaneous
CLND and UV (214 nm) detection.
2.3.4—
Proteins
Because of their nutritional importance, the separation and quantitative determination of edible proteins
in different species by various chromatographic methods have been extensively studied. Since each
living organism produces special (frequently highly individual) proteins that are characteristic for the
species, the determination of the protein composition can be used for the indirect identification of the
origin of the protein that is for the detection of adulterations. Since proteins are large molecules
containing both acidic and alkaline substructures, they cannot be successfully analyzed by using
traditional supports. Many efforts have been devoted to the development of special
Page 258
Figure 2.136.
column of a 10% hydrolyzed vegetable protein with simultaneous
supports for protein analysis such as new RP -HPLC supports [ 312] and size exclusion materials [313].
Various electrophoretic techniques have also found application in the analysis of proteins [314]. Gel
electrophoresis [ 315], ultrathin-layer focusing [ 316], two-dimensional electrophoresis [ 317], and recently
different capillary electrophoretic methods [ 318,319] have been frequently used in the analysis of
proteins.
2.3.4.1—
Milk Proteins
The composition and the relative amount of various protein components in milk considerably influence
its physicochemical, biochemical, and nutritional
Page 259
Figure 2.137.
column of extensively hydrolyzed casein with simultaneous
characteristics. The protein composition of milk exerts a marked impact on the milk processing
parameters too [ 320]. A wide variety of chromatographic and electrophoretic methods were developed
and used for the determination of the protein composition of milk and other dairy products. Thus,
anion-exchange chromatography [ 321,322] and capillary electrophoresis [323] successfully separated the
different protein fractions in milk. HPLC combined with electrospray ionization-mass spectrometry has
recently been used for the analysis of major bovine milk proteins [324]. Electron spray ionization mass
spectrometry (ESI-MS) was developed for the determination of the molecular mass of large and labile
compounds up to 200 kD [ 325,326]. The separation of milk proteins,
Page 260
paracaseinate, and corresponding whey proteins was carried out on a C8 column (Zorbax 300 SB, 150 ×
2.1 mm. I.D., particle size 5 µm) at 40°C. The flow rate was 0.3 mL/min. Eluent A and B were aqueous
0.1% TFA and 0.1% TFA in acetonitrile:water (80:20 v/v), respectively. Before the RP-HPLC
separation, the samples were reduced with 10 mmol/L dithiothreitol in 6 mol/L urea at 37°C for 1 h and
then were diluted twice with eluent A. The elution gradient was 0-5 min, 37-45% B; 5-15 min, 45-55%
B; 15-20 min, 55-80% B; and 20-22 min, 80% B.
Proteins were simultaneously detected with UV absorbance at 214 nm and with an electrospray
ionization mass spectrometer by splitting the effluent using a low dead volume T-connector. The
chromatograms of skim milk proteins and whey proteins using a dual detection system are shown in
Figures 2.138 and 2.139, respectively. It can be observed on the chromatograms that the protein profiles
are similar in the case of both detection systems; however, the relative sensitivities show marked
deviations because of the different principles
Figure 2.138.
Analysis by on-line RP-HPLC and ESI-MS
of skim milk proteins. Chromatographic peaks
(noted to 1-5) were detected by (A) total ion
current or by (B) UV absorbance at 214 nm.
Page 261
of detection. The molecular mass of various milk proteins determined by online LC/ESI-MS and the
theoretical molecular masses are given in Table 2.124. The data prove that RP-HPLC combined with
ESI-MS provides a unique possibility for the separation of milk proteins and for the exact determination
of the molecular mass. Both the low values of standard deviations and the negligible differences
between the measured and calculated molecular masses indicate the good reliability and reproducibility
of the method. Owing to its high resolution power, the method was proposed for the characterization of
milk from single cows; for the detection of the adulteration of human, goat, and sheep milks with
bovine milk; and for the study of the proteolysis of various milk proteins.
RP-HPLC has also been used for the detection of cow's milk in ewe's and goat's milk [ 327]. Samples
were prepared by adjusting the pH of 50 mL raw milk to 4.6 with 2 M HCL. After 15 min incubation at
room temperature, the mixture was centrifuged (2000 g, 20 min), and 5 mL of supernatant was diluted
Figure 2.139.
Analysis by on-line RP-HPLC coupled with
ESI-MS of whey proteins after milk-clotting
by chymosin. Chromatographic peaks
(noted to 1-5) were detected by (A) total ion
current or by (B) UV absorbance at 214 nm.
Page 262
TABLE 2.124. M τ Determination of Major Milk Proteins

by On-line LC/ESI-MS.
Average Standard Number of Theoretical Mτ
Proteins Observed Mτ (Da) Deviation Experiments
(Da) in This Work
κ-CN A-1P 19038.0 2.2 6 19037.3
κ-CN B -1P 19007.0 1.1 4 19005.5
αs2 -CN A-11p 25230.0 2.1 9 25228.4
αs1 -CN B -8P 23617.2 1.3 14 23614.8
β-CN B -5P 24092.0 1.7 6 24092.4
β-CN A 1-5P 24025.3 1.0 14 24023.3
β-CN A 2-5P 23984.8 0.7 14 23983.3
β-LG B 18278.3 2.2 5 18278.3
β-LG A 18364.8 1.6 5 18363.4
The average Mτ of a protein was calculated by summing up the corresponding masses of all residues in the
protein.
to 25 mL with 0.2 M phosphate buffer (pH 6.7). After 30 min, it was filtered and used for HPLC
analysis. Separation was carried out on a reversed-phase polymeric column at 40°C using gradient
elution (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in acetonitrile): 0-1 min, 35% B; 1-8 min,
38% B; 8-16 min, 42% B; 16-22 min, 46% B; 22-22.5 min, 100% B; 22.5-23 min, 100% B; 23-23.5
min, 35% B; and 23.5-35 min, 35% B. The flow rate was 1 mL/min, and the detection wavelength was
205 nm. The chromatograms of whey proteins of cow, ewe, and goat are shown in Figure 2.140. The
retention time of the cow milk proteins considerably differs from those of ewe and goat proteins; that is,
the difference in the retention behavior of proteins can be used for the detection of cows milk in the
milk of the two other species (Figure 2.141). The coefficient of variation of the retention times was
about 1.9%. Unfortunately, the method is not suitable for the differentiation between ewe and goat milk
because the retention times of the corresponding proteins are near to each other. It was established that
this HPLC method can detect 1% cow milk in both ewe and goat milk.
Whole sheep casein has also been separated into individual casein fractions by using HPLC [ 328].
Whole casein was prepared from skimmed sheep milk by acidification with 1 M HCl to pH 4.6. The
precipitate was washed twice with distilled water, redissolved by adjusting the pH to 7.0 with 1 M
NaOH, precipitated, washed, and redissolved again. The end product was lyophilized. Columns of
different dimensions filled with an anion exchanger (Mono-Q series) were used for the separation of the
proteins. Columns were equilibrated with buffer A (5.10-3 M Tris-HCl, pH 8.0, 4.5 M urea, 6.4 × 10-5 M
dithithreitol,
Page 263
Figure 2.140.
Whey protein chromatograms from (A) sheeps, (B) goats, and (C)
cow milk (α-LA: α -lactoalbumin; B -LG: β-lactoalbumin; SA: seroalbumin.
Page 264
Figure 2.141.
Chromatograms of three mixtures of (A) cow and goat milk, (percentages
of cow milk: 1%, 3%, and 10%) and (B) cow and sheep milk
(percentages of cow milk: 1%, 5%, and 10%).
(Repreinted from Reference [ 327] with permission from Chromatographia.)
0.12 M NaCl). Casein samples were dissolved in 5 × 10-3 M Tris-HCl (pH 8.0, 4.5 M urea, 12.4 × 10-4
M dithiothreitol) and applied to the columns. Gradient elution was used for the separation buffer B,
being identical with buffer A, but containing 0.32 M NaCl. The detection wavelength was 280 nm, and
the flow rate depended on the dimensions of the column. The purity of the κ-casein fractions was
verified by using polyacrylamide gel electrophoresis and amino acid analysis. The elution profiles of
sheep casein under various chromatographic conditions are shown in Figure 2.142. The data indicate
that the chromatographic conditions (column dimensions, flow rate, gradient) exert a considerable
impact on the separation of sheep casein fractions. It was found that 2 mg of pure κ-casein can be
isolated from 50 mg whole casein on a Mono-Q 10/10 column whereas separation on a Mono-Q 16/10
column yields 12 mg κ-casein from 250 mg whole casein. It was established that the method is less
time-consuming than the other preparative or semipreparative methods used for the isolation of sheep
κ-casein.
HPLC can be used, not only for the analysis of complex protein mixtures, but also for the preparation
on chromatographically pure individual protein fractions. Special HPLC techniques are suitable for the
separation of a given protein from a complicated mixture too [ 329]. Thus, subunit exchange affinity
chromatography was successfully used for the separation of β-lactoglobulin A [330]. The affinity
support was prepared by binding β-lactoglobulin A to
Page 265
Figure 2.142.
FPLC chromatography of sheep casein on Mono-Q columns. Whole sheep casein (5 mg/mL) dissolved in a
5.10 -3 M Tris -HCl, 4.5 M urea, 12.10-4 M dithiotreitol, p 8.0 buffer was applied on the column and eluted
with a gradient made of buffer A (5.10 -3 M Tris -HCl pH 8.0, 4.5 M urea in presence of 6.4.10 -5 M
dithiotreitol and 0.12 M NaCl) and buffer B (5.10-3 M Tris -HCl pH 8.0, 4.5 M urea in presence of 6.4.10 -5 M
dithiotreitol and 0.32 M NaCl). (a) Mono-Q 5/5; sample volume 200 µL; flow rate: 1 mL/min. (b) Mono-Q; sample
volume and flow rate as in (a). (c) Mono -Q 10/10; sample volume 10 mL; flow -rate: 2 mL/min. (d):
Mono-Q 16/10; sample volume 50 mL; flow rate: 4 mL/min.
(Reprinted from Reference [ 328] with permission from VV-GmbH Volkswirtschaftlicher Verlag.)
Page 266
CNBr-activated Sepharose 4B (S-CNBr -4B). A column was filled with 0.403 g of freeze-dried S-CNBr -
4B, hydrated, and then washed by 0.1 M HCl for 60 min at 2.5 mL/min at 20°C. The protein was
dissolved in the coupling buffer (0.1 M NaHCO3 containing 1.0 M NaCl, pH 7.0) and was recirculated
for 17 h at 4°C (flow rate 0.3 mL/min). After binding, the excess protein was washed away with the
same coupling buffer, and the remaining active groups were neutralized by 0.1 M ethanolamine (pH
9.5) for 24 h at 4°C. Samples containing various amounts of pure β-lactoglobulin were injected into the
column and eluted with a buffer. The kinetic parameters of the interaction of β-lactoglobulin in solution
with β-lactoglobulin bonded to the support were calculated. It was stated that this system represents an
ideal solution for the separation of this particular protein from whey. Unfortunately, no experiments
were carried out with real whey protein samples; therefore, the results and conlusions have to be
considered with caution.
Because of its high separation power and high selectivity, CZE has been extensively used for the
separation and characterization of a wide variety of proteins. It has been successfully used for the
separation and characterization of ewe milk proteins too [ 331]. For CZE analysis, milk samples were
diluted 1:4 v/v with a sample buffer (5 mM trisodium citrate dihydrate, 9 M urea, 30 mM D, L-
dithiothreitol). Acid and rennet caseins were prepared by traditional methods and were dissolved in the
sample buffer at the concentration of 0.8% w/v. Casein fractions were prepurified by FPLC on a Mono
S HR5/5 column using NaCl gradient (0-0.26 M NaCl) and urea-acetate buffer (0.02 M acetate, 6 M
urea, pH 5.0). The detection wavelength was 280 nm. The individual fractions were collected and
further analyzed with CZE and polyacrylamide agarose gel electrophoresis (PAAGE).
CZE was carried out in a 55 cm (50 cm to detection point) × 50 µm I.D. hydrophilically coated fused-
silica capillary. Voltage was applied using positive to negative polarity. The electrolyte was a citrate
buffer with 20 mM trisodium citrate dihydrate, 6 M urea, and 0.05% MHEC (pH adjusted to 3.0 with
concentrated citric acid). Samples were injected by using pressure (34,474 × 8 s). Separations were
carried out at 25 kV voltage at 40°C by UV detection at 214 nm. FPLC separated the acidic casein
sample in five fractions (0-4) (Figure 2.143). Subsequent PAAGE analysis of the fractions indicated
that fraction 0 does not contain any protein, fraction 1 consisted mainly of β-casein, fractions 2 and 3
were rich on κ-casein and contained small amounts of τ-and fastmoving αs2 -casein, whereas fraction 4
corresponded to αs-casein fractions. CZE separation of the fraction prepurified with FPLC is shown in
Figure 2.144. CZE separated the casein fractions well, and the retention times of various fractions were
29-36 min for αs-cn, 38-45 min for β-cn, 37 min for κ-cn, and 26 min for para-κ-casein. The protein
components of whey ( α- and β-lactalbumin, β-lactoglobulin) were also well separated by this CZE
method. As demonstrated in Figure 2.145, the main proteins of an individual milk sample
Page 267
Figure 2.143.
FPLC analysis of ewe milk. Preparative FPLC analysis of whole acid
casein. Mono S HR5/5 column; urea-acetate buffer (pH 5.0, 0.02
M acetate, 6 M urea); NaCl gradient, 0-0.26 M; UV detector,
280 nm. Abbreviations: 0-4 = FPLC fractions.
Figure 2.144.
CZE analysis of ewe milk. CZE analysis of FPLC fractions
and whole casein. Hydrophilically coated fused-silica capillary,
50 cm (to the detection point), 55 cm total length × 50
µm I.D.; electrolyte, pH 3.0; injection by pressure,
34,474 Pa × 8 s; applied voltage, 25 kV; T = 40°C;
UV detection, 214 nm.
Page 268
Figure 2.145.
CZE analysis of ewe milk. Identification of the major
components in individual milk. Hydrophilically coated
fused-silica capillary, 50 cm (to the detection point), 55 cm
total length × 50 µm I.D.; electrolyte, pH 3.0; injection by
pressure, 34474 Pa × 8 s; applied voltage, 25 kV;
T = 40°C; UV detection, 214 nm.
Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)
can be separated in one run. It was concluded that, because of its high separation power, this CZE
method can be used in the analysis of dairy products. It was further stated that the method requires less
manipulation and is easier to carry out than the corresponding RP -HPLC and PAAGE separation
techniques.
The folding and unfolding of the whey protein β-lactoglobulin B was extensively studied with
electrophoretic methods [ 332]. Since it has been previously shown that CZE can also be used for the
study of protein folding [ 333], a theoretical study used CZE as a tool to study the folding and unfolding
of β-lactoglobulin B [ 334]. A fused-silica capillary (total length 50 cm, separation length 35 cm, 50 µm
I.D.) was used for the study of β-lactoglobulin denaturation. The separation was monitored at 215 nm.
The electrophoretic buffer was 0.05 M tris (hydroxymethyl)aminomethane (pH adjusted to 8.2 with
HCl) containing 0.005% NaN 3. The injection was carried out electrokinetically (+5 kV; 5 s). The
working voltage was +15 kV. The capillary was washed between the runs with 1 M NaOH, water, and
buffer. The denaturation capacity of urea (8 M) and dithiothreitol (0.1 M) and their mixture was studied
by adding the denaturants to the buffer solution of β-lactoglobulin (0.2 mg/mL) at room temperature.
Samples were injected after various incubation times. Protein refolding was studied by dialyzing
denatured protein and letting it remain for various times before injection. The electropherograms of β-
lactogobulin B incubated in
Page 269
Figure 2.146.
Electropherograms of β-lactoglobulin B obtained after exposure
to 8 M urea solution for various times: (A) 2-min exposure time,
native peak area/nonnative peak area is approximately 62%;
(B) 18-min exposure time, native peak area/nonnative peak
area is 36%; (C) 31-min exposure time, native peak
area/non-native peak area is 28%; (D) 95-min exposure time,
native peak area/nonnative peak area is 16%.
Urea marks the elctroosmotic flow time.
the Preston Publications, A Division of Preston Industries,
8 M urea solution for various times are shown in Figure 2.146. The change in peak shape and mobility
of β-lactoglobulin B increases with increasing incubation time. The retention time of the denaturated
form of the protein becomes shorter, with the relative standard deviation of the retention time of the
nature protein being 1.24%. After removing the urea, the protein molecules return to the original state,
as shown in Figure 2.147. The treatment with dithiothreitol and dithiothreitol urea caused similar
changes in the elctrophoretic mobility of β-lactoglobulin B. It was concluded that this CZE method is
faster than the other ones used for the study of protein folding, and it can also be used for the study of
folding kinetics of various proteins.
2.3.4.2—
Meat Proteins
Similar to milk proteins, meat proteins are also characteristic for the species; therefore, their analysis
can be used for the detection of adulteration. The chromatographic analysis of meat proteins is
hampered by the generally unsatisfactory solubility of these proteins in eluent used for the
chromatographic
Page 270
Figure 2.147.
Electropherograms of β-lactoglobulin B obtained after removal
from 8 M urea solution for various times: (A) 65-min exposure
time, native peak area/nonnative peak area is approximately
28%; (B) 81-min exposure time, native peak area/nonnative
peak area is 46%; (C) 112-min exposure time, native peak
area/nonnative peak area is 64%; (D) 133-min exposure time,
native peak area/nonnative peak area is 65%. Absence
of buffer salts, caused by removal by dialysis,
marks the electroosmotic flow time.
Preston Publications, A Division of Preston Industries,
separation of proteins. It has been proven many times that not only the species, but also many other
factors such as the origin of the animals, the feeding, storage conditions, and more, can have a marked
impact on the composition and nutritional value of the derived proteins. Various electrophoretic
techniques are used preferably for the analysis of meat proteins. The effect of rearing conditions on the
soluble pork proteins was also investigated with SDS polyacrylamide gel electrophoresis (PAGE) and
isoelectric focusing [ 335].
Pigs of genotype Hungarian Large White (75%) and Mangalica (25%) were kept under natural and
traditional conditions (further ''control"). Pigs kept under natural conditions did not receive growth
promoters, hormones, and antibiotics (further "organic meat"). Meat samples were taken from the
Longissimus dorsi muscles (loins), ground, and homogenized with cold 0.6 M KCl at 20.000 rpm. The
mixture was centrifuged for 30 min at 8000 rpm and the supernatant lyophilized. Trypsin was used for
the hydrolysis of the lyophilized samples [(37°C, pH 7.5, 2 h with stirring, enzyme:meat protein ratio
(1:100)]. The hydrolysates were directly used for the investigations or lyophilized again. The
Page 271
Figure 2.148.
Densitograms of SDS -PAGE patterns of organic and
control meat proteins (MW: molecular weight).
The Netherlands.)
molecular mass was determined with SDS -PAGE (10% polyacrylamide, 0.1% SDS, 6 M urea). Thin-
layer isoelectric focusing was also used for the separation of water-soluble protein fractions. To
increase the information power of the isoelectric focusing, the zones were stained either with silver
nitrate or for esterase activity. The quantitative evaluation was carried out with a densitometer. SDS-
PAGE found marked differences between the protein composition of the "organic" and control samples,
as shown in Figure 2.148. The quantity of fractions from the organic sample was higher than that of the
control meat between 40.000 and 43.000 molecular mass. The statistical parameters of the evaluation of
SDS-PAGE results are compiled in Table 2.125. The statistical evaluation proved that the differences
between the SDS-PAGE results are highly significant and can be used for the differentiation of the
meats originated from different rearing conditions. Not only the protein pattern, but also the pattern of
the esterase activity of the meat enzyme system, showed marked differences, as substantiated in Figure
2.149. The differences between the isoelectric
Page 272
TABLE 2.125. Evaluation of SDS-PAGE Densitograms

of Organic and Control Meat Samples.
Molecular Masses of Proteins Corresponding
to the Major Peaks (Mw)
56,000 42,000 38,000 35,000
Organic x = 3.9 x = 10.7 x = 11.1 New peak
meat sample s = 0.14 s = 0.28 s = 0.85

Control x = 1.7 x = 4.0 x = 2.8 No peak
meat sample s = 0.14 s = 0.14 s = 0.14
The difference
is significant on the
probability level of 99% 99.9% 99% —
Molecular Masses of Proteins Corresponding

to the Major Peaks (Mw)
27,000 20,000 16,000
Organic x = 11.9 x = 6.7 x = 4.7
meat sample s = 0.28 s = 0.28 s = 0.28
x = 6.75 x = 6.75 x = 4.35 x = 3.05
meat sample s = 0.35 s = 0.21 s = 0.21
The difference is
significant on the
probability level of 99% 95% 95%
x = Arithmetic mean of the intensities of the bands from three SDS-PAGE densitograms;
s = standard deviation.
focusing profile of the tryptic hydrolysates were evaluated with principal component analysis.
Calculations proved that the organic and control meat can be distinguished significantly by this
combined method.
SDS-PAGE also successfully separated beef myofibrillar proteins [ 336]. Myofibrils were isolated by
homogenizing 2.5 g minced meat in 25 mL buffer solution (pH 7.6, 3°C, 0.25 M sucrose, 0.05 M Tris, 1
mM EDTA. The homogenizate was centrifuged, the supernatant discarded, and the solid rest
resuspended in 25 mL of 0.05 M Tris, 1 mM EDTA (pH 7.6, 3°C). The suspension was centrifuged
again, the supernatant decanted, and the treatment repeated with 25 mL of 0.15 M aqueous KCl. About
2.4 g of myofibrils were dissolved overnight at room temperature in 30 mL buffer (pH 7.0, 0.01 M
imidazole, 2% SDS, 2% mercaptoethanol). The solutions were filtered, and the protein content was
adjusted to 4.0 mg protein/milliliter and 0.2 mg bovine serum albumin/milliliter internal standard; 15%
sucrose and about 0.3 mg bromophenol blue were added.
Page 273
Figure 2.149.
Densitometric evaluation of the zones developed with protein
and esterase stain of electrophoretograms of soluble pork proteins.
The optimal separation of protein fractions in the molecular mass range of 30-50 kDa was carried out
on gels consisting of 8.0% total (T) acrylamide + N, N'-methylene-bis acrylamide with a cross -linking
(C) of 3.1% [bis:monomer (1:31)], 0.55% SDS, and 0.5% mercaptoethanol (ME) (pH 7.0).
Polymerization was catalysed with 0.3% N, N,N',N'-tetramethyl-ethylene-diamine (TEMED) and
0.031% ammonium persulphate. A slightly different gel composition was used for the separation of
protein fractions in the molecular mass range of 50-1000 kDa (4.6% T, 3.1% C, 0.55% SDS, 0.5%
ME). Polymerization agents were 0.3% TEMED and 0.039% ammonium persulphate. The
electrophoresis buffer (pH 7.0) contained 0.05 M imidazole and 0.1% SDS, and the separation was
carried out at 160 V. Gels were evaluated with a densitometer at a 600-nm wavelength; the relative
concentration of proteins was calculated using the absorption of the bovine serum albumin (BSA)
internal
standard as reference. Purified myosin, tropomyosin, actin, and troponin were used for the tentative
identification of the bands.
The effect of aging, heat treatment, and the addition of protease inhibitors to the protein samples was studied in
separate experiments. Myofibrillar proteins were separated in many fractions: myosin heavy chain, myosin light
chain 1 and 2, tropomyosin, titin, and filamin were identified. The concentrations of various protein fractions
unheated or heat treated are given in Table 2.126. It has been established that the solubility of the majority of
the myofibrillar proteins did not change after heat treatment. The significant decrease of the concentration of
titin was tentatively explained by the heat instability of this protein. Protease inhibitors exert no significant
effect on the electrophoretic profiles of myofibrillar proteins, indicating that proteolysis does not occur during
the sample preparation steps. Aging and electrical stimulation exerted a marked influence on the composition of
myofibrillar proteins as demonstrated by the data in Table 2.127. The data unambigously prove that both aging
and electrical stimulation exert a considerable influence of the composition of
TABLE 2.126. Comparison of Myofibrillar Protein Band Concentrations Solubilized With and Without Heating (expressed as
BSA equivalents/milligrams myofibrillar protein).
Unheated (n = 8) Heated (n = 8)
Protein Band Mean SD VC * Mean SD VC

Separation on 4.6%
Rod Gels
Titin 36.2 ** 1.8 5.0 31.5 *** 2.3
Filamin 5.3 0.3 5.7 4.9 0.5
Separation on 8%
Rod Gels
Myosin heavy chain 209.3 7.1 3.4 203.1 14.6
α-Actinin 19.2 0.8 4.2 18.6 0.6
Tropomyosin 20.6 1.7 9.2 19.8 1.7
Actin 135.5 4.6 3.4 139.0 5.1
Troponin -T 17.8 1.0 5.6 16.7 1.5
36 kDa 9.3 0.7 7.5 9.2 0.5
30 kDa 9.3 0.7 7.1 9.3 0.6
Myosin light chain 1 21.5 0.9 4.2 21.5 0.7
Troponin I 20.0 ** 0.7 3.5 21.0*** 0.9
Troponin -C 5.9 0.7 11.9 5.9 1.4

Myosin light chain 2 28.1 1.4 5.0 28.2 1.6
** Variation coefficient.
**,*** Means with different following letters differ significantly (p <0.05, t-statistic).
Page 275
myofibrillar proteins, which can modify the technological properties and nutritional value of the
proteins.
SDS-PAGE has also been used for the identification of red seaweeds (Gracillaria sp.) [ 337]. The study
was motivated by the fact that these seaweed species (G. verrucosa, G. compressa and G. foliigera) are
used as vegetables or as ingredients in food products and are also used for alga production [ 338]; their
identification with other methods is fairly difficult and time-consuming. Before extraction, the
seaweeds were washed, lyophilized, and homogenized (100 µm). Proteins were extracted from the
seaweed powder with distilled water (1:10 w/v) using sonification for 10 min and then were incubated
for 12 h at 4°C. Finally, the samples were centrifuged at 5000 g and the supernatant was used for SDS-
PAGE. It was established that the electrophoretic pattern of the seaweed species differs considerably
and can be successfully used for their differentiation and identification.
Electrophoretic techniques proved to be a suitable technique for the identification of fish and shellfish
species also [ 339,340]. Because of the modification
TABLE 2.127. Influence of Aging and Electrical Stimulation (ES) on Myofibrillar Protein Concentrations
(means of three determinations expressed as µg BSA -equivalents/mg myofibrillar protein ± SD).
Days Postmortem *
0 1 6 12
+ – + – + – + –
Titin 58.8 51.8 50.0 54.0 38.6 46.9 30.7 29.9
(5.5) (8.5) (2.3) (3.8) (3.2) (5.8) (2.3) (3.8)
Filamin 3.7 3.9 3.8 4.5 3.5 4.5 3.2 2.8

(0.4) (0.5) (0.2) (0.7) (0.7) (0.9) (0.1) (0.4)
Creatine
Phosphokinase 2.9 2.5 6.3 4.8 12.0 10.9 10.7 12.9
(0.6) (0.1) (0.6) (1.2) (1.3) (1.1) (6.0) (1.9)
Troponin -T 15.9 16.7 18.7 17.8 4.8 10.4 3.7 0.9

(0.1) (1.6) (0.9) (2.8) (1.3) (1.2) (3.8) (1.2)
Troponin -T2 5.7 5.3 7.2 5.6 4.3 4.4 2.8 1.8
(0.3) (0.9) (0.5) (1.9) (1.0) (0.8) (1.3) (1.6)
39 kDa 4.1 3.9 5.0 3.9 5.8 4.5 4.9 4.7

(0.2) (1.2) (0.2) (1.2) (0.8) (1.0) (2.5) (1.4)
36 kDa 1.0 0.8 1.5 1.7 10.2 11.1 18.7 19.1
(0.4) (0.6) (0.3) (0.8) (0.5) (1.1) (2.1) (2.6)
30 kDa 1.2 1.3 1.3 1.1 9.2 5.5 12.4 15.7

(0.3) (0.4) (0.3) (0.3) (0.8) (0.2) (1.3) (0.0)
* +, with electrical stimulation; -, without electrical stimulation.
Page 276
of the solubility of proteins during thermal treatment, cooked fish proteins were identified with using
SDS electrophoresis of SDS extract [ 341,342]. SDS-PAGE and isoelectric focusing have been used for
monitoring the authenticity of raw reformed breaded scampi (Nephrops norvegicus) [ 343]. Samples of
minced meat were homogenized with water (5:10 w/v) and centrifuged, and the supernatant was used
for the analysis. SDS extraction was carried out with 2% (w/v) SDS solution: 0.5 g meat was
homogenized with 2 mL of SDS solution and heated for 30 min at 60°C, centrifuged, and diluted ×20
with sample buffer containing 2-mercaptoethanol. The mixture was heated again in boiling water for 2
min, cooled, and centrifuged. The supernatant was stored at -18°C before use. It was established that
isoelectric focusing was not suitable for the differentiation between scampi (Nephrops norvegicus),
pacific scampi (Metanephrops andamanicus), and tropical shrimp (Penaeus indicus); however, the
presence of meat of some fish species such as cod (Gadus morhua), haddock (Melanogrammus
aeglefinus), and whiting (Merlangius merlangus) can be easily detected. SDS-PAGE profiles of
samples were similar but not identical and allowed for differentiation between scampi (Nephrops
norvegicus), pacific scampi (Metanephrops andamanicus) and tropical shrimp (Penaeus indicus). It was
concluded from the data that SDS-PAGE of the SDS extract of both raw and cooked samples is a
suitable method for the identification of these species.
Because of its good separation power, SDS-PAGE was found to be suitable for the differentiation
between mechanically recovered meat (MRM) and hand-deboned meat (HDM) in meat products [ 344].
MRM and HDM samples from beef, pork, lamb, chicken, and turkey were used in the experiments.
Water extract was prepared by homogenizing 1 g meat sample in 9 mL of chilled, deionized water. SDS
extract was prepared by adding 1 mL of 100 g/L SDS and 7 mL of water to 2 mL of homogenate.
Before SDS -PAGE, SDS and 2-mercaptoethanol were added to the samples to a final concentration of
10 g/L of each. Samples were heated at 60°C for 15 min, and then equal volumes of 100 g/L Ficoll and
0.2 g/L bromophenol blue were added. The gel contained 75 g/L acrylamide, 2 g/L N, N'-
methylenebisacrylamide in a Trisbicine buffer. Protein loading was 30 µg. After electrophoresis, gels
were stained overnight with Serva Blue G and then destained with acetic acid and methanol washing. It
was established that the relative intensities of some protein fractions are different in MRM and HDM
samples. These differences can be used for the identification of MRM and HDM in composite mixtures.
It was suggested that MRM can be detected in HDM at the range of 5-10% for red meat and 25% for
poultry meat.
The high separation capacity and low analysis cost of CZE has also been exploited to differentiate
flatfish species [ 345] on the basis of the composition of the sarcoplasmic proteins. The flatfish species
included in the investigation are listed in Table 2.128. Proteins were extracted by homogenizing 5 g
minced mus-
Page 277
TABLE 2.128. Common and Latin Names of the Flatfish Species Employed in the Study.
No. Common Name Latin Name
1 Turbot Scophtalmus maximus
2 Megrim Lepidorhombus whiffiagonis
3 Four spotted megrim Lepidorhombus boscii
4 Brill Scophtalmus rombus
5 Witch Glytocephalus cynoglossus
6 Sole Solea solea
7 European plaice Pleuronectes platessa
8 American plaice Hipoglossus platessoides
cle with 10 mL of cold distilled and deionized water and then centrifuged at 8000 g for 15 min at 4°C.
Supernatant was filtered and diluted with 30 mM phosphate buffer (pH 2.44) to give a protein
concentration of 1 mg/mL. Samples were prepared on the day of the analyis. A 57 cm × 75 µm I.D.
fused-silica capillary column was used for the separation. The capillary was treated with a 1 M NaOH
flush for 2 min; 0.1 M NaOH for 30 min; deionized water for 5 min; phosphate buffers 100 mM and 75
mM (pH 2.44) for 3 min; and 30 mM phosphate buffer (pH 2.44), which was the electrolyte buffer for
35 min. Injection was carried out by pressure injection during 3 s. The temperature of the capillary was
held at 25°C. The gradient voltage was 0-10 kV for 0.17 min, 10-15 kV for 22 min, and 15-25 kV for 1
min. Proteins were detected at 214 nm. Between analyses, the capillary was washed with 0.1 M NaOH
for 3 min and water for 1 min; then it was reconditioned with the electrolyte buffer for 10 min. At the
low buffer pH, proteins moved toward the cathode end. The injection of 0.01% dimethylformamide
indicated a negligible electroosmotic flow. The coefficient of variation of the migration time of proteins
varied from 0.14-6.43%, proving the good reliability of and reproducibility of the CZE method. The
electropherograms of the sarcoplasmic proteins of flatfish species are shown in Figure 2.150. The
electropherograms indicated that the protein profiles of flatfish species are different; therefore, they can
be used for the identification and differentiation of the species. It has been further established that the
separation power of CZE improves considerably by injecting more diluted samples. The protein peaks
of the various flatfish species are given in Table 2.129.
2.3.4.3—
Other Edible Proteins
The quantity and composition of proteins in various food grains exert a considerable influence, not only
on the nutritional value of the products, but also
Page 278
Figure 2.150.
Separation of sarcoplasmic proteins by CZE. 1 = turbot
(Scophtalmus maximus); 2 = megrim (Lepidorhombus
whiffiagonis) ; 3 = four spotted megrim (Lepidorhombus
boscii); 4 = brill (Scophtalmus rombus); 5 = witch
(Glytocephalus cynoglossus); 6 = sole (Solea solea);
7 = European plaice (Pleuronectes platessa); 8 = American
plaice (Hipoglossus platessoides).
(Reprinted with from Reference [ 345], copyright
© 1995 American Chemical Society).
Page 279
Figure 2.150
(continued). Separation of sarcoplasmic proteins by CZE.
1 = turbot (Scophtalmus maximus); 2 = megrim (Lepidorhombus
whiffiagonis); 3 = four spotted megrim (Lepidorhombus boscii);
4 = brill (Scophtalmus rombus) ; 5 = witch (Glytocephalus
cynoglossus); 6 = sole (Solea solea); 7 = European plaice
(Pleuronectes platessa); 8 = American plaice
(Hipoglossus platessoides).
Page 280
Figure 2.150
(continued). Separation of sarcoplasmic proteins by CZE.
1 = turbot (Scophtalmus maximus); 2 = megrim (Lepidorhombus
whiffiagonis); 3 = four spotted megrim (Lepidorhombus boscii);
4 = brill (Scophtalmus rombus) ; 5 = witch (Glytocephalus
cynoglossus); 6 = sole (Solea solea); 7 = European plaice
(Pleuronectes platessa); 8 = American plaice
(Hipoglossus platessoides).
Page 281
TABLE 2.129. CZE Migration Times for All of the Different Flatfish Species.
No. of Flatfish Species
Peak
No. 1 2 3 4 5 6 7 8
1 12.9
2 13.2 13.5 13.5
3 13.7
4 14.6 14.2 14.8 14.7 14.9 14.5 14.7 14.6
5 15.0 15.7 15.3 15.4 15.0 15.2
6 16.0
7 16.5 16.4 16.4
8 16.9 16.6 16.7 16.6
9 16.9 17.0 16.9 16.9 16.9 17.0
10 17.4 17.2 17.4 17.1 17.2

11 17.9 17.7 17.7 17.6 17.5
12 18.3 18.1 18.2 17.9 17.9 18.1
13 18.5 18.5 18.5
14 18.8 18.6 18.7 18.7

15 19.0
16 19.7 19.9 20.1
17 20.9 20.8 20.6 20.6 20.5 20.3
18 21.2 21.0
19 22.3
20 30.8
1 = turbot (Scophtalmus maximus); 2 = megrim (Lepidorhombus whiffiagonis ); 3 = four spotted megrim

(Lepidorhombus boscii); 4 = brill ( Scophtalmus rombus); 5 = witch (Glytocephalus cynoglossus); 6 = sole
(Solea solea); 7 = European plaice (Pleuronectes platessa ); 8 = American plaice (Hipoglossus platessoides).
on the processing parameters [ 346]. Many chromatographic and electrophoretic methods have been
developed for the separation of the protein fractions of various grains [ 347]. Thus, polyacrylamide gel
electrophoresis was used for the separation of wheat proteins [ 348] for the identification of cereal
varieties [ 349] and for the detection of gliadins [350].
Isoelectric focusing was applied to distinguish durum wheat from common wheat [ 351]. RP-HPLC has
also been frequently used for the analysis of various grain proteins such as reduced glutenin [ 352],
gluten [353], and wheat proteins [ 354]. RP-HPLC has been used for the separation of oat proteins too
[355]. Oat samples were defatted by n-butanol before the extraction of proteins. Proteins were
subsequently extracted with 1 M NaCl (salt-soluble fraction), 52% ethanol (alcohol-soluble fraction)
and 1% SDS in borate buffer (pH 10) (alkali-soluble fraction). Butyl-, octyl-, and octadecyl -coated
silica columns were used for the RP-HPLC separation of oat protein fractions. Gradient elution was
used: eluent A and B being 10% acetonitrile + 0.11% TFA and 90%
Page 282
acetonitrile + 0.09% TFA, respectively. Column temperature was set to 50-72°C, and the flow rate was
0.9 mL/min.
In order to determine the molecular mass of the various protein fractions, the same samples were also
analyzed with SDS -PAGE using standard proteins for calibration. The effect of column type, TFA
concentration and gradient elution on the optimization of the separation of protein fractions is
demonstrated for salt-soluble, alcohol -soluble, and alkali-soluble oat proteins in Figures 2.151-2.153,
respectively. It was established that the higher acetonitrile concentration was required for the elution of
salt-soluble oat protein fractions at a higher TFA concentration. The best separations were achieved
under conditions A, F, and G. The separation capacity of the C8 column was inferior to the other
Figure 2.151.
RP-HPCL of salt-soluble oat proteins. Elution profiles
were obtained at 60°C (A) on a Vidac 218TP54 column
with 0.2% TFA, (B) on a Supelcosil LC-308 column
with 0.1% TFA, and (C) 0.2% TFA; and on two
Vidac214TP5415 C 4 columns, designated as (a)
and (b), with 0.1% TFA (D, F), and 0.2% TFA (E, G).
Gradients: A, 26% acetonitrile (ACN), increasing linearly
to 34% at 3 min, to 46.8% at 50 min, and to 48.4% at 54 min;
(B, C, E, G) 26% ACN, increasing linearly to 34% at
3 min, to 41.2% at 40 min, and to 43.6% at 42 min;
(D) 26% ACN, increasing linearly to 31.6% at 4 min,
to 36.4% at 15 min, to 38% at 40 min, and to 42% at
42 min; and (F) 26% ACN, increasing linearly to 34% at
3 min, to 42% at 50 min, and to 43.6% at 52 min.
Page 283
columns, which showed slight differences in their retention characteristics. The separation profile
depended not only on the chromatographic conditions listed above, but also on the column temperature.
The optimum separation temperature depended on the protein fraction to be separated. The composition
of the protein fractions was characteristic of the oat cultivar; subsequently, it can help the identification
of cultivars, and it can be used for the detection of changes caused by various processing steps.
RP-HPLC has also been used for the detection of common-wheat flour in Durum-wheat semolina [ 356].
The investigation was motivated by the fact that Italian ''pasta" can be prepared only with Durum-wheat
semolina and the mixing of common-wheat flour is not allowed. Protein samples were prepared by
Figure 2.152.
RP-HPLC of alcohol-soluble oat proteins. Elution profiles
were obtained at 60°C (A) on a Vidac 218TP54 column
with 0.2% TFA, (B) on a Supelcosil LC-308 column with
0.1% TFA, and (C) 0.2% TFA; and on two Vidac214TP5415
C4 columns, designated as (a) and (b), with 0.1% TFA (D, F),
and 0.2% TFA (E, G). Gradients: (A, C, D, E) 26% ACN,
increasing linearly to 32.4% at 4 min, to 38% at 15 min, to 43.6%
at 40 min, and to 44.4% at 42 min; (B) 26% ACN, increasing to
32.4% at 4 min, to 35.6% at 15 min, to 41.2% at 40 min,
and to 42.8% at 42 min; (F) 26% ACN, increasing to 32.4% at 4 min,
to 40.4% at 42 min, and to 43.6% at 46 min; and (G) 26% ACN,
increasing to 30% at 3 min, to 36.4% at 10 min,
to 43.6% at 50 min, and to 44.4% at 52 min.
(Reprinted from Reference [ 355].)
Page 284
Figure 2.153.
RP -HPLC of alkali -soluble oat proteins. Elution
profiles were obtained at 60°C (A) on a Vidac
218TP54 column with 0.2% TFA, (B) on a
Supelcosil LC-308 column with 0.1% TFA, and (C)
0.2% TFA; and on two Vidac214TP5415 C 4
columns, designated as (a) and (b), with 0.1%
TFA (D, F), and 0.2% TFA (E, G). In all runs, the
ACN concentration was initially 26%, and increased
linearly to 34% at 3 min. The remainder of the gradients
were: (A) increase of ACN to 43.6% at 43 min and to
50% at 54 min; (B) increase of ACN to 37.2% at
15 min, to 41.2% at 35 min, and to 43.6% at 36
min; (C, D, E, G) increase of ACN to 41.2% at 40 min
and to 43.6% at 42 min; and (F) increase of ACN to
42.8% at 40 min and to 44.4% at 42 min.
extracting 10 g of flour, semolina, or ground pasta for 30 min at room temperature with 35 mL of 0.8 M
ammonium sulphate and 0.1 M magnesium sulphate solution (pH adjusted to 3.25). The mixture was
centrifuged and the supernatant was purified with solid-phase extraction: 0.5 mL of supernatant was
loaded into a prewetted C18 cartridge and then eluted with 0.5 mL of 15% methanol and 1 mL 30%
acetonitrile. The last 1 mL was used for the RP-HPLC separation. Preparative separation of protein
fractions was carried out on an S-Sepharose Fast Flow column (300 × 20 mm I.D., particle size 10 µm).
Common-wheat samples were separated isocratically using a 0.025 M sodium acetate buffer (pH 6.0).
Durum-wheat proteins were separated in the same eluent using a sodium chloride gradient (0-30 mM
NaCl in 360 min). The column was thermostatted
Page 285
Figure 2.154.
RP-HPLC of albumins of common wheat flour
(F) and Durum-wheat semolina (S).
permission from Elsevier Science—NL, Sara Burgerhartstraat
at 20°C, the flow rate was 1 mL/min, and the detection wavelength was 280 nm. A PLRP-S column
(150 × 4.6 I.D., 8 µm particle size) was used for the analytical separation of the fractions. Eluents were
water (A) and acetonitrile (B), both containing 0.1% v/v TFA. The gradient was in the percent of B: 0-
15 min, 27-31%; 15-17 min, 31-100%; 17-19 min, 100%; 21-30 min, 27% (column temperature 50°C,
flow rate 1 mL/min, 210 nm detection). The major fractions were collected and subjected to isoelectric
focusing. The RP -HPLC profiles of common-wheat flour and Durum-wheat semolina differed
considerably (Figure 2.154). It has been proven that this difference can be successfully used for the
detection of common-wheat flour in Durum-wheat semolina. The relationship between the peak height
of the added common-wheat protein and its concentration is significantly linear ( R = 0.98). The
coefficient of variation was lower than 4%, and 1% of common-wheat flour can be reliably detected in
the samples. Although the method is suitable for the detection of common-wheat flour in low-heat dried
pasta, it cannot be used for pasta dried at higher temperatures. In these instances, the resolution of
protein fraction in RP-HPLC was poor and cannot be used for the identification of common-wheat
flour. It was assumed that drying at a high temperature enhances the insolubilization of proteins and
increases the aggregation of albumins.
Page 286
Hydrophobic chromatography was also used in the analysis of proteins of plant origin. Thus, soybean
proteins were purified with hydrophobic chromatography carried out on Phenyl Sepharose CL-4B
column [ 357]. The chromatographic profiles of glycinin, one of the major proteins in soybean seeds,
and that of Kunitz soybean trypsin inhibitor are shown in Figures 2.155 and 2.156. It was concluded
from the data that hydrophobic chromatography is a promising method for the preparation of
chromatographically pure soybean proteins such as glycinin and Kunitz soybean trypsin inhibitor.
A combined two -step chromatographic method was used for the purification of hen egg white
ovomucin, lysozyme, ovotransferrin, and ovalbumin [ 358]. Samples were prepared by diluting egg
white with 2 volumes of 0.05 M Tris-HCl (pH 9) containing 0.4 M NaCl and 10 mM β-mercaptoethanol
and gently stirring overnight. Preliminary separation was carried out on a Superose 6 HR column; the
chromatograms are shown in Figure 2.157. The protein peaks were tentatively identified by using pure
proteins as reference substances. Peak 4 contained mainly ovalbumin, ovotransferrin, and ovomucoid,
while peak 5 contained lysozyme. It was assumed that ovomucin eluted in either peak 1, 2, or 3. SDS-
PAGE of the collected fractions indicated that peaks 1, 2, and 3 consist of
Figure 2.155.
Stepwise chromatography of glycinin
preparation (50 mg) on Phenyl Sepharose.
The column (11 × 1 cm) equilibrated with
(NH4)2SO 4 of ionic strength 2.1 containing
0.1% (v/v) of 2 -mercaptoethanol and
0.02% of NaN 3 adjusted to pH 8.0 with
NH 4OH. Elution with the same solution
and then with water. Elution rate: 24 mL/h.
(Reprinted from Reference [ 357 ] with
corresponding author.)
Page 287
ovomucin contaminated with ovalbumin and ovotransferrin, peak 4 is composed mainly from
ovalbumin and ovotransferrin, and peak 5 contain lysozyme. Peak 4, containing the overwhelming
majority of hen egg white proteins, was further separated on a Sepharose Fast Flow column (10 × 5 cm
I.D) using the gradient described in Table 2.130. Two buffers—(A) 0.05 M Tris-HCl (pH 9) and (B) A
containing 0.3 M NaCl—were used for increasing the stepwise gradient from 100% A to 100% B (flow
rate: 7.5 mL/min). The use of an anion-exchange column was motivated because a similar anion-
exchanger has been successfully used for the separation of ovalbumin from hen egg white proteins
[ 359]. The elution profile of peak 4 on the anion-exchange column is shown in Figure 2.158. Similar to
the results of SDS-PAGE, the separation on the anion-exchange column proves again the
inhomogeneity of the fractions separated with gel permeation chromatography. It was established that
peak A contained ovotransferrin and a minor glycoprotein, and peaks B and C contained pure
ovalbumin. The purity of peaks collected after the separation of hen
Figure 2.156.
Stepwise chromatography of
commercial Kunitz soybean trypsin
inhibitor preparations on Phenyl
Sepharose. The columns equilibrated
with (NH4 )2SO 4 solution containing
0.1% (v/v) of 2 -mercaptoethanol and
0.02% of NaN 3 adjusted to pH 8.0 with
NH 4OH. Elution with the same solution
and then with water and 1% SDS.
(a) Renal preparation (350 mg); column
105 × 2.8 cm; ionic strength of the initial
eluent (0.7); elution rate (30 mL/h). (b)
Serva preparation (24 mg); column
8.5 × 1.2 cm; ionic strength of the initial
eluent (0.2); elution rate (14 mL/h).
(Reprinted from Reference [ 357 ] with
permission from the publisher
Page 288
Figure 2.157.
(A) Chromatography of hen egg white proteins on a Superose
6 HR column (30 × 1 cm I.D.): 100 µL of the egg white
preparation containing ca. 6 mg protein were applied
to the column previously adjusted with 0.05 M Tris-HCl
buffer (pH 9) containing 0.2 M NaCl, using the Pharmacia
FPLC system at room temperature. Proteins were eluted
with the same buffer at a flow rate of 0.4 mL/min.
(B) Chromatography of hen egg white proteins on a
Superose 6 Prep Grade column (90 × 2.6 cm I.D.), using
0.05 M Tris-HCl buffer (pH 9) containing 0.2 M NaCl:
10 mL of egg white preparation containing ca. 615
mg of proteins were loaded on the column and protein
elution was performed at a flow rate of 2 mL/min.
kind permission from Elsevier Science—NL, Sara
egg white proteins on a Superose 6 Prep Grade column and on a Q Sepharose Fast Flow column was
checked on an analytical TSK-G3000 SW gel permeation column with the eluent consisting of 0.01 M
sodium phosphate buffer (pH 2.4) containing 0.2 M NaCl (ovomucin was chromatographed at pH 7).
The flow rate was 0.4 mL/min. The chromatograms are shown in Figure 2.159.
Page 289
TABLE 2.130. Parameters for the Elution of Gel Permeation Peak Four
Proteins from Q Sepharose Fast Flow Column.
0 100 0
25 100 0
40 60 40
85 60 40
95 55 45
115 55 45
125 50 50
175 50 50
185 45 55
235 45 55
280 0 100
Netherlands.
Because of the low mass requirement, low analysis time, and excellent separation characteristics, CE
has also found application in the separation of plant proteins. Thus, CE has been used for the
fractionation of gliadins and high molecular mass glutenin subunits of wheat [ 360]. Samples were
prepared by extracting crushed seeds, either with 70% ethanol (gliadins) or with 1% SDS
Figure 2.158.
Chromatography of proteins contained in peak 4
from gel permeation on a Q Sepharose Fast
Flow column (10 × 5 cm I.D.). Protein sample was
dialyzed in 0.05 M Tris-HCl buffer (pH 9) (A) and
applied to the column previously adjusted with the
same buffer. After thoroughly washing the column
(25 min) with buffer A, the proteins were
eluted by gradient elution.
Page 290
Figure 2.159.
Rechromatography using a TSK-G3000 SW gel filtration column of fractions
eluted from Superose 6 Prep Grade gel permeation column or fractions eluted
from Q Sepharose Fast Flow: (a) whole egg white; (b) ovomucin fractions (peaks 1,
2, and 3 from gel permeation); (c) proteins contained in peak 4 from gel permeation;
(d) lysozyme fraction (peak 5 from gel permeation); (e) ovotransferrin fraction
(peak A from the anion -exchange column); (f) ovalbumin fractions
(peak B or C from the anion-exchange column).
Page 291
and 1% 2-mercaptoethanol (total protein) for a few hours. After extraction the samples were
centrifuged, and the supernatants were acidifed with acetic acid (total protein samples were placed in
boiling water for 5-10 min before separation with CE). For the separation of gliadin fractions, the
capillary (72 cm total length, 50 cm separation length, 50 µm I.D.) was coated with the Micro-Coat
reagent that reverses the charge on the capillary inner surface. The capillary was equilibrated with an
aluminum lactate buffer consisting of 50 mg aluminum lactate and 650 µL of 90% lactic acid filled to
30 mL end volume (pH 2.25). Samples were injected hydrodynamically and separated at -10.000 V (7
µA). Detection wavelength was 200 nm. The CE analysis of SDS -soluble proteins was carried out using
the ProSort SDS -Protein analysis kit (capillary of 42 cm total length, 22 cm separation length, 55 µm
I.D.). The reagent for the separation of SDS -soluble proteins was modified by adding 500 µL of 75%
glycerol and 500 µL of methanol to 9 mL of the reagent. The detection wavelength was 215 nm. Both
capillaries were thermostatted at 30°C.
The electrophoregrams of gliadins of 10 wheat and 2 durum wheat cultivars are presented in Figure
2.160. It was found that the relative standard deviation of the reproducibility of the migration times is
better than 1%. The migration time of τ-gliadins were the shortest, followed by β- and α-gliadins. The
electrophoregrams of the 12 cultivars show marked differences, indicating that this method can help the
identification of these cultivars. The electrophoregrams of the SDS -soluble proteins of the same
cultivars are shown in Figure 2.161. In this instance, the relative standard deviation was <3%. The data
indicated that not only the CE separation of gliadin fractions, but also that of the high molecular mass
glutenin subunit, can promote the identification of the cultivars included in the experiments. It was
further stated that both the separation efficiency and the reproducibility of CE methods is superior to the
traditional SDS-PAGE method.
CE has also been used for the separation and quantitative determination of the major proteins in chicken
egg and cow's milk [ 361]. Egg white was dissolved in a buffer containing 75 mM sodium chloride, 20
mM potassium phosphate, and 0.01% sodium azide (pH 7.0) at a ratio of 1:50. Egg yolk was dispersed
in the same buffer at 1:40 dilution and was delipidated. Dimethylformamide (DMF) was used as a
neutral marker added to the samples at a concentration of 0.01% (v/v). Untreated fused-silica column
(25 cm total length, 18.5 cm to the detector window, 20 µm I.D.) was used for the separation of food
proteins. Detection was carried out at 200 nm. The capillary was thermostatted at room temperature
(about 23°C). Samples were introduced by pressure injection (15-30 s). The voltage depended on the
character of the proteins to be separated (10-20 kV). Borate buffers (pH 10.0) were used for the
separations. After each separation, the capillary was washed with 1.0 M NaOH and water (12 s each,
with pressure washing at 15 psi). Thereafter, the capillary was reequilibrated with the buffer (60 s with
pressure washing at 15 psi). The
Page 292
Figure 2.160.
Capillary electrophoresis of gliadins from single seeds of various wheat
varieties (YR: Yecoro rojo; S66: Scout 66; A: Anza; CS: Chinese Spring; E: Eagle;
C: Cheyenne; N.1: Newton Biotype 1; N.2: Newton Biotype 2; RR68.1:
Red River Biotype 1; RR68.2: Red River 68 Biotype 2; R: Rugby; V: Vic).
The capillary (72 cm total length, 50 cm separation length, and 50 µm I.D.)
was coated with the Micro -Coat reagent and equilibrated with an aluminium
lactate buffer (pH 2.3). Samples were injected hydrodynamically and
separated at -10.000 Volts (7 µA). The large negative peaks (at 11-12 min)
mark where uncharged molecules would migrate (electroendosmotic flow marker).
Page 293
Figure 2.161.
Size-based capillary electrophoresis for distinguishing complements and varietal
differences of high-molecular-mass glutenin subunits. The ProSort matrix was
modified by the addition of 75% glycerol and methanol (5% total volume of each).
Samples were injected into the capillary (42 cm total length, 22 cm separation length,
and 55 µm I.D.) electrokinetically and separated at -12.000 Volts. Electrophoregrams
are displayed for the cultivars: (YR: Yecoro rojo; S66, Scout 66: A: Anza;
CS: Chinese Spring; E: Eagle; C: Cheyenne; N.1: Newton Biotype 1; N.2: Newton
Biotype 2; RR68.1: Red River Biotype 1; RR68.2: Red River 68 Biotype 2: R: Rugby;
V: Vic). The high-molecular-mass glutenin subunits are identified
by the appropriate numbers on the patterns.
Page 294
protein fractions were identified by using authentic standards. The separation of egg white proteins
considerably depended on the experimental conditions, as demonstrated in Figure 2.162. The separation
was the best at the highest concentration of the borate buffer. Under these conditions, lysozyme was
also separated from the neutral marker. The separation of egg yolk proteins also depended on the
experimental conditions. The electropherograms of egg yolk proteins using buffers of various
concentrations are compiled in Figure 2.163. The slight differences between the separation patterns of
A and B was attributed to the lipid fraction present in the egg yolk. The best separation was achieved by
using the highest buffer concentration. The electropherograms of milk proteins are shown in Figure
2.164. Only a partial separation of fresh milk proteins was achieved under these experimental
conditions because β-lactoglobulin B and A migrated together with α-casein. Because of the thermal
denaturation of proteins during spray-drying, the electrophoregram of proteins of spray-dried milk
powder differ considerably from that of fresh milk proteins. It has been proposed that this difference
can be used for the detection of the adulteration of fresh milk with milk powder.
Page 295
Figure 2.162.
Electrophoregrams of egg white proteins.
(A) Conditions untreated fused-silica capillary,
25 cm × 20 µm I.D.; applied potential, 20 kV/17 µA,
80 mM borate at pH 10.0. Peaks: 1 = neutral marker
(DMF); 2 = conalbumin and globulins; 3 = ovomucoid
and ovalbumin. (B) Conditions: untreated
fused-silica capillary, 25 cm × 20 µm I.D.; applied
potential, 10 kV/17 µA, 200 mM borate at pH 10.0.
Peaks: 1 = neutral marker and lysozyme; 2 = conalbumin;
3 = globulins; 4 = ovomucoid and ovalbumin.
(C) Conditions: untreated fused-silica capillary, 25
cm × 20 µm I.D.; applied potential, 12 kV/17 µA, buffer,
300 mM borate at pH 10.0; peaks: 1 = lysozyme;
2 = neutral marker; 3 = conalbumin; 4 = globulins;
5, 6 = ovomucoid and ovalbumin.
The Netherlands.)
Page 296
Figure 2.163.
Electrophoregrams of egg yolk proteins. (A) Conditions
untreated fused-silica capillary, 25 cm × 20 µm I.D.;
applied potential, 20 kV/17 µA, 80 mM borate at pH 10.0.
(B) The lipid portion in yolk was extracted with SeroClear;
conditions as for (A). (C) Conditions: untreated fused-silica
capillary, 25 cm × 20 µm I.D.; applied potential, 10 kV/21 µA,
buffer 300 mM borate at pH 10.0. Peaks: 1 = neutral marker;
2 = yolk immunoglobulins; 3 = yolk lipoproteins.
Page 297
Figure 2.164.
(A) Electropherogram of the fresh nonfat milk proteins;
conditions untreated fused-silica capillary, 25 cm × 20 µm I.D.;
applied potential, 10 kV/17 µA, 250 mM borate at pH 10.0. Peaks:
1 = neutral marker; 2 = β-casein; 3 = α-lactalbumin; 4 = α-casein;
5 = β-lactoglobulin. (B) Electropherogram of the powder milk
proteins; conditions as for A. Peaks: 1 = neutral marker;
2 = denaturated β-casein aggregates; 3 = denaturated α-casein aggregates.
Page 299
Chapter 3—
Microcomponents in Foods
3.1—
Vitamins and Provitamins
Vitamins play a considerable role in maintaining human health. A wide variety of analytical methods
have been developed for their determination in various foods and food products. Because the volatility
of vitamins is negligible, mainly liquid chromatographic methods such as HPLC and micellar
electrokinetic capillary chromatography have been used for the separation and quantitation of vitamins.
Ascorbic acid influences the quality of foods, i.e., increases the shelf life of soft drinks and juices,
exerts many physiological and biochemical functions, and shows antioxidant capacity. Because of its
considerable importance, many chromatographic methods were applied for the analysis of ascorbic acid
in different matrices [ 362]. A new HPLC method was developed for the determination of ascorbic acid
in soft drinks and apple juice using tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy) 32+) electroluminescence
[363]. Samples were filtered and diluted before HPLC analysis, and no other pretreatment was applied.
Separation of ascorbic acid was carried out on an octadecylsilica column (250 × 4.6 I.D.), using
aqueous 15 mM NaH 2 PO4-K2HPO4 buffer (pH 6.5). The flow rate was 0.3 mL/min. The eluted ascorbic
acid was mixed with 0.5 mM (Ru(bpy)3 2+) and oxidized at +1.5 V (vs. Ag/AgCl). The interference of
citric acid was avoided by adding 10 -4M tetrabutylammoniumtetrafluoroborate (Bu 4NBF4) to the eluent.
Typical chromatograms for the detection of ascorbic acid in soft drinks and apple juice are shown in
Figure 3.1. The chromatograms clearly show that the method allows the separation of ascorbic acid
from the other components in soft drinks and apple juice, the reproducibility of the luminescence
intensity being 1.5% at 50 µM ascorbic acid. The mean and standard deviation of the ascorbic acid
content of samples and the result of spike test are compiled in Table 3.1. It was
Page 300
Figure 3.1.
Typical chromatogram for detection of softdrinks and apple
juice: (A) ascorbic acid; (a) vitamin C—drink A;
(b) vitamin C —drink B; (c) vitamin C —drink C; (d) apple juice
(100%). Eluent: 0.015 M NaH2PO 4 - K 2HPO4,
+ 10-4M tetrabutylammoniumtetrafluoroborate (pH 6.5).
Flow rate, 0.3 mL/min; carrier solution, 0.5 m Ru(bpy) 32+, 0.2
M NaAc; flow rate, 0.3 mL/min; potential, +1.5 V (vs. Ag/AgCl),
glassy carbon working electrode 22.1 mm2; separation column,
5C 18 AR (250 × 4.6 mm); sample volume, 20 µL, temperature, 25 °C.
The Japan Society for Analytic Chemistry, Tokyo.
Page 301
TABLE 3.1. Results of Ascorbic Acid Determination in Beverages (n = 5).

Spike Test
Nominal Found Added Found Recovery
Beverage Value (µg/mL) (µg/mL) µg/mL µg/mL (%)
Vitamin-C drink A 952 903.5 ± 4.8 1000 1847 94 ± 5.2
Vitamin-C drink B 2860 2799 ± 4.3 2000 4859 102 ± 3.5
Vitamin-C drink C Not labeled 86.3 ± 2.2 100 184.5 98 ± 2.6
Apple juice (100%) Not labeled 59.4 ± 3.8 100 162.8 105 ± 4.5
Reprinted from Reference [ 363] with permission from the Japan Society for Analytical Chemistry, Tokyo.
established that the method is relatively simple, the detection limit is 10 pmol ascorbic acid, and the
calibration curve is linear between 0.06-80 nmol. Owing to the sensitivity and selectivity, the method
was proposed for the quantitation of ascorbic acid in soft drinks and apple juice, and it can probably be
employed for the analysis of the ascorbic acid content of other beverages.
The quantity of ascorbic acid in infant formulas was determined by both HPLC and voltammetry, and
the results were compared [ 364]. Samples were prepared for HPLC analysis by suspending 5 g of infant
formula in 100 mL of 1% w/v metaphosphoric acid. The homogenate was centrifuged for 10 min (2000
g); then the supernatant was filtered, and 1 mL of filtrate was diluted to 5 mL with the same
metaphosphoric acid solution and injected to the octadecylsilica column. Eluent was aqueous potassium
phosphate buffer (0.1 M, pH 3.5). Flow rate was 1 mL/min and the detection wavelength 245 nm. The
quantity of ascorbic acid in infant formulas determined by HPLC and voltammetry is listed in Table
3.2. The differences between the ascorbic acid contents determined by HPLC and voltammetry were not
significant at the significance level of 95%. The corrected sample variance of the HPLC method was
2.9 and 1.3 for milk-based and soya-based infant formulas, respectively. It was established that both
methods can be successfully used for the determination of the ascorbic acid content in infant formulas;
however, the voltammetric method is easier to carry out.
Since the vitamin activity of isoascorbic acid (IAA) is markedly lower than that of ascorbic acid (AA),
the simultaneous determination of AA and IAA is of considerable importance. Various HPLC methods
were developed for the separation and quantitation of AA and IAA in processed meat [365], animal
issue [366], wine and beer [ 367], orange juice [ 368], candies and soft drinks [ 369], infiltrated apple and
potato tissue [ 370], and other food products [ 371]. RP-HPLC has been employed for the separation and
quantitative determination of AA acid, dehydroascorbic (DHAA), IAA, and dehydroisoascorbic acid
(DHIAA) in food and animal tissue [372]. Samples were homogenized with
Page 302
TABLE 3.2. Comparison of Ascorbic Acid Contents (milligrams per 100 g) Obtained by the
Voltammetric Method and HPLC Method on Milk-Based Infant Formula and Soya-Based
Infant Formula.
Milk-Based Infant Formula Soya -Based Infant Formula
Sample Voltammetric HPLC Voltammetric HPLC

Number* Method Method Method Method
1 50.93 45.97 43.09 39.72
2 50.98 46.86 44.38 42.61
3 51.22 45.01 44.32 41.71
4 49.56 48.03 42.20 43.36
5 48.55 46.45 50.00 43.43
6 49.05 45.35 45.37 41.99
7 43.56 49.61 45.00 43.09
8 52.76 48.03 48.84 42.40
9 47.73 47.82 49.89 42.61
10 48.52 44.94 39.61 44.94
X ± SD 49.28 ±2.53 46.81± 1.55 45.27 ± 3.41 45.59 ± 1.35
d 2.48 2.68
s 3.51 3.83
D/(s/n 1/2 ) 2.23 2.22
tn-1 2.26 2.26
* Mean of differences; s, standard deviation of the differences; n, number of replicates.

cold 17% metaphosphoric acid, centrifuged, and filtered. For the determination of AA and IAA, 500 µL
of supernatant were mixed with 115 µL of 45% K2HPO4 buffer (pH 9.8) to reach the final pH of 7.1.
The mixture was thermostated at 25°C for 30 min, and then diluted to 2 mL final volume with 0.85%
metaphosphoric acid, and an aliquot of 100 µL was diluted to 10 mL with the eluent. For the
determination of DHAA and DHIAA, 1% homocysteine was added to the 45% K2 HPO4 buffer, the rest
of the procedure being the same as in the case of AA and IAA separation. Two PLRP-S columns (250 ×
4.6 mm I.D.; particle size 5 µm) connected in series were used for the analysis. The eluent was 20 mM
monobasic sodium phosphate monohydrate containing 0.17% metaphosphoric acid (final pH 2.2).
Columns were held at 22°C and 5°C for food and tissue analysis, respectively, and the corresponding
flow rates were 0.7 mL/min and 0.6 mL/min. An amperometric detector was used at +0.7 V. AA and
IAA were well separated even from animal issues, as demonstrated in Figure 3.2. The concentrations of
AA, IAA, DHAA, and DHIAA in animal tissues and various food products are presented in Tables 3.3
and 3.4 respectively. Adrenal gland accumulated the highest level AA, IAA, DHAA, and DHIAA
whereas their concentration in plasma was
Page 303
Figure 3.2.
Chromatograms of tissues from rats fed a diet containing 10% of isoascorbic
acid (IAA). In panel A is liver, in panel B is spleen, and in panel
C is adrenal. In the latter tissue, there was an unidentified
peak with a retention time of 17.5 min.
markedly lower. The HPLC method described above is very sensitive (0.5 ng/20 µL injection) and can
be employed for the analysis of these acids in many different types of foods and food products.
Not only HPLC but also CE has been frequently applied for the analysis of ascorbic acid. Thus, CE was
employed for the analysis of ascorbic acid in fruit beverages [ 373] and juices [ 374]. A rapid MECC
method was developed for the determination of total L-ascorbic acid in fruits and vegetables, and the
results were compared with those obtained by HPLC [ 375]. Samples for MECC analysis were prepared
by homogenizing 40 g of fruits or vegetables with 160 mL of 3% metaphosphoric acid, diluted to 200
mL with 3% metaphosphoric acid, and filtered. An aliquot of 5 mL was mixed with 0.5 mL of D-
erythorbic acid (600 µg/mL) as internal standard and diluted to 10 mL with aqueous D, L-dithiothreitol
(0.2%) to prevent oxidation. Solution was passed through an octadecylsilica cartridge equilibrated with
methanol and water. The first 5 mL were discarded, and the rest was used for MECC. Samples for
HPLC were prepared in a similar manner, but no internal standard was
Page 304
TABLE 3.3. Ascorbic Acid, Dehydroascorbic Acid, Isoascorbic Acid, and Dehydroisoascorbic Acid Content of Plasma and
Selected Tissues of Rats Fed a Diet Containing a 10% of Isoascorbic acid. *
Tissue Total AA AA DHAA Total IAA IAA DHIAA
Plasma 9.09 6.48 2.61 53.52 40.38 13.14
±1.65 ±1.27 ±0.52 ±14.69 ±12.11 ±6.89

Spleen 376.6 257.8 118.6 456.6 299.4 157.2
±56.8 ±32.0 ±31.1 ±116.4 ±89.9 ±30.7

Liver 114.8 101.6 13.2 123.2 102.2 21.2
±33.8 ±38.0 ±7.7 42.8 ±31.7 ±15.2

Adrenal 1794.4 1505.2 289.0 1983.6 1465.4 518.2
±418.9 ±530.0 ±156.6 ±433.0 ±558.8 ±512.9

* The values are mean ± SD and are expressed in µg/g of tissue or mL of plasma. There were five rats in the group.
added and the samples were not passed through the C18 cartridge. MECC analysis was carried out in
fused-silica capillary columns (65 cm × 75 µm I.D. for fruits and 75 cm × 75 µm I.D. for vegetables,
effective lengths being 40 and 50 cm, respectively). The buffer was 0.05 M sodium deoxycholate, 0.01
M sodium borate, and 0.01 M potassium dihydrogen orthophosphate. Samples were loaded under
vacuum. Separation conditions were +25 kV, 28°C and 254 nm detection wavelength. Capillaries were
washed with running buffer for 2 min between analyses. HPLC separation was carried out on an
octadecylsil-
TABLE 3.4. The Content of Ascorbic Acid and Isoascorbic Acid and Their Corresponding Oxidized Forms in
Selected Foods.*
Food Total AA AA DHAA Total IAA IAA DHIAA
Skimmed 1% milk ND — — ND — —
Condensed milk 1.47 0.79 0.68 ND — —
Evaporated milk (2%) 19.60 16.80 2.80 ND — —
Cherry cheesecake 10.55 4.40 6.15 ND — —
Diet product,
chocolate 22.75 10.34 12.41 ND — —
Iced tea ND — — 10.43 7.34 3.09
Cooked ham tr — — 72.16 68.99 3.17
Sausage 1.44 0.63 0.81 12.81 7.63 5.18
Chicken spread 5.13 4.24 0.89 42.13 29.64 12.67
Fruit preservative 8433.0 7767.0 667.0 ND — —

* Values are expressed as mg/100 g or mL and represent the average of duplicate determinations; individual
values did not differ by more than 3% from the average value; tr: trace.
Page 305
Figure 3.3.
Separation of (1) L-ascorbic acid and (2) D -erythorbic acid for (A)
a standard solution, (B) blueberry extract before C 18 Sep-Pak cartridge
clean-up, and (C) blueberry extract after C 18 Sep-Pak cartridge
clean-up. The x-axis gives the migration time.
ica column (eluent: 0.2% aqueous orthophosphoric acid; flow rate: 1 mL/min; detection wavelength:
254 nm). The electropherograms in Figure 3.3. clearly show that the prepurification of samples on a C 18
cartridge considerably improve the separation efficiency of MECC. The peaks of L-ascorbic acid and
the D-erythorbic acid internal standard are well separated (Figure 3.3.). The L-ascorbic acid contents of
fruits determined by both MECC and HPLC are given in Table 3.5. The data show that the quantity of
L-ascorbic acid determined by both methods is highly similar, and the values of relative standard
deviations are low for both methods. These results indicate that both methods can be successfully used
for the separation and quantitative analysis of L-ascorbic acid in fruits. The results of the analysis of
vegetables are listed in Table 3.6. Also, in the case of vegetables, both methods produced similar results
and very low coefficients of variation, suggesting that they can also be
Page 306
TABLE 3.5. Comparison of the Levels of Total L-Ascorbic Acid Determined by MECC
and HPLC for Fruits Extracted with 3% Metaphosphoric Acid and Stabilized with
Aqueous 0.2% D, L-Dithiothreitol and Showing the Repeatability Data (% CV) between
Instruments.
Total L-Ascorbic Acid (mg/100 g) % CV
Sample MECC HPLC MECC HPLC

Orange 31 31 1.2 0.7
Lemon 34 34 0.6 2.1
Lime 34 33 1.6 1.2
Grapefruit 34 34 0.5 1.2
Blueberry 8 7 0.6 0.8
Strawberry 48 46 1.3 0.6
Raspberry 22 21 1.1 1.2
Kiwi 82 80 1.1 1.1
Kiwi (peeled) 101 99 0.8 0.7
Tomato 14 14 0.6 2.1
Pineapple 14 14 1.3 1.6
Honeydew melon 21 19 0.9 0.9
Watermelon 6 5 0.9 3.1
Rock melon 38 36 1.2 0.3
Mango 18 18 1.8 2.2
Apple 5 5 3.4 0
Green grape 2 2 3.1 2.7
Peach 8 6 3.3 1.6
Dark plum 3 1 2.1 0
Apricot 8 10 2.0 2.2
used for the determination of total L-ascorbic acid in vegetables. It was established that MECC is faster
and more cost-effective than HPLC, and the precision and repeatability of the MECC method are the
same. These findings indicate that MECC can be a valuable substitute for HPLC in the analysis of total
L-ascorbic acid in fruits and vegetables.
A similar MECC method was developed for the simultaneous determination of total ascorbic acid and
D-erythorbic acid in beers, wines, and fruit drinks, and the results were compared with those obtained
with HPLC [ 376]. Because it was established that buffers containing sodium dodecylsulfate or
cetyltrimethylammonium bromide were not adequate for some products, samples were prepared for
MECC with sodium deoxycholate: 2 mL of wine and 5 mL degassed beer were mixed with 0.5 mL of
nicotinic acid internal standard (1 mg/mL) and diluted to 10 mL with D, L-dithiothreitol (2 mg/mL)
reducing agent. For fruit juices, 0.5 mL was mixed with 1 mL of D-erythorbic acid (300 µg/mL) and
diluted to 10 mL with dithiothreitol. Apple juice was diluted
Page 307
TABLE 3.6. Comparison of the Levels of Total L-Ascorbic Acid Determined by MECC and
HPLC for Vegetables Extracted with 3% Metaphosphoric Acid and Stabilized with Aqueous
0.2% D, L-Dithiothreitol and Showing the Repeatability Data (% CV) between Instruments.
Total L-Ascorbic Acid (mg/100 g) % CV
Sample MECC HPLC MECC HPLC

Asparagus 3 3 2.2 2.2
Green bean 11 12 1.1 1.2
Beetroot 3 3 2.0 1.7
Broccoli 100 96 1.1 0.5
Brussel sprouts 130 122 1.3 0.2
Green capsicum 89 87 1.7 0.2
Carrot 10 10 1.4 1.7
Cauliflower 70 71 1.0 0.2
Red chili 200 188 1.1 0
Cucumber 9 8 1.3 0.4
Lettuce 3 3 1.3 1.6
Onion 8 8 0.9 1.4
Potato (peeled) 19 21 1.0 1.7
Potato 15 15 1.6 0.5
Sweet corn 2 1 2.1 3.2
Zucchini 23 20 0.8 0.5
Snow peas 52 47 0.8 0.5
Radish 21 20 0.7 0.8
with 3% metaphosphoric acid. Samples for HPLC were prepared by diluting wines and fruit juices
fivefold with 3% metaphosphoric acid and further diluted twice with D, L-dithiothreitol. Degassed
beers were diluted twofold with D, L-dithiothreitol. MECC separation was carried out on a fused-silica
capillary column (75 cm × 75 µm I.D., effective length 50 cm). Operating voltage was +25 kV, column
temperature 28°C, and detection wavelength 254 nm. Samples were loaded under vacuum. Buffer was
prepared by dissolving 2.16 g sodium deoxycholate in 100 mL of 0.02 M potassium orthophosphate and
0.02 M sodium tetraborate 1:1 v/v (pH 8.6). HPLC separation was carried out on a C18 column using
0.2% (v/v) aqueous orthophosphoric acid (flow rate: 1.0 mL/min, detection wavelength 254 nm).
Electropherograms of beer, wine, and fruit juice samples are shown in Figure 3.4. The method
effectively separated L-ascorbic acid, D-erythorbic acid, and the internal standard nicotinic acid. The
quantity of acids in beer, wine, and fruit juice samples determined by both MECC and HPLC are given
in Table 3.7. Except in apple juice, the amount of L-ascorbic acid determined by both methods were
similar, and the
Page 308
Figure 3.4.
Separation of L-ascorbic acid (peak 1), D-erythorbic acid (peak 2),
and nicotinic acid (peak 3) in (A) beer, (B) wine, (C) fruit juice, and (D)
a standard solution. A phosphate-tetraborate buffer modified
with sodium deoxycholate was used.
Publications, A Division of Preston Industries, Inc.)
relative standard deviation was low for both methods. These findings indicate that MECC can be
employed for the quantitative determination of L-ascorbic acid in wine, beer, and fruit juices with the
same accuracy as with HPLC. Various HPLC techniques have also been used for the determination of
other vitamins in foods. Thus, HPLC with fluorometric detection was extensively employed for the
quantitative determination of vitamin B6 in raw and fried chicken [ 377] and in other food products
[378,379].
Page 309
TABLE 3.7. Comparison of MECC and HPLC Data for Samples Using a Sodium
Deoxycholate Modified Buffer.
MECC HPLC
L-Ascorbic Acid % CV* L-Ascorbic Acid
Beer samples**
Ale 53 3.8 59
Light A 57 0.8 56
Lager A 14 1.8 17
Light B ND ND
Light C 1 2
Lager B 16 20
Lager C ND ND
Draught A 41 45
Draught B 14 16
Wine samples **
White burgundy 123 1.9 117
R. Riesling A 95 1.6 91
R. Riesling B 92 0.7 97
R. Riesling C 62 65
Chardonnay 78 77
Chablis 30 34
Fruit juice samples**

Orange fruit drink 22 1.5 22
Tropical juice 100% 47 0.8 45
Pine/orange fruit drink 30 1.5 29
Orange juice A 69 63
Tropical fruit drink ND ND
Orange juice B 33 30
Apple juice 39 47
Apple juice *** 45 47

* The coefficient of variation (% CV) was obtained from seven replicate injections.
**The following levels of ascorbic acid were found in the samples: beer and wine,
milligrams per liter; fruit juice, milligrams per 100 mL.
*** This sample was diluted with 3% metaphosphoric acid before dilution with D, L-
dithiothreitol (2 mg/mL).
Reprinted from Reference [ 376] with permission from Preston Publicaitons, a Division
of Preston Industries, Inc.
A collaborative study involving 12 participants was carried out on the determination of vitamin B6 in
foods by HPLC [ 380]. Ground samples (2.5 g if the vitamin B6 content was higher than 2.0 µg/g; 5 g if
the vitamin content was lower) were mixed with 25 mL of 0.05 M sodium acetate, 2.5 mL of 1 M
glyoxilic acid, 400 µL of ferrous sulfate solution (36.56 g of ferrous sulfate dissolved in 10 mL of 0.05
M sodium acetate), and 20 mg of acid phosphatase. The mixture was continuously shaken overnight at
37°C. After incubation, the
Page 310
mixture was cooled and diluted to 50 mL with distilled water, filtered, or centrifuged. A 5-mL aliquot
was mixed with 4.5 mL of 0.1 M sodium borohydride and shaken; then 0.5 mL of glacial acid was
added. The solution was filtered and used for HPLC. Separation was carried out on an octylsilica
column (250 mm × 5 mm I.D., particle size 5 µm) connected to an octadecylsilica guard column (4 mm
× 4 mm I.D., particle size 10 µm). Isocratic eluent was acetonitrile-0.05 M potassium dihydrogen
phosphate (4:96 v/v) containing 0.5.10.3 M sodium heptane sulfonate or 0.3.10.-3 M sodium octane
sulfonate; pH was adjusted to 2.5 with orthophosphoric acid. The flow rate was 1 mL/min, and the
excitation and emission wavelengths were 290 nm and 395 nm, respectively. The vitamin B6 content of
foodstuffs was also measured by the microbiological method. The results of the collaborative study are
compiled in Table 3.8. The relative standard deviation considerably decreased when the concentration
of vitamin B6 increased in the foodstuffs. The results of HPLC analysis were similar to those obtained
with the microbiological method. The statistical results indicate that this rapid and simple HPLC
method can replace the microbiological method for the determination of vitamin B6 in foods for
nutritional purposes.
HPLC was also successfully employed for the separation and quantitative determination of vitamin B1
[ 381], B2 [ 382], and their mixture [ 383] in various foodstuffs. The various isomers of vitamin B12
(cyanocobalamins) present in fresh milk, pasteurized milk, sterilized milk, and milk reconstituted from
TABLE 3.8. Summary Statistics * for Collaborative Data on LC Determination of Vitamin B6 (µg/g of pyridoxol)
in Various Foodstuffs.
Tube-
Baby Cereal Feeding Chocolate Cereal Powdered
Material Food Biscuit B Yeast Solution Powder A Milk
n 12 12 12 12 12 12 12 12
n' 11 10 11 12 11 11 12 11
N 32 29 31 36 33 33 35 33
X 0.6 1.4 2.2 5.3 5.5 6.7 15.0 32.8
X' 1.0 1.9 2.4 5.4 5.3 6.0 16.1 35.5
Sr 0.1 0.2 0.2 0.4 0.2 0.3 1.0 0.9
RSDr 18 13 10 8 4 4 6 3
SR 0.2 0.5 0.7 1.4 0.7 0.8 1.8 4.3
RSDR 30 35 30 26 13 12 12 13
*Symbols used: n = number of participants; n' = number of participants retained; N = number ofresults; X (µg/g
pyridoxol) = material mean measured by HPLC; X' (µg/g pyridoxol) = materialmean measured by the
microbiological method; Sr = standard deviation of repeatability;
RSDr = relative standard deviation of repeatability; S R = standard deviation of reproducibility;
RSDR = relative standard deviation of reproducibility.
Page 311
milk powder were also separated by an RP-HPLC method [ 384]. Cyanocobalamins were extracted from
the samples by mixing 25 mL of milk with 2-4 mL of 0.1 M HCl (pH 4.6). The mixtures were held at
120°C for 10 min and then filtered. The pH of the filtrate was adjusted to 5.5 with 0.1 M sodium
hydroxide and diluted to 50 mL with distilled water. Cyanocobalamins were preconcentrated on an
octadecylsilica cartridge equilibrated with 2 mL of acetonitrile and washed with 6 mL of distilled water.
The filtrate was passed through the cartridge, and the cartridge was washed with 12 mL of water.
Cyanocobalamins were eluted with 8 mL of acetonitrile:water (1:1 v/v) and separated on an octylsilica
column. Gradient elution began with an acetonitrile:aqueous ammonium phosphate solution (pH 3.0;
5:95) increasing the acetonitrile concentration to 30 vol% in 16 min. The concentration of vitamin B12
isomers was determined by radioassay. The concentration of cyanocobalamins and the standard
deviation of the measurement are given in Table 3.9. The recovery of the HPLC-radioassay method
varied considerably according to the type of cyanocobalamin and the milk product, being the lowest for
cyanocoblamin from pasteurized milk (28.3%).
Many efforts have been devoted to the development of an HPLC method for the determination of the
concentration of folates in different foodstuffs such as milk and dairy products [ 385], fruits and
vegetables [ 386], and meat products [ 387]. The HPLC method was compared with the radioprotein-
binding method for the determination of folates in milk and blood samples [ 388]. Milk samples were
prepared by adding 1% sodium ascorbate, heating in boiling water for 15 min, and then cooling. Three
milliliters of the supernatant were mixed with 0.4 mL of human plasma conjugase and 0.1 M phosphate
buffer (pH 6.0) containing 0.5% sodium ascorbate and then incubated at 37°C for 1 h in dark. After
centrifugation, the samples were passed through a strong anionexchange column equilibrated with one
column volume of methanol and water. The column was washed with two column volumes of water,
and the folates were removed with 1 mL of a 10% solution of sodium chloride
TABLE 3.9. Cobalamin Concentrations (nmol/L) in Milk and Some Milk Products.
Hydroxo- Cyano- Adenosyl- Methyl -
Milk cobalamin cobalamin cobalamin cobalamin
Fresh 0.35 ± 0.05 bf 0.20 ± 0.04 dh 1.08 ± 0.13 ae 0.31 ± 0.04 ag
Pasteurized 0.24 ± 0.05 ch 0.72 ± 0.07 ae 0.45 ± 0.02 bf 0.29 ± 0.007abg
Sterilized 0.17 ± 0.02 dh 0.59 ± 0.04 be 0.36 ± 0.08 cf 0.30 ± 0.007ag
Reconstituted 0.60 ± 0.04 ae 0.40 ± 0.04 bf 0.31 ± 0.04 cdg 0.26 ± 0.007bh
The values shown are the mean ± standard deviation of six repetitions. The different
letters indicate significant (p < 0.01) differences in columns (a –d) and in rows (e –h).
Reprinted from Reference [ 384] with permission from Lavoiser Abonnements.

Page 312
containing 1% sodium ascorbate. Folates were separated on a 30 × 4.0 mm I.D. guard column and on a
250 × 4.0 mm I.D. analytical column (both filled with octadecylsilica; particle size 5 µm). Columns
were thermostatted at 27°C, and the eluent was 8% acetonitrile in acetic acid (pH 2.3). Folates were
detected by their fluorescence, the excitation and emisson wavelengths being 310 nm and 352 nm,
respectively. Typical chromatograms of nonconjugated and conjugated folates are shown in Figure 3.5.
Tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-CH3 THF) were well separated from each other
and from the impurities present in the milk samples; 5-methyltetrahydrofolate (5-CHOTHF) was not
detected in the samples. The average recovery of 5-CH3 THF, THF, and 5-CHOTHF was 106%, 70%,
and 0%, respectively. The detector response was linear for each folate (THF = 1.4-28.7 pmol; 5-
CH3THF = 0.08-35.8 pmol; 5-CHOTHF = 2.9-116.1 pmol). The results obtained by the HPLC and the
radioprotein-binding assay (RPBA) are compiled in Table 3.10. It was concluded from the experiments
that the HPLC method is rapid and sensitive and the linearity and precision are satisfactory; therefore,
the method can be employed for the separation and quantitative determination of folates in milk.
Water-insoluble vitamins have also been frequently analyzed with HPLC. Thus, a rapid procedure was
developed for the HPLC measurement of the concentration of β-carotene in milk [ 389]. Carotenes were
extracted from 6 mL of
Figure 3.5.
(a) Representative HPLC chromatogram of nonconjugated milk
folates. (b) Representative HPLC chromatogram of conjugated
milk folates, diluted 10 times compared to the milk sample shown
in (a) THF: tetrahydrofolate; 5-CH 3 THF: 5-methyltetrahydrofolate.
Page 313
milk by mixing it with 3 mL of 2-propanol and by extracting the mixture twice with 4.5 mL of hexane.
Phase separation was facilitated by centrifugation. The pooled extracts were washed with 3 mL of 0.47
M sodium sulfate and evaporated under nitrogen to 2 mL. Saponification was carried out by dissolving
50-100 mg of lipids in 1 mL of 95% ethanol containing 12.5 mg/mL pyrogallol as antioxidant. One
milliliter of 60% KOH in water was added, and the sample was incubated at 30°C for 30 min. Then 1
mL of water was added, and the mixture was extracted with 2 mL of hexane; the organic phase was
dried under nitrogen and redissolved in 300 µL hecane:dichloromethane (95:5 v/v). The triglyceride
content was controlled by TLC [silica plates; eluent hexane:diethyl ether (7:3 v/v); detection 10%
phosphomolybdenic acid in diethyl ether:ethanol (1:1 v/v)]. Separation was carried out on two
ChromSep ChromSpher PAH columns (100 × 3 mm I.D.) connected in series. The isocratic mobile
phase was acetonitrile:methanol:dichloromethane (80:14:6 v/v). Flow rate was 0.7 mL/min, and the
detection wavelength was 450 nm. The chromatograms demonstrate that β-carotene is well separated
from α-carotene and from the other components present in the sample when the sample was subjected
to saponification (Figure 3.6). The method showed good validation parameters: linearity range 0.67-4
µg/mL, the highest concentration corresponding to about 24 µg/g milk fat; limit of quantitation 30
ng/mL; repeatability of the whole method 5.3%; recovery 100 ± 6%. It has been stated that the HPLC
method employs small solvent volumes and mild saponification conditions and is rapid and easy to
carry out. It was further assumed that the method probably can be used for the determination of β-
carotene content in other dairy products.
Since the vitamin A activities of retinol isomers differ considerably, a microbore column method was
developed for their separation [ 390]. Samples
TABLE 3.10. Comparison between HPLC and RPBA, Analyzing Folates in Low-Fat Milk. The RPBA
Estimates Have Been Calculated Using the PGA (kit) Calibrant and the In-house 5 -CH 3THF and PGA
Calibrants.
HPLC RPBA
Sample n 5-CH 3THF THF PGAkit PGA 5-CH 3THF
Low-fat milk* 10 22.6 ± 1.8** 2.6 ± 8.8** 79.6 ± 8.8** 74.0 ± 8.2** 51.0 ± 5.6**
(ng/mL) 10 45.6 ± 4.6*** nd *** 47.7 ± 9.8*** 44.3 ± 9.1*** 30.6 ± 6.3***
* 0.5% fat.
** Before conjugase treatment.
*** After conjugase treatment
Mean ± SD.
PGA - pteroylmonoglutamic acid.
Page 314
Figure 3.6. (A) Separation of (1) lutein, (2) α-, and (3) β-carotene
on two ChromSep ChromSpher PAH glass columns (100 × 3 mm I.D.,
particle size 5 µm, Chromapack, Netherland) connected in series.
The mobile phase was acetonitrile: methanol:dichloromethane
(80:14:6, v/v) at a flow rate of 0.7 mL/min. The detector was set at 450 nm.
(B, C) Comparison between chromatograms of (3) β-carotene
for a (B) saponified sample and a nonsaponified sample.
permission from Elsevier Science—NL, Sara Burgerhartstraat
Page 315
(1.0-10.0 g) were homogenized, and 30 mL of water were added to the solid samples. Saponification
was carried out by mixing 40 mL of the homogenized sample with 12 mL of 60% aqueous KOH, 80
mL of absolute ethanol, 0.5 mL of 1% ethanolic tert-butylhydroxytoluene, and 0.5 g of AA to prevent
oxidation. Samples were stirred at room temperature for 16 h. After saponification, the solute was
diluted to 250 mL with water and ethanol to obtain a final water:ethanol ratio of 1:1 v/v. An aliquot of
20 mL was added onto a Kiselguhr cartridge and was extracted with 50 mL of light petroleum after 20
min. The eluate was evaporated and redissolved in 2-50 mL of isooctane, depending on the
concentration of vitamin A in the original sample. Geometrical retinol isomers were separated on a
guard (7 × 2 mm I.D.), and an analytical column (100 × 2 mm I.D.), both filled with silica gel (particle
size 3 µm). Eluents were n-hexane containing low concentrations of 1-octanol. Because the maximum
absorption wavelength of retinol isomers showed marked differences, a diode array detector was
employed. The method successfully separated 7 retinol isomers are 11-cis-, 11, 13-di-cis-, 13-cis-, 9,
13-di-cis-, 9-cis-, 7-cis-, and all-trans-retinol—the relative retention times with respect to all-trans-
retinol being 0.510, 0.568, 0.672, 0.740, 0.877, 0.924, and 1.000, respectively. Typical chromatograms
of milk samples are shown in Figure 3.7. The concentration of various retinol isomers in liver and liver
products and milk and dairy products are given in Tables 3.11 and 3.12, respectively. The majority of
retinols was in the all-trans form in both liver and milk products; however, the concentration of other
retinols markedly depended on the type of product and on the heat treatment. It was established that
heat treatment and other processing steps result in the decrease of vitamin A activity, either by
degradation of retinol or by the formation of cis-isomers by the isomerization of all-trans-retinol.
Vitamin A palmitate and β-carotene content in margarine and in margarines mixed with the fat
substitute sucrose polyester were also measured by an HPLC method [ 391]. Samples were prepared by
dissolving 10 g of margarine in 50 mL of methylene chloride. The solution was filtered and diluted to
100 mL with methylene chloride. Acylglycerols and sucrose polyester were separated on four µStyragel
100 GPC column connected in series using methylene chloride as eluent at a flow rate of 1 mL/min.
Separation was followed with UV (313 nm) and refractive index detectors. The fractions containing
sucrose polyester and vitamins were evaporated before further HPLC analysis. Sucrose polyester was
measured on an octadecylsilica column (250 × 4.6 mm I.D.), the eluent being a gradient of methylene
chloride, acetonitrile, and 2-propanol; the flow rate changed between 1.0 mL/min and 1.5 mL/min.
Solutes were detected with an evaporative light scattering detector (pressure 2.0 bar, temperature 40°C).
Vitamins were separated on the same column with acetonitrile:methylene chloride:methanol (700:300:2
v/v/v mobile phase). The flow rate was 1.0 mL/min and the detection wavelength 313 nm and 436 nm
for vitamin A
Page 316
Figure 3.7.
Chromatograms obtained from extracts of the unsaponifiable matter of (a)
untreated and (b) microwave -heated UHT milk. Stationary phase,
3-µm silica gel; column, 100 × 2 mm I.D.; mobile phase, 0.25% 1-octanol in
n-hexane; flow rate, 0.4 mL/min; detection wavelength, 325 nm. Peaks:
1 = 11-cis-; 2 = 11, 13-di-cis-; 3 = 13-cis-; 4 = 9, 13-di-cis-;
5 = 9-cis-; 6 = all-trans-retinol.
and β-carotene, respectively. The gel permeation chromatogram of a sucrose polyester-margarine

mixture is shown in Figure 3.8. It was established that the presaparation of sucrose polyester and
acylglycerols is necessary for the interference-free determination of both SPE and vitamins. The
quantity of solutes determined by this combined method is compiled in Table 3.13. It was concluded
from the data that this combined method is suitable for the simultaneous determination of SPE, vitamin
A palmitate, and β-carotene in margarine.
Page 317
TABLE 3.11. Isomer Distribution in Liver and Liver-Containing Products (%) and Influence of Thermal
Treatment.
All-trans-
Food Sample 11-cis- 11, 13-di -cis- 13-cis- 9, 13-di-cis- 9-cis- 7-cis- retinol
Beef liver (raw) ND ND 15.3 1.8 5.5 ND 77.4
Beef liver (after

conventional
heating) ND ND 16.1 3.3 8.4 ND 72.2
Beef liver (after
microwave heating) ND ND 15.7 3.4 8.4 ND 72.5
Sheep liver (raw) tr tr 9.3 1.6 5.5 ND 83.6
Pig liver (raw) tr 2.1 4.6 0.5 2.7 ND 90.1
Infant food
(manufacturer 1) 4.7 ND 23.3 6.0 9.0 tr 57.0
Infant food
(manufacturer 2) 13.2 ND 3.9 tr 2.7 tr 80.2
Calf liver sausage 1.2 ND 8.7 1.3 3.2 tr 85.6
TABLE 3.12. Retinol Isomer Distribution in Milk and Dairy Products (%) and Influence of Thermal Treatment.
All-trans-
Food Sample 11-cis- 11, 13-di-cis- 13-cis- 9, 13-di-cis- 9-cis- 7-cis- retinol
Pasteurized milk
(untreated) ND ND 4.9 ND ND ND 95.1
Pasteurized milk (after
conventional heating) tr tr 9.8 ND 5.4 ND 84.4
Pasteurized milk (after
microwave heating) tr tr 6.8 ND 6.4 ND 86.6
UHT milk (untreated) tr tr 6.8 ND 3.6 ND 89.6
UHT milk (after
conventional heating) tr tr 12.3 2.5 5.3 ND 79.9
UHT milk (after
microwave heating) tr tr 14.6 3.2 7.5 ND 74.7
Pasteurized milk
(fermented) 9.8 tr 9.0 0.8 3.1 tr 77.3
Sourcream 7.3 tr 3.7 tr 2.3 ND 86.7
UHT = ultra heat treatment
Page 318
Figure 3.8.
Gel permeation chromatogram of the extract of sucrose polyester-margarine
blend eluted with a mobile phase of methylene chloride. The solid trace is UV
detection at 313 nm for vitamins, and the broken trace is RI detection for sucrose
polyester. Horizontal lines on the chromatograms indicate the collection
points for the sucrose polyester and vitamins.
TABLE 3.13. Analysis of Vitamin A Palmitate, β-carotene, and Sucrose Polyester from Sucrose Polyester-Margarine Blends.
Sample
5% Sucrose 10% Sucrose 20% Sucrose

Control Polyester Polyester Polyester
Vitamin A (IU/kg) 25906 23763 25313 25509 25006 24943 25942 26715
β-carotene (IU/kg) 11446 10461 11873 11489 11491 10940 11572 11333
Total (IU/kg) 37352 34224 37186 36999 36496 35883 37514 38044
Sucrose Polyester found — — 5.22 5.09 10.10 10.10 21.80 21.80
(g/100g)
Sucrose Polyester added 0 0 5.27 4.99 10.10 10.10 20.70 20.60
(g/100g)
Percent Sucrose Polyester — — 99.1 102 100 100 105 106
recovered:
Note: The values found for vitamins were corrected for the Sucrose Polyester that was added.
Page 320
HPLC methods for the determination of more than one vitamin in various foodstuffs have also been
developed [ 392]. The HPLC methods employed for the measurement of the concentration of various
vitamins in milk and dairy products have been recently reviewed [ 393]. Water-insoluble vitamins were
separated and quantitatively determined in margarine by using a silica column [ 394]. The aqueous phase
of margarines was separated, and the fat phase was dissolved in n-hexane and used for HPLC analysis.
Because of their different retention characteristics, the mixture of water-insoluble vitamins can be
separated only with a gradient elution. Separation was carried out on a silica column (150 × 4.6 mm
I.D., particle size 5 µm). Some chromatograms are shown in Figure 3.9. It was established that similar
methods using silica column and gradient elution are suitable for the determination of the lipid-soluble
vitamins originally present or added to margarines.
A directly coupled sample treatment-HPLC method was developed for the on-line automatic
determination of liposoluble vitamins in milk [ 395]. The
Figure 3.9.
Determination of vitamins A in margarines from different countries. A = Portugal;
B = Finland; C = Ireland; D = Germany; E = England; F = England; MAB = internal
standard; VAA = vitamin A acetate; VAPr = vitamin A propionate;
VAPa = vitamin A palmitate.
(Reprinted from Reference [ 394] with permission from the publisher
Page 321
sample preparation set-up is shown in Figure 3.10. The hydrolysis reagent consisted of 50 mL of
ethanol, 15 mL of 60% aqueous NaOH solution, and 5 mL of 10% AA. The washing solution and the
elution solvent were water:methanol (6:4) and methanol, respectively. HPLC separation was carried out
on an octadecylsilica precolumn (15 × 3.2 mm I.D., partical size 7 µm) and analytical column (220 ×
4.6 mm I.D., particle size 5 µm). The mobile phase was methanol:2.5 mM acetic acid-sodium acetate
(99:1 v/v). Both
Figure 3.10.
Schematic diagram of the on-line hydrolysis and elution with
HPLC system (P: peristaltic pump; R: hydrolysis reagent; S:
sample; Ac: 2.5 M acetic acid; W: washing; E: elution;
C: C 18 cartridge; L: 100 µL loop).
Page 322
electrochemical (+1300 mV) and UV detectors (280 nm) were used for the detection of vitamins.
Owing to the different absorption wavelength of vitamins, the use of an electrochemical detector was
found to be more advantageous than that of the UV detector. Vitamins A, D3, and E were well separated
under these chromatographic conditions, as demonstrated in Figure 3.11. The detection limits for
vitamins A, D3, and E were 3.49.10 -8, 1.77.10-6 and 3.11.10-7M (0.10 ng, 6.8 ng, and 1.34 ng injected),
respectively. The precision of the method was 3.8%, 5.0%, and 3.7% for vitamins A, D3, and E,
respectively; the coefficient of correlation between the amount of vitamins and the detector signal was
over 0.999 in each instance. The concentration of vitamins determined by the method and quoted by the
supplier are presented in Table 3.14. The recoveries varied from 88-105%, and the day-to-day precision
of the method was vitamin A, 3.9%; vitamin D3 , 6.8%; and vitamin E, 5.4%, showing the good
reproducibility of the method. The concentration of vitamin A
Figure 3.11.
Chromatogram obtained after application of the
proposed method to a sample of milk powder.
(Reprinted from Reference [ 395], copyright ©
1995. With kind permission from Elsevier
Page 324
3.2—
Miscellaneous Bioactive Microcomponents
The use of various methods for the separation of phenol derivatives with marked biological activity is
an important area of chromatographic research. The phenolic compounds in linseed or flax (Linum
usitatissum) were separated with a combined liquid chromatographic-thin-layer chromatographic
method [ 396]. These compounds show mild antioxidant effects. The scheme for the separation of
flaxseed phenolics is shown in Figure 3.12. Defatted flaxseed was extracted with 80 vol% ethanol (1:5
w/v) at 65-70°C for 1 h; then it was evaporated to dryness and resuspended in butanol:water (1:1 v/v).
After overnight incubation at room temperature, the butanol layer was separated and evaporated again
to dryness. One gram of solid residue was dissolved in 8 mL of methanol and applied to a Sephadex
LH-20 column (16/100). Phenolic
Figure 3.12.
Scheme for chromatographic separation of flaxseed phenolics.
Page 325
compounds were eluted with methanol and collected in fractions of 8 mL, and their absorption was
measured at 280 nm. After the color reactions for sugars and phenols, the absorption was also
determined at 490 nm and 726 nm, respectively. Eluates were pooled in four fractions, the methanol
was removed, and the weight of the residue was determined. The first two fractions were separately
redissolved in 2 mL of methanol and separated on an octylsilica column (310 × 25 mm I.D., particle
size 40-63 µm) using methanol-water mixtures as eluents containing 15%, 60%, 80%, and 100%
methanol. The last two fractions were also redissolved in 2 mL of methanol and applied to a silica
column (450 × 10 mm I.D., 70-230 mesh). The eluent was chloroform:methanol:water (65:35:10 v/v/v).
Each fraction was further separated with silica TLC using the same eluent as silica column
chromatography. Spots were detected with an aqueous ferric chloride-potassium ferricyanide solution.
The various chromatographic profiles of flaxseed phenolic are shown in Figures 3.13-3.15. The data
clearly show that the flaxseed phenolics cannot be well separated on an LH-20 column, and the
fractions collected probably contain more than one phenolic compound. The further column
chromatographic separations also resulted in different fractions, showing the complexity of the solute to
be analyzed. Even the fractions separated by octylsilica and silica columns contained various phenolic
compounds that can be separated with
Figure 3.13.
Sephadex LH-20 chromatography of
flaxseed phenolics.
Page 326
Figure 3.14.
RP-8 chromatography of fractions (A) I and (B) II from Sephadex LH-20 column.
Page 327
Figure 3.15.
Silica gel chromatography of fractions (A) III and
(B) IV from Sephadex LH-20 column.
TLC (Table 3.16). It was concluded from the data that flaxseed contains a high number of phenolic
compounds. The UV spectra suggests but does not prove that lignans and their derivatives may be the
most important phenolic compounds present in flaxseed.
Eugenol (4-allyl-2-metoxyphenol) exhibits antioxidant, antiseptic, and antimicrobial activity; therefore,

its determination is of considerable importance. A simple RP -HPLC method was developed for the
separation and quantitative determination of eugenol in cloves [ 397]. Eugenol was extracted from dried
cloves by mixing 50 g of ground cloves (preferably 250-µm particle size) with 300 mL of 95% ethanol
and stirring the mixture at 50°C for 2 h. The extract was filtered and used for RP-HPLC analysis
without any other prepurification step. Separation of eugenol from the coextracted compounds was
performed on a 300 × 3.9 mm I.D. octadecylsilica column using a methanol:water (80:20 v/v) mixture
as the mobile phase. The flow rate was 0.7 mL/min, and the detection wavelength was set to 280 nm.
The identification of eugenol was carried out in a GC-MS system: 25 m × 0.22 mm I.D. capillary
column coated with methyl 5% phenyl silicone (film thickness 0.33 µm). The column temperature was
programmed from 80°C to 250°C at 7°C/min and held for 15 min. Helium was used as the carrier gas.
Eugenol was well separated from the other coextracted compounds in the RP -HPLC method, as
demonstrated in Figure 3.16. The comparison of the retention time of pure standard with the
chromatogram of the clove extract indicated that the peak eluted with the retention time of 5.54 min
contains the eugenol fraction. The gas chromatogram of the extract is shown in Figure 3.17. Mass
spectrometry indicated that the peaks eluted at the retention times of 13.95, 15.27, 15.85,
Page 328
TABLE 3.16. R f Values of Phenolic Compounds in Different Fractions of Flaxseed Extract.
Fractions Rf
I-1 0.37, 0.33, 0.0
I-2 0.84, 0.75, 0.65, 0.48, 0.42, 0.26, 0.0
I-3 0.75, 0.26
I-4a 0.96, 0.75, 0.51
I-4b 0.88, 0.84, 0.30
I-4c 0.88
II-1 0.71, 0.33, 0.26
II-2a 0.84, 0.75, 0.65, 0.59, 0.48, 0.42
II-2b 0.96, 0.75, 0.65, 0.59, 0.48
II-3a 0.88
II-3b 0.74, 0.57
III-1 0.78, 0.75, 0.63, 0.55, 0.42
III-2 0.75, 0.55, 0.42
III-3 0.36, 0.23
III-4 0.23
IV-1 0.78
IV-2 0.78, 0.75, 0.55, 0.42
IV-3 0.75, 0.43, 0.29
IV-4 0.24
and 16.84 min correspond to eugenol, caryophyllene, β-humulene and eugenol acetate, respectively. It
was established that this rapid and simple RP-HPLC procedure can be used for routine analysis and
quality control. The advantage of the method is that it avoids the risk of thermal modification of
eugenol, which can occur during GC analysis.
Phenolics in wine have also been extensively investigated generally using an octadecylsilica column
[398,399], and the performance of HPLC and TLC in the quantitative analysis of phenolic compounds in
wine has been compared [ 400]. Phenolics were separated from red wine by applying 25 mL of wine to a
column (259 mm × 17 mm I.D.) filled with 5 g of Polyamid-6 (Serva) equilibrated with acidified water
(pH 2.5). The column was washed with 100 mL of distilled water, and phenolics were eluted with 150
mL of 30% methanol (pH 2.5). The eluate was concentrated in vacuum to 5 mL and filled up to 10 mL
with 15% methanol. Pigments were separated on a similar column (100 mm × 10 mm I.D.) by applying
10 mL of wine. The column was washed with 25 mL of distilled water, and the pigments were removed
with a 25-mL mixture of n-butanol:acetic:water (4:1:5 v/v/v). The eluate was concentrated to 3-4 mL
and filled up to 5 mL with methanol. HPLC separation was performed on an octadecylsilica column
(125 mm × 4 mm I.D.) using water:methanol (85:15 v/v), and pH was adjusted to 2.5 with HClO3. The
flow rate was 1 mL/min, and
Page 329
Figure 3.16.
High-performance liquid chromatogram of alcohol
clove extract detected at 280 nm. Column, µBondapak
C18 (300 × 3.9 mm I.D.), mobile phase, methanol:water
(80:20); flow rate, 0.7 mL/min; injection amount, 20 µL.
permission from AOCS Press.)
Figure 3.17.
Gas chromatogram of ethanol clove extract. Eugenol
peak at retention time of 13.95 min.
(Reprinted from Reference [ 397 ] with permission
from AOCS Press.)
Page 330
phenolics were detected at 280 nm. TLC of phenolic was carried out on the cellulose layer with
water:methanol (80:20) eluent (pH 2.5). Spots were visualized by immersing the plate in 5% FeCl3
solution. TLC of pigments was performed on silica layers with n-butanol:butylacetate:formic acid:water
(13:5:2:3 v/v) eluent. Spots of phenolic and pigments were evaluated with a chromatogram
densitometer at 700 nm and 540 nm, respectively. The results of HPLC and TLC analyses are compiled
in Table 3.17.
In the majority of cases, the results of HPLC and TLC are commensurate, indicating that both methods
can be used for the analysis of phenolics in wines. A TLC densitogram of color pigments is shown in
Figure 3.18. Pigments are well separated on the TLC plate, proving that this TLC method can be used
for the separation and quantitative determination of the color pigments in wines.
Nonvolatile phenolic acids were separated by HPLC using traditional [ 401] and microbore columns
[402]. Samples were prepared for HPLC by evaporating the alcohol under vacuum below 30°C, and 1
mL or 1.5 mL of phosphate
TABLE 3.17. Comparison of HPLC and TLC Analysis of 11 Wine Probes (mg/mL).
No. of Gallic Protoca- Cate- Chlorogenic Epica-
Wine Method Acid techuic Acid chin Acid catechin
1 HPLC 13.0 2.0 6.0 12.5 ND
TLC 12.0 ND 4.0 12.0 6.0
2 HPLC 30.0 6.0 95.0 8.0 95.0
TLC 26.0 6.0 98.4 10.0 100.0
3 HPLC 20.0 6.5 69.0 10.0 50.0
TLC 24.0 5.2 72.0 10.0 50.0
4 HPLC 17.0 6.0 40.0 10.0 32.0
TLC 16.0 7.2 38.0 12.0 30.0
5 HPLC 21.0 6.0 50.0 8.0 50.0
TLC 24.0 6.0 50.0 8.0 48.0
6 HPLC 12.0 5.0 37.5 7.5 40.0
TLC 12.0 4.8 38.4 8.0 42.0
7 HPLC 42.5 8.0 35.0 10.0 45.0
TLC 48.0 10.0 38.0 8.8 44.0
8 HPLC 17.0 6.0 49.0 10.0 60.0
TLC 16.0 6.0 52.0 12.0 58.0
9 HPLC 41.0 6.0 49.0 7.5 32.0
TLC 18.0 6.0 48.0 7.2 36.0
10 HPLC 34.5 6.5 105.0 7.0 *

TLC 32.0 6.0 100.0 6.0 *
11 HPLC 32.0 10.0 52.5 7.0 23.0
TLC 30.0 12.0 24.0 6.0 20.9
Reprinted from Reference [ 400] with permission, copyright © 1995, Wissenschaftliche

Page 331
buffer (pH 8.0) was added to 1 mL or 2 mL of red or white wine, respectively. The mixture was
extracted with 3 × 1.5 mL of diethyl ether; the collected organic phase was dried with anhydrous
sodium sulfate, evaporated to dryness under nitrogen in the dark at maximum 30°C, and dissolved 50
µL of methanol (alkaline extract). The extracted wine was acidified with 6 M HCl to pH 1.0 and
extracted with 3 × 1.5 mL of diethyl ether saturated with polyethylene glycol 20,000. After drying the
organic extract with anhydrous sodium sulfate, it was divided into two parts and evaporated under
nitrogen. One part was dissolved in 50 µL of methanol (acid extract). The second part was dissolved in
2 mL of water and applied to a strong anion-exchange cartridge. The cartridge was washed with water,
and the solutes were eluted with 14 mL of phosphate buffer (pH 2.4)-sodium perchlorate solution. The
eluate was passed through an octadecyl silica cartridge and removed from the cartridge with 2 mL of
methanol. The eluate was dried and evaporated as described above, and the rest was dissolved in 50 µL
of methanol. The scheme of the sample preparation is shown in Figure 3.19. Depsides were hydrolyzed
by adding 2 mL of 2 M NaOH to 2 mL of wine after the evaporation of alcohol. The mixture was
Figure 3.18.
TLC densitogram of color pigments of a Bordeaux 1990 wine.
(Reprinted from Reference [ 400] with permission, copyright © 1995,
Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany.)
Page 332
Figure 3.19.
Sample treatment for extraction of nonvolatile compounds from wine.
held at room temperature for 48 h in the dark and then acidified to pH 1.0 and treated as the acidic
extract. Separations were performed on octadecylsilic columns (250 × 1.1 mm I.D., 500 mm × 1.1 mm
I.D., particle size 5 µm). The mobile phase consisted of (a) methanol:aqueous phosphate buffer (pH 2.4)
(95:5) and (b) phosphate buffer (pH 2.4):distilled water (5:95). The gradient was 0 min, 100% B; 67
min, 56% A; 70 min, 100% A. Phenolic compounds were detected at 280 nm, and the flow rate was 40
µL/min.
The chromatograms of various extracts are shown in Figure 3.20. The chromatograms clearly show that
the method of sample preparation and extraction exert a considerable effect on the quality of HPLC
separation and on the composition of the phenolic compounds. The recoveries of the acid extract were
between 75.6% and 88.6% with RSDs of 3.5% and 11.5%, repectively. The concentration of phenolic
acids in alcoholic beverages are given in Table 3.18. The data prove that the HPLC method is suitable
for the separation and quantitative determination of phenolic acids in alcoholic beverages as red and
white wines, vermouth, martini, and beer. They further indicate that the composition of phenolic acids
considerably depends on the type and character of alcoholic beverages.
Page 333
Figure 3.20.
Chromatogram of Corvo red wine (A = acid extract; B = anionic extract;
C = acid extraction after alkaline hydrolysis) detected at 280 nm. Peaks:
1 = gallic acid; 2 = 3,4-dihydroxybenzoic acid; 3 = vanillic acid; 4 = caffeic
acid; 5 = syringic acid; 6 = cis-p-coumaric acid; 7 = trans-p-coumaric acid;
8 = ferulic acid; 9 = depside of caffeic acid; 10 = depside of p-coumaric acid
or ferulic acid. Column ODS2 (250 × 1.1 mm I.D.); flow rate, 40 µL/min; mobile
phase, A = methanol:aqueous phosphate buffer (pH 2.4) (95:5), B = phosphate
buffer (pH 2.4):distilled water (5:95), with the gradient 0 min,
100% B; 67 min, 56% A; 70 min, 100% A.
Page 334
TABLE 3.18. Phenolic Acid Concentrations (µg/L) in Wine,

Vermouth, and Beer.
Salice Vermouth
Corvo Chianti Salentino Cerveteri Porto (red Verdicchio
Acid* (red) (red) (red) (red) (white) Martini) (white)
1 455.1 409.1 217.7 290.5 182.8 — —
2 72.8 120.7 79.2 52.5 81.2 45.2 15.0
3 33.7 24.6 17.0 22.4 42.4 29.5 7.3
4 83.7 47.3 91.7 86.7 27.7 — 14.4
5 70.4 24.8 40.0 23.1 81.5 52.6 3.8
6 2.3 5.1 5.0 2.9 5.4 3.3 3.8
7 26.3 10.3 15.6 19.8 14.1 5.1 13.2
8 11.4 9.8 7.2 12.5 7.4 4.6 7.2
Vernaccia Frascati Pinot

San Gimignano Superiore Robola Grigio Cerveteri Corvo
Acid* (white) (white) (white) (white) (white) (white)
1 — — — — — —
2 51.3 24.2 64.9 12.6 14.1 61.0
3 7.9 5.4 4.6 4.2 4.5 5.1
4 41.0 23.4 38.8 27.6 49.6 15.1
5 9.9 3.2 — 4.3 3.6 4.4
6 5.4 4.3 12.1 3.4 4.1 3.4
7 13.3 14.3 60.8 11.6 4.4 7.1
8 10.8 13.3 21.6 11.7 3.2 9.3
Acid* Moretti Beer Ciro (white) Fontana Candida (white)
1 — — —
2 — 10.9 21.0
3 16.1 5.7 4.6
4 4.2 24.3 12.7
5 2.2 3.0 3.1
6 — 3.9 4.6
7 17.1 2.6 8.9
8 40.4 6.7 8.3

* 1 = gallic acid; 2 = 3,4-dihydroxybenzoic acid; 3 = vanillic acid; 4 = syringic acid; 5 = caffeic acid;
6 = cis-p-coumaric acid; 7 = trans-p-coumaric acid; 8 = ferulic acid.
The highly efficient CE methods have also been employed for the analysis of phenolic compounds
[403]. CE was successfully applied for the separation of phenolic compounds in honey [ 404] and Spanish
red wines [ 405]. Noncolored phenolic compounds (phenolic acids and others) were determined in
Portuguese red wine by HPLC and CE, and the results of the methods were compared [ 406]. Phenolics
were extracted from 5 mL of wine fermented from Vitis
Page 335
vinifera var. Roriz grapes with 5 mL of diethyl ether. The organic phase was separated, evaporated to
dryness, and redissolved in 1 mL of aqueous 0.6% perchloric acid containing 2 vol% methanol (HPLC)
or in 0.5 mL of methanol (CE). HPLC separation was performed on a C18 column (100 mm × 2.1 mm
I.D., particle size 5 µm) at 40°C. Eluents were aqueous 0.6% perchloric acid and methanol. Methanol
concentration varied from 2% to 62% in 60 min. CE analysis was performed on a fused-silica capillary
(50 cm × 75 µm I.D.) at 30°C. Running buffer was 0.1 M sodium borate (pH 9.5). Separations were
carried out at 20 kV (average current 97 µA). Hydrodynamic injection was applied for 2 s. Detection
wavelength was 280 nm for both methods. The separation profiles of HPLC and CE analyses are shown
in Figure 3.21. The elution order of phenolics was highly different in HPLC and in CE. Because HPLC
detected more fractions than CE, it was assumed that, for this special application, HPLC is superior to
CE. The concentrations of phenolics determined by both methods are listed in Table 3.19. The
concentration values determined by both methods showed reasonable agreement for most of the
phenolic compounds; however, in some cases, marked deviations were found. It was concluded that for
this special application HPLC is more accurate, sensitive, and precise; therefore, its use for the
quantitative analysis of phenolics in red wines is proposed.
Many other phenolic and diphenolic compounds and their derivatives were investigated by different
chromatographic techniques. Thus, isoflavones and their conjugates were determined in soybean
products using either a diode array [ 407] or a mass spectrometric detector [ 408], and phytoestrogens
were separated on an octadecylsilica column with fluorometric [409] or diode array detection [ 410]. An
isotope dilution GC-MS method was developed for the determination of isoflavonoids, coumestrol, and
lignans in samples of plant-derived foods [ 411].
Samples for analysis were prepared by mixing 50 mg of milled or freeze-dried sample with 500 µL of
distilled water and allowed to interact overnight. Fatty samples were extracted before with 2 × 2.5 mL
of hexane. For the enzymatic hydrolysis, 1 mL of Helix pomatia juice was treated with 1% charcoal in 9
mL of 0.66 M acetate buffer (pH 4.1) overnight with shaking. The next day the mixture was
centrifuged, and 2500 Fishman unit of Helix pomatia supernatant and 1 mL of 0.3 M acetate buffer (pH
4.1) were added to the samples. Labile metabolites were protected by adding 2.5 mg of AA. Deuterated
internal standards of formononetin, biochanin A, daidzein, genistein, and coumestrol were also added to
the samples before hydrolysis. The mixtures were incubated at 60°C for 2 h. One milliliter of
hydrolysate was mixed with 5 mL of cold diethyl ether twice. The combined organic phases were
evaporated to dryness under nitrogen and redissolved in 0.5 mL of methanol. The water phase was
subjected to acid hydrolysis: 500 µL of 6 M HCl was added to reach the final concentration of 2 M, and
the hydrolysis was performed at
Page 336
Figure 3.21.
Separation of red wine phenolic compounds with (A) HPLC and with
(B) CE. 1 = Gallic acid; 2 = 3,4-dihydroxybenzoic acid;
3 = 4-hydroxyphenethyl alcohol; 4 = cis-caffeoyl tartaric acid;
5 = (+)-catechin; 6 = vanillic acid; 7 = trans-coumaroyl tartaric acid;
8 = caffeic acid; 9 = syringic acid; 10 = p-coumaric acid;
11 = (-)-epicatechin; 12 = myricetin; 13 = quercetin;
14 = kaempferol; 15 = isorhamnetin.
100°C for 2.5 h. The hydrolysate was cooled at room temperature, and the pH was adjusted to 3-5 with
10 M NaOH (300 µL), and the deuterated standards were added. The samples were evaporated to
dryness and redissolved in 0.5 mL of methanol. Neutral lipids were removed by passing through the
sample on a column filled with the free base form of DEAE -Sephadex (15 × 5 mm I.D.). Neutral lipids
were eluted with methanol and the diphenols with 5 mL
Page 337
TABLE 3.19. Comparison of Quantitative Determination of Phenolic Compounds in

Red Wine by HPLC and CE.
Peak No. Compound HPLC CE
1 Gallic acid 25.0 ± 0.62 29.0 ± 2.84
2 3,4-dihydroxybenzoic acid 6.7 ± 0.27 9.0 ± 0.75
3 4-hydroxyphenethyl alcohol 105.8 ± 5.81 104.0 ± 1.8.10 -8
4 Cis-caffeoyl tartaric acid 2.1 ± 0.51 NQ
5 (+) -catechin 1.7 ± 0.02 6.4 ± 9.8.10 -8
6 Vanillic acid 8.0 ± 1.60 8.2 ± 0.41
7 trans-coumaroyl tartaric acid NQ 0.5 ± 3.2.10 -8
8 Caffeic acid 5.4 ± 0.03 26.0 ± 2.78
9 Syringic acid 8.4 ± 0.27 4.0 ± 0.50
10 p-coumaric acid 4.0 ± 0.30 3.2 ± 0.29
11 (–)-epicatechin 7.7 ± 0.06 21.2 ± 1.0.10 -8
12 Myricetin 27.0 ± 1.02 ND
13 Quercetin 51.4 ± 1.25 ND
14 Kaempferol 0.4 ± 0.02 ND
15 Isorhamnetin 3.2 ± 0.90 ND
Results are mean values of two determinations, expressed as mg/L ± SD. NQ = Not
quantified.
of 0.1 M acetic acid in methanol. Organic acids and chromogens were removed by passing through the
eluate of the free base form DEAE-Sephadex on a column filled with DEAE-Sephadex in the acetate
form (30 × 5 mm I.D.). The column was eluted first with 4 mL of methanol and then 7 mL of 0.2 M
acetic acid in methanol. The first fraction contained anhydrosecoisolariciresinol, secoisolariciresinol,
and matairesinol, while formononetin, daidzein, genistein, and coumestrol were eluted in the second
fraction. Fractions were evaporated to dryness and silylated by adding 100 µL of reagent
[pyridine:hexamethyldisilazane:trimethylchlorosilane (9:3:1 v/v/v)]. Silylation was performed at room
temperature (30 min or overnight), and then the samples were dried again and redissolved in n-hexane.
The flow diagram of the sample preparation is shown in Figure 3.22. GC separation was carried out on
a vitreous silica column (12.5 m × 0.2 mm I.D., film thickness 0.25 µm). Helium was used as the carrier
gas; the column temperature was 100°C for 1 min and then increased to 280°C at 30°C/min. The
retention times, recovery, and between-assay imprecision (CV%) are given in Table 3.20. The high
recovery values and the relatively low imprecision indicate the reliability and precision of this isotope
dilution (ID)/GC/MS method. The sensitivity of the method was high, the detection limit being 2-3
µg/100 g foodstuff. The results of the analyses of food samples are presented in Table 3.21. It was
proposed that this specific, quantitative, reproducible, and sensitive method can be
Page 338
Figure 3.22.
Flow diagram of the method for the determination of lignans and isoflavonoids in food.
Page 339
TABLE 3.20. The Relative Retention Times in I.D./GC/MS/SIM, Recovery and Between-Assay
Precision for Isoflavonoids, Coumestrol, and Lignans and Their Deuterated Homologs.
Compound Relative
Phytoestrogen Time Retention
Fraction (min) Time Recovery (%) CV (%)
Formononetin 0.802 85.2 10.2
D 3-Formononetin 0.800
Bichanin A 0.841 96.2 13.2
D 4-Biochanin A 0.839
Daidzein 1.003 109.2 4.5
D 4-Daidzein* 8.821 1.000
Genistein tri-TMS 1.029 105.3 9.4
di-TMS 1.050
D 4-Genistein tri-TMS 1.028
di-TMS 1.049
Coumestrol 1.168 97.6
D 4-Coumestrol 1.165
Lignan fraction
ANHSEC 0.916 98.6 **
D 8-ANHSEC 0.914
SECO 0.946 98.6 ** 8.2
D 6-SECO 0.945
Matairesinol 1.001 104.6 14.6
D 6-Matairesinol 8.732 1.000

* Reference compound.
** For the sum of ANHSECo and SECO because of the conversion.
employed for the simultaneous determination of phytoestrogens and lignans in foods. Similar studies
are important in populations with marked risks of cancer and coronary heart diseases.
HPLC has also been employed for the analysis of flavonoids [412] using diode array [ 413] or
coulometric array detection [ 414] and β-cyclodextrinbonded stationary phase [ 415]. Determination of
flavonoids by HPLC has been proposed as a control method for the detection of the adulteration of
grapefruit and sour orange juice [ 416]. The prominent flavonoids were determined in orange and
grapefruit concentrates with RP-HPLC [ 417]. Samples were prepared by adding 1 mL of rhoifolin (1.8
mg/mL) internal standard to 1 mL of grapefruit concentrate. The mixture was centrifuged, and the pellet
was washed with 2 × 1 mL of methanol and centrifuged. The combined supernatant was diluted with 3
mL water and filtered. The procedure was modified for orange juices: 1 g of concentrate was mixed
with 0.4 mL of internal standard solution and with 3 mL of methanol. After mixing and heating at 55°C
for 15 min, the samples were centrifuged. The pellet was washed with 3 mL
TABLE 3.21. Quantitative Results in Selected Foods.
Food Item Formononetin Biochanin A Daidzein Genistein Coumesterol SECO
Granola candy bar, 4.1 2.2 51.9 78.3 tr 20.5
U.S.A.
9-Grain bread, U.S.A. 2.4 ND 7.6 10.5 ND 70.7
Crisp bread I, Finland 2.9 ND 13.1 5.4 tr 42.0
Crisp bread II, Finland tr ND tr tr tr 40.1
Finn crisp bread, Finland 2.5 tr 5.5 9.8 tr 27.7
Sunflower seeds 26.0 tr tr tr tr 609.5
Country rye bread, tr ND tr tr ND 1152.0
Finland
Lapacho tea 7.5 35.9 18.1 28.6 ND 2670.0
(Tecoma heptaphylla)
Flax seed 0 0 0 0 0 369900.0
Soy flour 30.2 74.4 67369 96914 0 130.4
(Soyolk flour, Spillers,
UK)
* Sum of ANHSEC and SECO.
** Values confirmed using gas chromatography.
Note. All results expressed in µg/100 g.
Page 341
of methanol:water (2:1 v/v) and treated in a similar mode. The supernatants were collected, diluted with
4 mL of water, and filtered. Separation of flavonoids was performed on a C18 column (250 × 4.6 mm
I.D., particle size 5 µm) with diode array detection. The mobile phase consisted of water:acetonitrile:2-
propanol:formic acid (158:23:19:0.2 v/v) with a flow rate of 0.6 mL/min. Flavonoids (narirutin,
hesperidin, naringin, and rhoifolin) were well separated in this HPLC system without any interference
with each other (Figure 3.23). The average recoveries were hesperidin 106%, narirutin 84%,
Figure 3.23.
Chromatograms of orange juice concentrate and
grapefruit concentrate extracts. All chromatograms
are shown with absorbances at 283 nm, whereas
peak areas used for quantitative analysis were
integrated at 283 nm for flavanone analytes and 335 nm
for rhoifolin. Chromatograms show (in order of
increasing retention) narirutin, hesperidin, naringin,
and rhoifolin (internal standard). C 18 column; mobile
phase of water:acetonitrile:2-propanol:formic
acid (158:23:19:0.2); flow rate = 0.6 mL/min.
The Netherlands.)
Page 342
and rhoifolin 90% from orange juice concentrate and naringin 100%, narirutin 84%, and rhoifolin 91%
from grapefruit juice concentrate. The concentration of flavonoids in two orange juice concentrates
were 120 and 150 mg/100 g hesperidin and 24 and 30 mg/100 g narirutin. The concentration of
narirutin and naringin in two grapefruit concentrates were 62 and 68 mg/100 g and 200 mg/100 g (for
both samples), respectively.
Because many studies indicated that resveratrol may have some protective effect against atherosclerosis
and coronary heart disease, both GC and HPLC methods were developed for its determination in grapes
[ 418] and wines [ 419,420,421]. The cis and trans isomers of resveratrol were separated with a GC-MS
method [422]. Octadecylsilica cartridges were used for SPE of resveratrol isomers from wines.
Cartridges were consecutively equilibrated with 3 mL of ethyl acetate, 3 mL of 96% (v/v) ethanol, and
2 × 3 mL of 10% v/v ethanol. One milliliter of wine was applied to the cartridge and dried for 45 min
under vacuum with nitrogen. Resveratrol was removed with 2 mL of ethyl acetate; the first 1 mL was
used for GC-MS. Separation was performed on a DB-17 capillary column (15 × 0.25 mm I.D., film
thickness 0.15 µm). Initial oven temperature was 150-160°C for 6 min, increased to 290° C at 20°
C/min, held for 2 min, raised to 305°C at 25°C/min, and held for 5 min. The retention times of cis- and
trans-veratrol were 4.3 min and 5.7 min, respectively. The recovery of the method varied from 92.2-
97.5%, and the detection limit was 10 µg/L. The precision of the method determined for two wines was
0.33 ± 0.02 (CV = 6.1%) and 4.34 ± 0.23 (CV = 5.3%) mg/L. The concentrations of cis-, trans-, and
total resveratrol in wines are shown in Figure 3.24. The investigations proved that cis-resveratrol is
present in commercial wines from every wine-producing region. The data indicate that the resveratrol
concentration of wines from different countries shows considerable differences. These differences are
probably caused by the cultivar, climate, soil composition, fungal infection and wine-making
techniques.
The resveratrol glucosides and isomers were also separated by a direct injection HPLC method [ 423].
Separation was performed with a Lichrosphere 100 CN column (250 × 4 mm I.D., particle size 5 µm)
using water:acetonitrile:methanol (90:5:5 v/v) eluent. The flow rate was 1 mL/min, and the resveratrols
were detected at 306 nm. Untreated wine was directly injected onto the column. Typical chromatograms
of untreated red wine and red wine treated with α- and β-glucosidase are shown in Figure 3.25. The
HPLC system separates the cis and trans isomers of resveratrol and its glucosides well; the coefficients
of correlation between the detector response and the concentration of solutes were between 0.983 and
0.999, showing the good linearity of the relationship. The recovery of trans- and cis-resveratrol were
105% and 100%, respectively. The detection limits were: cis-resveratrol = 0.20 µmol/L; cis-resveratrol
glucoside = 0.30 µm/L; trans-resveratrol = 0.11 µmol/L; and trans-resveratrol glucoside = 0.16 µmol/L.
The resveratrol and resveratrol glucoside
Page 343
Figure 3.24.
Concentrations of cis-resveratrol (top), trans-resveratrol (middle),
and total resveratrol (bottom) in commercial red wines from various
countries and regions as follows: (A) Canada, Ontario (n = 38);
(B) United States, California (n = 62); (C) Australia (n = 39);
(D) South America (n = 25); (E) South Africa (n = 11);
(F) United States, Oregon (n = 14); (G) France, Burgundy (n = 44);
(H) France, Bordeaux ( n = 40); (I) France, Rhône Valley (n = 40);
(J) France, Beaujolais (n = 18); (K) France, Midi (n = 17);
(L) Italy (n = 69); (M) Spain and Portugal ( n = 29);
(N) central Europe (n = 16); (O) Switzerland ( n = 6).
Columns and vertical bars represent mean and SD,respectively.
publisher and the corresponding author,
copyright © 1995, American Chemical Society.)
Page 344
Figure 3.25.
Chromatograms of red wine directly injected into the HPLC. The left-hand
trace is that of an untreated sample showing peaks A (cis-resveratrol
glucoside), B (trans-resveratrol glucoside), C (cis-resveratrol),
D (trans-resveratrol). The right-hand trace is that of the same
wine treated with β-glucosidase for 12 h, showing a disappearance
of peaks A and B and an increase in peaks C and D.
Full-scale absorbance was 0.010 with attenuation of 4.
contents of some red wines are given in Table 3.22. The concentration of resveratrol isomers and their
glucosides showed marked variations among the wines. This finding indicates that the cultivar, soil, and
weather conditions exert a considerable influence on the resveratrol concentration. Since the biological
activity of various resveratrol isomers and glucosides may be different, this method provides a useful
tool for the more complete assessment of the biological efficacy of individual red wines.
Resveratrol and other phenolic compounds in wine were determined in one run with an RP-HPLC
method [424]. Wines mixed with internal standard were directly injected on the RP-HPLC system
(analytical column: 250 mm × 4 mm I.D., guard column: 4 mm × 4 mm I.D.; both octadecylsilica, 5
µm). Mobile phase constituents were (A) acetic acid, (B) methanol, and (C) water. Initial eluent
composition was 5% A, 15% B, 80% C (flow rate 0.4 mL/min); after 5 min: 5% A, 20% B, and 75% C
(flow rate 0.5 mL/min); at 30 min: 5% A, 45% B, and 50% C (flow rate 0.5 mL/min) held for 10 min.
Typical chromatograms are shown in Figure 3.26. The method showed good validation
Page 345
TABLE 3.22. Concentrations of Resveratrol Isomers and Glucosides in Some Commercial Red Wines (µmol/L).
Glucoside Resveratrol
Vintage Country/Region cis trans Total cis trans Total
1993 Côtes du Rhone (France) 4.9 7.2 12.1 5.8 9.8 15.6
1993 Côtes du Rhone (France) 8.6 7.7 16.3 5.7 10.3 16.0
1993 Burgundy (France) 13.8 7.7 21.5 12.4 20.2 32.6
1992 Burgundy (France) 4.5 0.7 5.2 10.7 6.2 16.9
1991 Bordeaux (France) 1.7 3.2 4.9 17.5 33.4 50.9
1990 Bordeaux (France) 1.7 4.7 6.4 3.4 15.4 18.8
1991 Spain 3.6 14.6 18.2 0.1 9.2 9.3
1991 Spain 8.8 5.6 14.4 1.6 5.0 6.6
1990 Chile 0.6 2.2 2.8 0.2 3.6 3.8
1992 Chile 5.4 5.2 10.6 3.8 6.9 10.7
1992 Oregon (USA) 12.3 0.8 13.1 13.0 1.4 14.4
1992 Oregon (USA) 8.0 ND 8.0 10.9 7.3 18.2
1992 Niagara (Canada) 6.5 ND 6.5 14.9 25.4 40.3
1991 Niagara (Canada) 28.6 9.3 37.9 3.1 5.3 8.4
1992 California (USA) 0.9 2.0 2.9 0.3 2.0 2.3
1991 California (USA) 2.6 2.6 5.2 2.1 4.5 6.6
parameters (lowest coefficient of correlation of the relationship between concentration and peak area =
0.980; recoveries between 95.2% and 105.5%; RSD between 0.5% and 5.7%), proving the reliability of
the measurements. The concentration of phenolics in various wines determined by this method are listed
in Table 3.23. In agreement with other data, the composition of phenolics highly depends on both the
cultivar and the vintage. It was further established that phenolics show acceptable stability in open
bottles for a week, and their concentration decreased by 10-40% in 6 weeks.
The presence of various inositol phosphates in foods can modify the uptake of various metal ions by
forming ion complexes. Because of their considerable biological importance, HPLC methods were
developed for their determination using macroporous polymer column [ 425] or ion-exchange
chromatography [ 426,427]. Several isositol phosphates such as hexakis-, tetrakis- and four
pentakisphosphates were separated and quantitatively determined in coffee and malt by ion-exchange
HPLC [ 428]. Samples were extracted either with hot bidistilled water or with cold 2.4% HCl,
centrifuged, and purified on a Dowex (Cl - form) column and on an octadecylsilica cartridge. HPLC
separation was performed on a precolumn (20 × 4.4 mm I.D.) and on an analytical column (120 × 4.6
mm I.D.), both filled with polystyrol-divinylbenzol-based anion exchange. Mobile phase components
were: (A) bidistilled water, (B) 80 mmol/L
Page 346
Figure 3.26.
Chromatogram of wine showing the peaks for all eight phenolics measured by
this method. (A) Absorbance monitored at 280 nm. Peak 1, catechin; peak 2,
epicatechin. (B) Absorbance monitored at 306 nm. Peak 1, trans-polydatin;
peak 2, rutin; peak 3, trans-resveratrol; peak 4, cis-polydatin;
peak 5, cis-resveratrol; peak 6, quercetin.
aqueous sodium nitrate solution (pH adjusted to 2.8 with HNO3), and (C) 41.2 mmol/L aqueous
magnesium nitrate. Initial composition was 20% A, 80% B, 0% C to 20% A, 0% B, 80% C in 50 min
and held for 10 min. The flow rate was 1 mL/min. Inositolphosphates were detected by postcolumn
derivatization using the aqueous solution of 2% HClO 4 and 0.2% Fe(III)ClO4. The flow rate was 0.5
mL/min. Reaction column was 250 × 4.6 mm I.D. Both analytical and reaction columns were
thermostatted at 30° C. Solutes were detected at 310 nm. Inositolphosphates were well separated on the
anion-exchange column, as demonstrated in Figure 3.27. Inositolphosphates are well separated
according to the number of phosphate groups, but the separation of the various isomers containing the
same number of phosphate groups cannot be achieved with this method. The validation parameters of
the method are summarized in Table 3.24. The data prove that no significant difference can be
established between the efficiency of the hot aqueous and cold HCl extraction. The validation
parameters indicate that the method is reliable and precise; therefore, it can be successfully used for the
determination of inositolphosphates in coffee and malt.
TABLE 3.23. Phenolic Composition of Representative Commercial Wines.
Wine Catechin Epicatechin trans Polidatin trans Resveratrol cis Polidatin
1991 Chianti (Italy) 34.1 22.9 2.20 1.28 1.00
1990 Chianti 39.6 22.7 1.15 1.17 1.26
1990 Chateauneuf (France) 50.3 23.2 3.59 4.68 2.03
1991 Chateauneuf (France) 55.2 23.5 2.66 3.82 1.32
1990 Medoc (France) 48.9 38.1 0.72 1.72 0.49
1990 Medoc 58.2 30.6 0.15 2.32 0.18
1989 Burgundy (France) 127.0 38.4 2.34 2.27 0.98
1989 Burgundy 136.0 50.7 2.22 1.17 1.12
1990 California Cabernet
Sauvignon 33.5 21.4 0.24 0.30 0.10
1991 California Cabernet
Sauvignon 44.4 23.1 1.86 2.23 1.52
1993 Australian Shiraz 44.2 34.9 1.45 2.62 0.19
1991 Australian Shiraz 33.9 33.7 0.72 2.26 0.32
1991 Oregon Pinot Noir 122 41.6 0.84 0.50 1.74
1991 Oregon Pinot Noir 119 38.6 0.83 0.45 2.05
1993 Beaujolais (France) 31.5 24.2 1.72 2.29 2.64
1993 Beaujolais 30.7 64.4 1.76 2.69 2.90
1988 Barolo (France) 23.6 16.7 0.98 0.31 1.17
1989 Barolo 59.0 34.3 1.46 1.04 0.79
Reprinted from Reference [ 424], copyright © 1996. With kind permission from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV
Netherlands.
Page 348
Figure 3.27.
HPLC chromatogram of inositolphosphates (IP3 = inositoltriphosphate;
IP4 = inositotetraphosphate; IP5A = 1,2,3,4,6-inositolpentaphosphate;
IP5B = 1,2,3,4,5-(or 1,2,3,5,6-)inositolpentaphosphate;
IP5C = 1,2,4,5,6-(or 2,3,4,5,6-)inositolpentaphosphate;
IP5D = 1,3,4,5,6-inositolpentaphosphate; IP6 = inositolhexaphosphate).
(Reprinted from Reference [ 428] with permission, copyright © 1993,
Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany.)
Not only anion-exchange but also octadecylsilica column were used for the separation of
inositolphosphates [429]. Lupinus albus and L. luteus, Lens culinaris var. Magda 20, Cicer arietinum
Desi and Kabuli, Vicia faba cv Alameda and cv Brocal were freeze-dried and ground to pass through a
100-mesh sieve. An aliquot of 0.5 g was extracted with 20 mL of 0.5 M HCl at room temperature for 2
h and then centrifuged. The supernatant was filtered, evaporated to dryness under vacuum (35°C),
redissolved in 15 mL of 25 mM HCl, and passed through a strong anion-exchange column. The loaded
column was washed with 10 mL and 5 mL of 25 mm HCl. Inositolphosphates were eluted with ten 1-
mL solutions of 2 M HCl. The solvent was evaporated, and the residue was dissolved in 0.5 mL of the
mobile phase. Separation was carried out on an octadecylsilica column (250 × 4.6 mm I.D., particle size
5 µm) thermostatted at 45°C. Eluent was prepared by mixing 515 mL of methanol, 485 mL of water, 8
mL of tetrabutylammonium hydroxide (40% in water), 1 mL of 5 M sulfuric acid, 0.5 mL of 91%
formic acid, and 100 µL of phytic acid hydrolysate (6 mg/mL). The pH was adjusted to 4.3 with 8 M
sulfuric acid. The flow rate was 1.2 mL/min. Solutes were detected by their refractive index. Some
chromatograms showing the separation of phytic acid
Page 349
TABLE 3.24. Recovery, Coefficient of Variation, Detection Limit and Limit of Quantitation.
Inositolphosphate
IP3 IP4 IP5 IP6
Recovery (%)
Aqueous extract 99.7 99.6 96.9 89.3
HCl extract 95.4 100.6 97.1 94.2
Coefficient of variation (%)
Malt, aqueous extract 8.4 5.3 4.8 4.5
Malt, HCl extract 5.6 5.3 3.6 5.2
Roasted coffee, aqueous extract 5.9 3.2 4.5 4.4
Roasted coffee, HCl extract 6.1 4.2 4.0 3.1
Green coffee, HCl extract nd nd nd 2.9
Detection limit (mg/kg) 34 55 41 55
Limit of quantitation (mg/kg) 62 101 72 116
Reprinted from Reference [ 428] with permission, copyright ©; 1993, Wissentschaftliche

hydrolysates are shown in Figure 3.28. The contents of inositol phosphates in different legumes are
given in Table 3.25. The content of inositol phosphates showed high variation between the species and
also within the species. The method was proposed for the determination of the effect of food processing
on the phytate content and on the production of lower inositol phosphates.
The antinutrients vicine and convicine were also determined in faba beans by HPLC [ 430]. Samples for
HPLC analysis were prepared by extracting 1.0-2.5 g of ground faba bean seeds (Vicia faba var. Dino)
twice with 0.1 M NaOH for 10 min. The mixture was centrifuged, and the pH of 15 mL of supernatant
was adjusted with 1 M HCl to 4.2. Precipitated proteins were separated by centrifugation, and the pellet
was washed with 30 mL of distilled water (pH 4.2) and filtered. The supernatant and filtrate were
combined and filled up to 50 mL with distilled water. The sample was diluted tenfold before injection.
Separation was performed on a 150 × 3.3 mm I.D. amino column with acetonitrile:water (70:30 v/v) as
the mobile phase. The flow rate was 1 mL/min, and the solutes were detected at 273 nm. Vicine and
convicine separated well from the internal standard uridine and from each other during the
chromatographic process (Figure 3.29). Highly significant linear correlations were found between the
concentration of solutes and the detector response in the concentration range of 0.01-0.05 mg/mL.
Using this method, it was established that the quantity of vicine and convicine considerably decreased
during the ripening process (vicine = 0.243 g/kg; convicine = 0.094 g/kg).
It was indicated many times that the endogenous thiol and disulphide content of barley markedly
influences its dormancy, resulting in the modification
Page 350
Figure 3.28.
RP-HPLC of a phytic acid hydrolysate. (a) L. albus; (b) L. culinaris;
(c) Ph. vulgaris var. Guerniquesa; (d) C. arietinum type Kabuli;
(e) V. faba var. Alameda; and (f) ion -pair chromatography. Samples
were injected (20 µL) onto a Spherosorb column (250 × 4.6 mm, 5 µm)
and heated at 45°C. Mobile phase was 0.12 M formic acid and 0.8%
tetrabutylammonium in 51.5% methanol. The pH was adjusted to 4.3
with sulfuric acid. Flow rate: 1.2 mL/min. Detector: Refractive index.
IP3 = inositol triphosphate; IP4 = inositol tetraphosphate;
IP5 = inositol pentaphosphate; and IP6 = inositol
hexaphosphate (phytic acid).
TABLE 3.25. Inositol Phosphate Content (% DM) of Different Legumes Samples.
Sample* IP3 IP4 IP5
Lupin
L. luteus — — 0.06 (0.008) 0.717 (0.120)
L. albus — — — 0.341 (0.028)
Lentils
L. culinaris
var. Magda 20 0.016 (0.008) 0.019 (0.006) 0.048 (0.005) 0.244(0.047)
Beans
Ph. vulgaris
var. Tolosana 0.009 (0.000) — 0.020 (0.005) 0.564 (0.137)
Ph. vulgaris
var. Guerniquesa 0.009 (0.001) — 0.019 (0.004) 0.628 (0.013)
Chickpeas
C. arietinum
var. Desi 0.027 (0.008) 0.011 (0.001) 0.068 (0.003) 0.389 (0.015)
C. aretinum
var. Kabuli 0.025 (0.009) — 0.031(0.002) 0.379 (0.009)
Fababeans
V. Faba
var. Alameda 0.016 (0.003) 0.047 (0.008) 0.212 (0.015) 0.610 (0.030)
V. Faba
var. Brocal 0.020 (0.012) 0.013 (0.003) 0.075 (0.002) 0.345 (0.056)
* Two replicate extracts of each sample and analyzed in duplicate with HPLC (mean values with their standard deviations expressed on a d
Netherlands.
Page 352
Figure 3.29.
HPLC chromatograms of (A) the mixture of standards and of (B) the
extract of faba beans: 1 = uridine, 2 = vicine, 3 = convicine.
Polish Journal of Food and Nutrition.)
of the malting process. Owing to its considerable practical importance, an HPLC method was developed
for the separation and quantitative determination of these compounds in barley [ 431]. Samples were
prepared for HPLC analysis by peeling lemmae from 100 g of barley and adding 1 mL of 1 mM N-
acetyl-cysteine and 1 mL of 0.5 mM cystamine (both dissolved in 6% sulphosalicylic acid) internal
standard, 2 mL of 40 mM Na2 EDTA, and 2 mL of 3% sulphosalicylic acid containing 20 mM of Na2
EDTA. The samples were homogenized and 0.2 g of acid washed; insoluble polyvinyl polypyrrolidone
was added, stirred, and centrifuged. Supernatant was used for further investigations. Thiols and
disulphides were separated on a column of p-hydroxymercurybenzoyl agarose beads synthesized from
Sepharose 6B beads. Extracts were added to the column 35 × 7 (or 70 × 7 mm I.D.,) and disulphides
were eluted with 5 mL of icecold water at a flow rate 0.15 mL/min. Bonded thiols were removed from
the column with 2-4 mL of 0.1 M dithiotreitol (DDT) containing 30 mM phosphate buffer, pH 8.0, and
2 mM Na2 EDTA. The column was thermostatted with the DDT solution overnight and then eluted with
5 mL of water at 0.1 mL/min. Eluate was extracted with 3 × 15 mL of ethyl acetate to remove excess
DDT. Disulphide fraction was purified by diluting the eluate to 0.5% sulphosalicylic acid concentration
and passed through two silica-benzene sulphonic acid columns (1 g support in each) equilibrated with 2
bed volumes
Page 353
Figure 3.30.
Separation by HPLC of the DTNB derivatives of thiols from an extract
of barley embryos. (Cys: cysteine; CG: cysteinyl glicine;
τ-GC, τ-glutamylcysteine; GSH: glutathione; N -ac: N-acetyl cysteine;
TNB: thionitrobenzoate ion; DTNB: unreacted
5, 5'-dithiobis(2-nitrobenzoic acid).
methanol and 5 bed volumes of 0.5% sulphosalicylic acid. Columns were separately washed with 5 mL
of 0.5% sulphosalicylic acid and joined again and disulphides were eluted with 25 mL of 2 M
dipotassium hydrogen phosphate containing 2 mM Na2 EDTA and 20% methanol. Eluted disulphides
were reduced at room temperature with 1 mL of 3 mM DDT for 1 h. Derivatization of thiols was
performed with 5, 5'-dithiobis(2-nitrobenzoic acid, DTNB) by adding 1 mL of 10 mM DTNB in 0.1 M
phosphate buffer (pH 8.0) containing 2 mM Na2 EDTA to the thiol solution. After 5 min, the pH was
adjusted to 5.0 with formic acid. Thiols were separated on an octadecylsilica column (250 × 4.0 mm
I.D., particle size 10 µm) thermostatted at 30°C. DTNB derivatives were detected at 330 nm. Solvents
A and B were 0.023 M ammonium formate (pH adjusted to 5.0 with 10% formic acid) and methanol,
respectively. The gradient was 0-10 min, 2% B; 10-18 min, 2%-10% B; 18-23 min, 10%-50% B; 23-25
min, 50% B; 25-30 min, 50%-2% B; 35 min, 2% B. Thiols were well separated under these RP-HPLC
conditions, as demonstrated in Figure 3.30.
Page 354
It was stated that the method, while laborious, is repeatable and reliable and can be employed for the
extraction, separation, and quantitative determination of thiols and disulphides in barley. Many efforts
have been devoted to the development of rapid and reliable HPLC methods for the separation and
quantitation of organic acids in various food products [ 432]. HPLC has also been used for the analysis
of organic acids in technical sugar solutions [ 433], juices [ 434], musts [ 435,436], wines [ 437], and other
beverages [ 438]. In order to increase the separation efficiency of RP-HPLC, the interfering molecules
can be eliminated by using sample pretreatment on an ion-exchange resin [ 439,440]. The effect of
gradient elution [ 441] and the derivatization of the organic acids in juices [ 442] and wines [ 443] on the
separation efficiency have also been determined. Various agents were used for the derivatization of
carboxylic acids such as p-nitrobenzyl [ 444] and phenacyl [445,446] esters. The use of a two-column
technique did not find frequent application in the separation of carboxylic acids [ 447]. Ion
chromatography using both isocratic [ 448] and gradient elution modes [449] has also been used for the
analysis of carboxylic acids. CE can also be used for the separation of carboxylic acids in wines [ 450]. It
has been proven many times that carboxylic acids can also be determined by GC after the appropriate
derivatization process [ 451,452,453]. Thus, methyl [ 454] and silyl [ 455] derivatives were separated by
various GC methods.
Since it has been proven many times that tartaric acid prevents the recurrence of calcium oxalate stones,
a rapid RP-HPLC method was developed for its determination in wines [456]. Wines were applied onto
the column without any pretreatment. Separation was performed on a C18 column (250 × 4.6 mm I.D.)
using water:methanol:0.05 M phosphoric acid (69:1:30 v/v) as eluent (flow rate, 0.8 mL/min; column
temperature, 22 ± 1°C; detection wavelength, 210 nm). Some typical chromatograms are shown in
Figure 3.31. The RP-PLC method separated the organic acid well without any interference from the
other components of wines. The recoveries of tartarate varied between 93.9 and 106.4, with a RSD of
4.4-5.7%. The RSD values of within run and between run variations were between 3.95-6.45% and
3.23-7.92%, showing the reliability of the method. The quantitative results of wine analyses are given
in Table 3.26. It was concluded from the data that the composition of organic acids in wine is
considerably different, resulting in different biological efficacy. It was further established that the
quantity and quality of organic acids also influence the organoleptic characteristics of wine. A low
quantity of malic acid, a higher concentration of lactic acid, and a low amount of tannic acid exert a
favorable influence on the organoleptic characteristics.
The measurement of the quantity of organic acids in fruit juices can also be used for the determination
of the authenticity of the product. A gradient ion chromatographic method was developed for the
determination of the organic acid profile of fruit juices [ 457]. Juices were diluted with redistilled water,
Page 355
Figure 3.31.
HPLC profile of a (A) Rossese di Finale Ligure and a
(B) Beaujolais wine. Column: LiChrosorb RP-18 (250 × 4 mm I.D.);
mobile phase: water:methanol: 0.05 M phosphoric acid (69:1:30);
flow rate: 0.8 mL/min; temperature: 22°C. Peaks: 1 = tartaric acid;
2 = malic acid; 3 = lactic acid; 4 = acetic acid; 5 = tannic acid.
Page 356
TABLE 3.26. Determination of Major Carboxylic Acids in Wines by HPLC.

Acid Concentration (g/L)
Wine Tartaric Malic Lactic Acetic Tannic
Beaujolais (F) 2.21 1.74 1.19 — 0.42
Chablis (F) 1.35 0.24 2.61 — 0.01
Cinque Terre (I) 2.61 0.61 1.22 — —
Clos Reginu (F) 1.19 0.88 2.85 — 0.31
Cortese (I) 2.56 0.56 1.86 — 0.02
Dolcetto (I) 1.86 — — 0.86 0.32
Liebfraumilch (D) 0.72 1.91 2.84 — 0.01
Malaga (E) 0.97 1.27 0.91 — 0.06
Moscato (I) 0.89 2.41 — 0.73 —
Nebblolo (I) 1.44 — 1.22 — 0.25
Pigalo (I) 1.32 1.05 0.47 0.25 —
Poggese (I) 2.71 0.91 — — —
Rosè (GR) 1.78 0.55 3.12 — 0.14
Rossese (I) 2.88 — 1.34 — 0.37
Santorini (GR) 1.52 0.73 0.61 — —
Sherry (E) 1.96 0.93 3.95 — 0.03
Vermentino 1.91 — 2.66 0.21 —
depending on the concentration of organic acids; filtered; and used for analysis. Separation was
performed on a guard (OmniPac Pax-500, 40 × 4 mm I.D.) and on an analytical column (OmniPac Pax-
500, 250 × 4 mm I.D.). Separation was carried out at room temperature, and solutes were detected with
a conductivity detector. The gradient is shown in Table 3.27. Chromatograms of fruit juices are shown
in Figure 3.32. The relative standard deviation of the reproducibility varied between 1.55% and 2.88%,
showing the reliability of the method. The determination limit considerably depended on the type of
solutes, being the highest (20 mg/mL) for quinic and shikimic acids and the lowest (2 mg/mL) for
chloride and nitrate. The concentrations of citric acid, isocitric acid, and malic acid in juices are given
in Tables 3.28-3.30. The concentration of various organic acids depended markedly on the provenance,
the variety, and the extraction technology of the fruits; therefore, the exact knowledge of the organic
acid composition of fruit juices may help quality control and the determination of the genuineness of
juices.
A combined HPLC -carbon isotope ratio measurement method has also been developed for the analysis
of the main organic acids in fruit juices and was used for their authenticity control [ 458]. The
performance of ion chromatograph, HPLC, and enzymatic analysis was compared for the quantitative
determination of organic acids in sugar factory products [ 459]. Ion chromato-
Page 357
TABLE 3.27. Eluent Gradient for Ion Chromatography.

Time (min) Eluent A (%) Eluent B (%) Eluent C (%)
0 100 0 0
4 100 0 0
15 0 100 0
19 0 100 0
27 0 0 100
39 0 0 100
40 100 0 0
Equilibration time between each injection: 15 mn. A = 0.60 mM NaOH in
water:ethanol:methanol (66.5:20:13.5, v/v); B = 20 mM NaOH in water:ethanol (65:35, v/v); C
= 60 mM NaOH in water:ethanol (65:35, v/v).
graph used a sulfonic column (approximately 9% styrene-divinyl benzene support), an isocratic mobile
phase (2 mM HCl), a flow rate of 0.8 mL/min, and a conductivity detector without thermostatting the
column. Samples were dissolved in the eluent without any pretreatment. HPLC separation was
performed on another sulfonic column thermostatted at 50°C, the mobile phase being 5 mM H2SO 4. The
flow rate was 0.6 mL/min, and the detector wavelength was set to 210 nm. Propionic and pyroglutamic
acids were not separated by these methods, but the other organic acids were well separated by both
methods, as demonstrated in Figure 3.33. The organic acid composition of 11 molasses samples
determined by ion chromatograph, HPLC, and enzymatic method are presented in Table 3.31. The data
show considerable deviations depending on the method. It was established that the best agreement
occurs between the results of enzymatic analysis and ion chromatography. Moreover, enzymatic
analysis was able to prove that only L-malic acid is present in the samples. It was concluded that, in this
special case, enzymatic analysis is superior to both HPLC and ion chromatography.
Not only organic, but also inorganic, compounds have been determined in different food products.
Because of its simplicity and rapidity, capillary electrophoretic techniques have been frequently used
for such analyses. Thus, various ions were determined in apple and orange juices [ 460], green tea [461],
apple vinegar and mineral water [ 462], and parmesan cheese [463]. A validated method was developed
for the separation and quantitative determination of K+, Na +, Ca 2+, Mg2+, and Mn2+ in solid natural
products using capillary electrophoresis [ 464]. Solid samples were digested by mixing 0.4 g of sample
with 2 m of HNO3, 0.5 mL of H2O2, and 1 mL of water and treated in a microwave oven under different
conditions. Digests were diluted 50-fold and injected hydrostatically to CE. Separation of ions was
performed on fused-silica
Page 358
Figure 3.32.
Chromatogram of (A) orange juice (sample
dilution 1:10; (B) apple juice (sample dilution 1:10);
and (C) grape juice (sample dilution 1:25).
The Netherlands.)
Page 359
TABLE 3.28. Citric Acid Content (g/L) in Different Fruit Juices.

Juice Min. Average Max. n SD
Orange juices (data referred to 11.2 °Bx)
Italy Blond I pressing 10.42 20.05 26.62 15 4.68
Italy Red I pressing 12.29 14.92 19.36 10 2.69
Italy Mix I pressing 13.09 18.21 24.21 14 3.44
Italy Blond II pressing 2.24 9.24 16.27 7 4.69
Italy Red II pressing 5.12 8.28 12.99 7 2.59
Italy Mix II pressing 8.04 10.01 14.03 7 1.98
Mediterranean Basin 7.67 11.53 14.94 15 2.01
Brasil-I pressing 6.32 9.01 17.24 58 1.80
Brasil-II pressing 1.64 7.83 12.86 37 2.03
Others South America 4.64 11.44 17.99 20 3.20
U.S.A and Cuba 3.57 8.36 19.40 34 3.70
Grapefruit juices (data referred to 10.0°Bx) 8.33 15.94 26.68 28 3.89
Apple juices (data referred to 10.0°Bx)
Germany 0.03 0.08 0.14 42 0.02
Italy 0.02 0.06 0.13 28 0.02
Others 0.03 0.06 0.11 14 0.02
Grape juices (data referred to 16.0° Bx)
France 0.15 0.24 0.41 21 0.06
Italy 0.17 0.39 0.85 31 0.15
Cherry juices (data referred to 14.0° Bx) 0.05 0.23 0.62 40 0.14
Blackcurrant juices (data referred to 14.0° 21.15 29.63 65.44 67 5.37

Bx)
TABLE 3.29. Isocitric Acid Content (mg/L) in Different Fruit Juices.

Orange juices (data referred to 11.2 ° Bx)
Italy Blond I pressing 154 201 232 11 25
Italy Red I pressing 103 127 153 5 21
Italy Mix I pressing 149 166 206 7 22

Italy Blond II pressing 120 145 188 3 37
Italy Red II pressing 83 97 114 4 13
Italy Mix II pressing — — — — —
Mediterranean Basin 69 162 282 15 58
Brasil-I pressing 51 83 185 58 21
Brasil-II pressing 16 73 119 36 21
Others South America 51 112 178 20 35
U.S.A and Cuba 28 94 228 31 42
Grapefruit juices (data referred to 10.0° 121 227 389 27 69

Bx)
Cherry juices (data referred to 14.0° Bx) tr 63 145 40 45
Blackcurrant juices (data referred to 12.5° 128 246 424 40 73

Bx)
Page 360
TABLE 3.30. Malic Acid Content (g/L) in Different Fruit Juices.

Orange juices (data referred to 11.2 ° Bx)
Italy Blond I pressing 1.02 1.76 3.63 15 0.63
Italy Red I pressing 0.92 1.53 2.70 10 0.51
Italy Mix I pressing 1.07 1.61 2.12 14 0.36
Italy Blond II pressing 0.94 1.36 1.98 7 0.34
Italy Red II pressing 0.69 1.13 1.96 7 0.40
Italy Mix II pressing 0.88 1.03 1.27 7 0.14
Mediterranean Basin 0.19 1.16 2.12 15 0.48
Brasil-I pressing 1.31 2.02 4.33 58 0.64
Brasil-II pressing 1.11 1.72 2.53 37 0.34
Others South America 1.22 1.68 2.22 20 0.27
U.S.A and Cuba 0.41 1.16 2.40 34 0.47
Grapefruit juices (data referred to 10.0° 0.18 0.52 1.00 28 0.19

Bx)
Apple juices (data referred to 10.0° Bx)
Germany 4.18 7.14 11.48 43 1.43
Italy 2.64 4.04 5.35 28 0.66
Others 3.45 5.17 8.44 14 1.48
Grape juices (data referred to 16.0° Bx)
France 1.49 3.62 9.67 21 1.81
Italy 1.99 4.48 9.62 31 1.87
Cherry juices (data referred to 14.0° Bx) 11.92 18.17 22.71 40 2.92
Blackcurrant juices (data referred to 12.5° 0.90 1.89 3.07 67 0.58

Bx)
capillaries (60 cm × 75 µm I.D.) using a positive voltage of 20 kV. Between injections, the capillary
was washed with 0.1 M KOH and distilled water for 1 min and with the electrolyte buffer for 3 min.
Background electrolyte was prepared by mixing 5 mL of 500.0 mM imidazole, 25 mL of 130.6 mM 2-
hydroxyisobutyric acid, and 5.5 mL of 50.0 mM 18-crown -6 with 100 mL of methanol and diluted to
500 mL with distilled water. The pH of the buffer was adjusted to 4.5 ± 0.4 with 1 M HCl. The
electropherograms of total diet, tea, and oyster tissue are shown in Figure 3.34. Baseline separation of
the ions was achieved independently of their relative concentration. The limit of detection of the
individual ions was: K+ = 500 µg/g; Na+ = 170 µg/g; Ca 2+ = 220 µg/ g; Mg2+ = 70 µg/g; and Mn2+ = 280
µg/g. The limit of quantitation was: K+ = 2.5 mg/g; Na + = 520 µg/g; Ca 2+ = 730 µg/g; Mg2+ = 340 µg/g;
and Mn2+ = 700 µg/g. The certified and measured values of ion concentrations are given in Table 3.32.
The sensitivity of the method was limited by the excess of acid, which deteriorated the separation
capacity. However, the method makes possible the rapid and precise determination of some inorganic
cations in food products without complicated sample pretreatment procedures.
Page 361
Figure 3.33.
(A) Ion chromatogram and (B) high-performance liquid chromatogram of sample
solution of molasses 2%. Peaks: 1 = citric; 2 = malic; 3 = lactic;
4 = formic; 5 = acetic; 6 = pyroglutamic + propionic; uk = unknown.
TABLE 3.31. Comparison of Enzymatic Analysis, HPLC, and Ion Chromatography for Organic Acids in Molasses.
Citric (mg/g) Malic (mg/g) Lactic (mg/g) Formic (mg/g) Acetic (mg/g)
Enz. HPLC IC Enz. HPLC IC Enz. HPLC IC Enz. HPLC IC Enz. HPLC
1 140 140 90 241 140 178 2111 1720 2256 277 225 264 594
2 210 400 174 357 520 260 3058 2420 3254 305 240 268 411
3 205 180 138 255 60 190 2707 1920 2588 238 160 216 543
4 246 200 142 317 240 246 2076 1620 2324 345 180 324 647
5 163 400 80 305 540 244 2214 2720 2524 340 200 326 690
6 98 480 36 163 80 118 1304 1000 1552 223 160 222 623
7 188 140 96 335 240 170 3444 3040 3980 309 180 282 673
8 181 180 126 217 120 187 1601 1900 1931 264 220 266 642
9 159 140 105 217 80 168 2674 2320 3090 235 140 221 1078
10 244 380 53 364 700 284 2610 2620 2817 248 300 227 702
11 169 160 200 326 160 263 2220 1940 2686 307 180 299 434
PCA = pyroglutamic acid.
Netherlands.
Page 363
Figure 3.34.
Electropherograms of reference material and tea digests (50-fold dilution.
Peaks: 1 = K+; 2 = Na +; 3 = Ca2+; 4 = Mg2+; 5 = Mn 2+; 6 = Zn2+; 7 = H +
and NH4+; 8 = unidentified. Experimental conditions: hydrostatic injection
from 10 cm for 20 s, applied voltage, +20 kV; I = ±5.4µA; background electrolyte,
5 mM imidazole 6.5 mM HIBA 0.55 mM 18 -crown-6 20% methanol (pH 4.5).
The Netherlands.)
Page 364
TABLE 3.32. Comparison of CE Results* and Certified or Recommended Values.

Reference Material Value K (%) Na (%)
Total diet CE 0.596 ± 0.046 0.592 ± 0.005
(NIST SRM 1548) Certified value 0.606 ± 0.028 0.625 ± 0.026
Oyster tissue CE 0.762 ± 0.016 0.398 ± 0.015
(NIST SRM 1566a) Certified value 0.790 ± 0.047 0.417 ± 0.013
Fish tissue CE 8.795 ± 0.346 1.905 ± 0.379
(IAEA MA-B-3/TM) Recommended value 9.00 - 10.0 2.00 - 3.60
Bovine liver CE 1.096 ± 0.073 0.226 ± 0.006
Pine needles CE 0.346 ± 0.017
(NIST SRM 1575) Certified value 0.37 ± 0.02

Citrus leaves CE 1.762 ± 0.026

Reference Material Value Ca (%) Mg (%)
Total diet CE 0.174 ± 0.006 0.0555 ± 0.0041
Oyster tissue CE 0.188 ± 0.006 0.109 ± 0.005
(NIST SRM 1566a) Certified value 0.196 ± 0.019 0.118 ± 0.017
Fish tissue CE 3.455 ± 0.070 1.227 ± 0.114
(IAEA MA-B-3/TM) Recommended value 3.18 - 3.60 1.04 - 1.20
Bovine liver CE 0.0131 ± 0.0004 0.0589 ± 0.0036
Pine needles CE 0.391 ± 0.012 0.108 ± 0.002
(NIST SRM 1575) Certified value 0.41 ± 0.02
Citrus leaves CE 3.123 ± 0.021

*The CE results were obtained from the analysis of three independent digests within one day. Each
digest was injected twice. The SD was estimated as the range of the three digestion means divided
by 1.91.
Experimental conditions: hydrostatic injection from 10 cm for 20 s; applied voltage, +20 kV; I = ±
5.4 µA; background electrolyte, 5 mM imidazole–6.5 mM HIBA–0.55 mM 18 -crown -6–20% (v/v)
methanol (pH 4.5).
3.2.1—
Aroma and Flavor Components
3.2.1.1—
Spices
Because of its appealing characteristic flavor and fragrance, cinnamon is frequently used in various
food products. Its considerable commercial importance necessitated the development of accurate and
precise analytical procedures for quality control.
Page 365
Many chromatographic techniques such as TLC [ 465] and GC [ 466] have found application in the
analysis of cinnamon. The presence of 2-hydroxycinnamaldehyde in commercial cinnamons has been
proven by a combined GC-TLC method [ 467]. About 500 mg of a powdered cinnamon sample was
mixed with 4.5 g of anhydrous sodium sulfate and was extracted with supercritical carbon dioxide (300
atm, 70°C, 30 min static, and 30 min dynamic extraction). The sample was collected in 4.0 mL of ethyl
acetate and concentrated to 2 mL under nitrogen. The isolation of 2-hydroxycinnamaldehyde was
performed by refluxing 85 g of powdered cassia with 500 mL of acetonitrile for 4 h. After cooling, the
acetonitrile was decanted, concentrated to 20 mL, and mixed with 20 mL of sodium carbonate solution
(5% w/v). The mixture was extracted with 2 × 20 mL of dichloromethane, acidified with 1 N HCl, and
extracted again with 2 × 20 mL of dichloromethane. The last two combined extracts were evaporated to
about 1 mL and applied to silica gel preparative TLC plates. The plates were developed with
chloroform:hexane: methanol (59:39:2 v/v). The band with an RF value of approximately 0.22 was
scraped, eluted with dichloromethane, and evaporated to dryness. GC separation was carried out in a
dual-oven gas chromatograph under the following conditions: oven 1 contained a fused-silica open
tubular column (25 m × 0.25 mm I.D., coated with 1.0 µm film of DB-5), temperature program from
100 to 250°C at 5°C/min. Oven 2 contained a fused-silica capillary column (30 m × 0.25 mm I.D.,
coated with 0.25 µm film of DB-225), temperature program from 100°C to 180°C at 5°C/min and held
at this temperature. The carrier gas was hydrogen, and the injector and detector temperatures were 285°
C and 300°C, respectively.
TLC analysis of the extracts was carried out on spacer-bonded propanediol layers using
chloroform:hexane:methanol (59:39:2 v/v) as eluent. Spots were evaluated with a TLC scanner at 300
nm either in the absorption mode or in the fluorescence mode (excitation 365 nm, emission >400 nm).
The GC profile of cinnamon extract is fairly complicated and the quantitative evaluation of the minor
components such as 2-hydroxycinnamon is difficult (Figure 3.35). The TLC method successfully
separated 2-hydroxycinnamaldehyde from the other coextracted compounds, as demonstrated in Figure
3.36. Since the extracts of cinnamon and cassia contain a low amount of fluorescence compounds, the
detection and quantitation method is highly selective. The concentrations of 2-hydroxycinnamaldehyde
in various commercial samples are given in Table 3.33. The data indicate that the concentration of 2-
hydroxycinnamaldehyde is higher in Cassia than in Cinnamon species that can be used for the
differentiation of these species.
Gas chromatography has also been used for the determination of the botanical origin of cinnamon [ 468].
Samples were prepared by solvent-assisted SPE as described above and with SPME: 10 mg of
powdered sample was heated in a test tube of 15 mL volume at 70°C for 15 min. The SPME syringe
was
Page 366
Figure 3.35.
Separation by series-coupled column gas chromatography of the semivolatile
compounds extracted by solvent-assisted SFE from a sample of powdered
cinnamon from Sri Lanka. The elution position of 2-hydroxycinnamaldehyde is
marked with an arrow; other major components are identified as follows:
1 = trans-cinnamaldehyde; 2 = cinnamyl alcohol; 3 = eugenol;
4 = cinnamyl acetate; and 5 = 2-methoxycinnamaldehyde.
equilibrated with the sample at the same temperature for 5 min and then thermally desorbed in the
injection port of the gas chromatograph for 2 min. Separations were performed on the same dual-oven
gas chromatograph as above. The temperature gradient for both columns was 2 min held at 40°C, to
150°C at 40°C, and held for 1 min, to 180°C at 3°C, and held to the end of the separation. It was
established that both the type and dimensions of the fiber coating exert a considerable influence on the
yield of semivolatile compounds. Moreover, the chromatograms of the samples obtained by SPME
(Figure 3.35) and solvent-assisted supercritical fluid extraction (Figure 3.36.) are highly different,
indicating the considerable impact of sample preparation on the quantitative results of the analysis.
Principal component analysis indicated that cinnamon and cassia can be distinguished by measuring the
relative concentrations of eugenol, benzyl benzoate, coumarin, and δ-cadinene.
The flavor composition of vanilla has also been extensively studied. Various TLC methods were
employed for the determination of the authenticity of natural vanilla extracts [ 469], for the separation of
vanillin and related flavor
Page 367
Figure 3.36.
Determination of 2 -hydroxycinnamaldehyde by TLC and
fluorescence scanning densitometry of extracts from
powdered cinnamon and cassia obtained by solvent-assisted
SFE. Identification: (A) Kroger brand cassia and (B) Davul
Kurundu true cinnamon. The peak marked × is
2-hydroxycinnamaldehyde.
compounds in foods [ 470], and for the identification of 5-(hydroxymethyl)-2-furfurol in vanilla extracts
[471]. The use of planar chromatographic methods for the determination of flavor compounds in
cinnamon and vanilla was reviewed [ 472]. RP-HPLC has found application in the analysis of vanilla
too. Thus, it has also been used for the determination of the authenticity of natural vanilla extracts [ 473]
and for the separation of vanillin and related phenolic components [ 474]. The performance of various
extraction methods were tested for the simultaneous isolation of glucovanillin and vanillin before their
HPLC separation [ 475]. Method 1: 2 g of vanilla were extracted with 200 mL of 47.5% ethanol in a
Soxhlet apparatus for 24 h heated at 200°C. After extraction, the volume was filled to 250 mL and then
reduced 2.5-fold in a rotary
Page 368
TABLE 3.33. Concentrations of 2-Hydroxycinnamaldehyde in the Inner Bark of

Mercantile Samples of Cinnamon.
Brand Name Country of Purchase Concentration (mg/g)
True cinnamon
Podi kurundu Sri Lanka 0.020
Davul kurundu Sri Lanka 0.096
Weli kurundu Sri Lanka 0.080
Sainsbury's UK 0.071
Cassia
McCormick USA 0.373
French's USA 0.132
Victoria USA 0.104
Kroger USA 0.360
Trader's choice USA 0.226
Food club USA 0.225
Ottugi Korea 0.430
Unclassified
Schwartz UK 0.206
Assi Korea 0.791
evaporator. Method 2: 0.2 g of vanilla was homogenized with 6 mL of solvent (method 2a: 95%
ethanol; method 2b: 47.5% ethanol; method 2c: 100% methanol; method 2d: 80% methanol) and then
filtered and washed three times with 1 mL of solvent. The extract was filled up to 10 mL with the
solvent. Method 3: 6 mL of solvent were added to 0.2 g vanilla, heated in boiling water and then
continued the homogenization and extraction as in method 2 (method 3a: 5 min heating + 47.5%
ethanol extraction; method 3b: 20 min heating + 47.5% ethanol extraction; method 3c: 5 min heating +
80% methanol extraction; method 3d: 20 min heating + 80% methanol extraction). Method 4: 0.2 g of
vanilla was macerated for 24 h (150 rpm) at 60°C in 9 mL of 47.5% ethanol (method 4a) or 80%
methanol (method 4b). After maceration, the mixture was filtered, the cake rinsed with the same
solvent, and adjusted at 10 mL of final volume. Flavor compounds were separated on a 250 × 4.6 mm
I.D. C 18 column (particle size 10 µm). The mobile phase consisted of water acidified with 1.25% acetic
acid (v/v) (solvent A) and of methanol (solvent B). The gradient was from 95% A to 90% A in 5 min;
hold for 5 min; to 65% A in 30 min; hold for 10 min. The flow rate was 1 mL/min. A typical
chromatogram is shown in Figure 3.37. The RP-HPLC method successfully separated the flavor
substances extracted from vanilla pods, and no interference from other constituents was observed. The
efficiencies of the various methods for the extraction of glucovanillin and vanillin from vanilla cured
beans are given in Table 3.34. The data clearly show that the recovery of both glucovanillin and
Page 369
Figure 3.37.
RP -HPLC of a vanilla extract from cured Bourbon beans:
(1) glucovanillin; (2) p-hydroxybenzoic acid;
(3) p-hydroxybenzaldehyde; (4) vanillic acid;
(5) vanillin; and (6) ethyl vanillin internal standard.
publisher and the corresponding author,
vanillin depends considerably on the method of extraction. It was proposed that the future development
of an optimalized extraction procedure may increase the accuracy of the RP -HPLC determination of
glucovanillin and vanillin.
The composition of ginger oil has also been frequently investigated with various chromatographic
techniques. The characteristic odorants in fresh rhizomes of ginger (Zingiber officinale Roscoe) were
identified by aroma extract dilution analysis and modified multidimensional GC-MS [476]. The
odorants were isolated from the fresh rhizomes of ginger by grating 3 kg of the sample with 300 mL of
distilled water and extracting the slurry with 3 L of hexane for 1 h with continous stirring. After
finishing the extraction, hexane was removed at 40°C, and the residue was filtered and dried over
anhydrous sodium sulfate. The oily residue (approximately 6 g) was dissolved in 15 mL of hexane and
applied to a column (180 × 20 mm I.D.) filled with silica. Odorants were eluted with 200 mL of hexane
(hexane fraction) and dichloromethane (O-fraction). Both fractions were dried over anhydrous sodium
sulfate, and the solvent was evaporated in a rotary vacuum evaporator. GC-olfactometry was performed
by separating the odorants on a fused-silica column (30 m × 0.53 mm I.D., film thickness of Supelco
wax 1 µm). The column temperature changed from 80°C to 210°C at 3°C/min. Injector and detector
temperatures were 250°C. A thermal conductivity detector was used, and the outlet was connected with
a glass sniffing port. The flavor dilution factor indicating the con-
Page 370
TABLE 3.34. Extraction of Vanillin and Glucovanillin from Vanilla Cured Beans.
Java Bourbon
Extraction
Method Vanillin* (%) Glucovanillin (%) Vanillin (%) Glucovanillin (%)
1 100.0 ± 7.0 100.0 ± 12.1 100.0 ± 1.5 100.0 ± 10.2
2a 61.4 ± 1.3 32.6 ± 4.2
2b 73.8 ± 3.0 72.1 ± 9.2
2c 69.1 ± 2.6 59.6 ± 9.8
2d 78.4 ± 2.9 72.4 ± 7.5
3a 84.9 ± 0.9 61.1 ± 3.0
3b 100.1 ± 1.6 68.0 ± 8.3 95.9 ± 1.5 88.5 ± 4.6
3c 88.3 ± 1.4 75.0 ± 6.2
3d 98.5 ± 2.6 71.5 ± 11.1 97.2 ± 2.1 80.3 ± 4.8
4a 107.5 ± 2.0 74.8 ± 6.4 101.1 ± 1.9 77.4 ± 4.5
4b 108.7 ± 1.7 78.3 ± 10.9 102.1 ± 1.8 80.0 ± 4.0

* Percentage relative to method 1 (Soxhlet); mean ± SD for three to six replications.
Reprinted with permission from Reference [ 475], copyright © 1995, American Chemical
Society.
tribution of the component to the characteristic flavor of the product was determined by aroma extract
dilution analysis. The identification of the components was carried out by a multidimensional GC-MS
system using the Kovats retention indices and by IR and nuclear magnetic resonance (NMR) spectra.
The results are presented in Tables 3.35 and 3.36. It was concluded from the data that the many
compounds play a considerable role in the formation of the typical odor of fresh ginger, and this
combined method seems to be very promising for the isolation and identification of odorants in
complicated mixtures.
Not only GC but also liquid chromatographic methods found application in the analysis of ginger. Thus,
countercurrent chromatography was employed for the isolation of gingerols from the powdered root
[477], and they were separated by an RP-HPLC method [ 478]. Pungent compounds were isolated from
commercial ginger by supercritical fluid extraction and separated first with high-speed countercurrent
chromatography using ethyl acetate:hexane:methanol:water (2:3:3:2) as the lower phase. Fractions were
detected at 282 nm. RP-HPLC separation of the solutes was performed on a C 18 column (250 × 4.6 mm
I.D., 10 µm particle size, end-capped) using gradient elution. Eluents A and B were
acetonitrile:water:acetic acid (50:50:1) and acetonitrile:methanol:acetic acid (80:19:1), respectively.
The linear gradient was 0-100% B in 30 min. The flow rate was 1 mL/min, and the fractions were
detected at 282 nm. High-speed countercurrent chromatography could not separate 10-gingerol and 6-
shogaol from each other (Figure 3.38), but the other compounds were isolated with acceptable purity (6-
gingerol 99%; 8-gingerol
Page 371
TABLE 3.35. Potent Odor Compounds in the O-part of the Extract from the Fresh Ginger Rhizomes.
No. Compound RI(DB-WAX) Odor Decsription* FD Factor
1 1,8-cineol 1214 Camphoraceous 15
2 Bornyl-methyl ether** 1252 Earthy, musty 11
3 2-heptanol 1273 Mushroom-like, herbaceous 13
4 Unknown 1320 Green, fruity, melon-like 11
5 Unknown 1352 Mushroom-like 11
6 (E)-2-octenal*** 1377 Green, nutty, burdock-like, fatty 12
7 Citronellal 1425 Japanese pepper tree-like 11
8 Decanal 1447 Green, waxy 11
9 Linalool 1484 Floral 20
10 2-undecanone 4- 1543 musty, dusty, green 14

terpineol camphoraceous
11 (E)-2-decenal*** 1590 Fatty, green 11
12 Citronellil acetate 1607 Musty, dusty, rosy 16
13 Neral 1630 Citrus-like, peely 15
14 Borneol 1642 Dry-camphoraceous 16
15 Unknown 1652 Musty, dusty 10
16 Geranial 1686 Citrus-like 17
17 Geranyl acetate 1711 Floral, rosy 14
18 Nerol 1753 Floral 10
19 Geraniol 1788 Floral, rosy 20
20 (E)-2-dodecenal*** 1807 Fatty, green 11
21 Zingiberenol 2037 Metalic, lemony 11
22 Isoeugenol 2250 Spicy, floral 13

* Odor descriptor assigned during aroma extract dilution analysis.
** Tentatively identified.
*** Newly identified in the aroma extract from ginger.
Reprinted with perimssion from Reference [ 476], copyright © 1995, American Chemical Society.
83%). The RP-HPLC separation of the components of commercial ginger extract is shown in Figure
3.39. The RP-HPLC method allows the baseline separation of each component and the internal standard
vanillin. The method was proposed for the analysis of the pungent principles in ginger and ginger
products.
The volatile components of coriander oil was studied with a GC-MS method using static and dynamic
headspace analysis [ 479]. The pigment and aroma composition of turmeric has also been extensively
investigated by chromatographic methods. Supercritical fluid extraction and chromatography [ 480] and
GC-MS [481] were equally employed for the determination of the volatile and semivolatile components
of turmeric. Liquid chromatography-mass spectrometry (LC-MS) and GC-MS were used together for
the characterization of powdered turmeric samples [ 482]. Samples for LC -MS were prepared by
Page 372
TABLE 3.36. Volatile Odor Compounds in the O-part of the Extract from the Fresh Ginger
Rhizomes Identified by a Modified Multidimensional GC-MS.
Peak RI(OV-
No. Compound Odor Description 101)
1 2-octanol Mushroom-like 987
2 2,6-dimethyl-5-heptenal Green, fruity, melon- 1039

like
3 2-nonanone Fruity, fatty 1093
4 2-(3'-methyl-2'butenyl)-3-methylfuran Green, minty 1099
5 Nonanal Floral, waxy, green 1102
6 2-pinen -5-ol * Musty, dusty 1106
7 Cis-rose oxide Floral 1115
8 Trans-rose oxide Floral 1128
9 2-octyl acetate* Fruity, floral 1132

10 (Z)-3,7-dimethyl -3,6-octadienal* Green 1151
11 Camphene hydrate Camphoraceous 1152
12 Isoborneol Musty, dusty 1157
13 2-(2',3'-epoxy -3'-methylbutyl)- 3- Green, earthy, citrus- 1161
methylfuran* like
14 (E)-3,7-dimethyl -3,6-octadienal* Green 1166

15 4-terpineol Earthy, musty 1175
16 Neral Citrus-like, peely 1222
17 Geranial Citrus-like 1249
18 2-undecanone Fruity, rosy 1269
19 Citronellyl acetate Floral, rosy 1335
* Newly identified in the aroma extract from ginger.
Reprinted with perimssion from Reference [ 476], copyright © 1995, American Chemical
Society.
vortexing 150 mg of powdered turmeric with 5 mL of water; the slurry was centrifuged and the water
decanted. The solid residue was vortexed with 10 mL of methanol and centrifuged again. One milliliter
of the extract was diluted with the solution of the internal standard (4-fluoro-4'-hydroxyben-
zophenone). Turmeric powder was mixed with activated Chromosorb W HP in 1:10 w/w for GC
analyses and was filled into a stainless steel tube. Desorption was carried out at 220°C for 5 min. An
octadecylsilica column (250 × 4.6 mm I.D., particle size 5 µm) was used for RP-HPLC. The three main
curcuminoids (curcumin, demethoxycurcumin, bis-demethoxycurcumin) were separated with gradient
elution solvents A and B, being 1% citric acid (pH adjusted to 3.0) and acetonitrile, respectively. The
other solutes were separated by a different gradient elution, solvents A and B being 50 mM ammonium
acetate + 5% acetic acid and acetonitrile, respectively. The gradient was in both cases 50% B for 10
min, to 80% B in 30 min. The flow rate was 1 mL/min. GC separations were performed on a DB-1
capillary column (60 m × 320 µm I.D., film thickness 0.25 µm). The temperature program was from -
20°C to 150°C at 20°C/min and then to 280°C at 5°C/min. A typical RP-HPLC chromatogram
Page 373
Figure 3.38.
High-speed, countercurrent chromatogram of ginger extract.
Figure 3.39.
HPLC chromatogram of a commercial ginger extract. Column; Spherisorb
ODS1 10 µm, partially endcapped, 250 × 4.6 mm; precolumn: Spherisorb ODS1
10 µm partially endcapped, 20 × 4 mm; eluent A: acetonitrile:water:acetic acid
(50:50:1); eluent B: acetonitrile:methanol:acetic acid (80:19:1), gradient
linear from 0 -100% B in 30 min, flow: 1 mL/min, detection 282 nm.
Page 374
of the turmeric extract is shown in Figure 3.40. The various components of turmeric are well separated
from each other and from the internal standard, proving the good separation capacity of the method.
The concentration of the main curcuminoids in five turmeric powders determined by RP-HPLC are
given in Table 3.37. The regression coefficients of the linear correlations between solutes and detector
response were 0.9989, 0.9996, and 0.9999 for curcumin, demethoxycurcumin, and bis-
demethoxycurcumin, respectively. The standard deviation was very low in each instance, indicating the
high precision of the method. The results of GC analysis of the same turmeric powders are shown in
Table 3.38. The samples contained many compounds, the con-
Figure 3.40.
Thermospray LC-MS chromatogram of turmeric.
Column, Supercosil LC-18, dp 5 µm, 250 × 4.6 mm I.D.;
mobile phase A, 50 mM NH4OAc-5% HOAc;
mobile phase B, acetonitrile; gradient, initial 50% B
(hold 10 min), 80% B at 30 min (hold 10 min);
massa scanned, 150-550 amu at 3.2 s/scan;
source temperature, 255°C; control temperature, 136 °C.
The Netherlands.)
Page 375
centration of the identified and not identified compounds being highly different.
Because of its advantageous characteristics, supercritical carbon dioxide extraction coupled with GC
has been extensively used in aroma research [ 483]. Thus, this method was employed for the analysis of
the volatile oils of Thymus vulgaris [ 484], essential oils in aromatic plants [485], and cloves [ 486] and for
the study of the absorption of aroma compounds by low-density polyethylene [ 487]. SFE-GC has also
been employed for the analysis of caraway fruits [ 488]. Samples were ground under liquid nitrogen to
avoid the loss of highly volatile components and were subsequently extracted with CO2 at 50°C at 9.7
MPa for 2 min. The solutes were trapped in a DB-1 fused-silica capillary column (3 m × 0.32 mm I.D.,
film thickness 0.25 µm) chilled to -40°C. Helium was used to remove CO2 and to wash the solutes to
the head of the analytical column (DB-5, 30 m × 0.32 mm I.D., df = 0.25 µm) immersed in liquid
nitrogen. Separation was carried out by a temperature program: from 40°C to 150°C at 5°C/min, to
220°C at 25°C. The schematic diagram of the on-line SFE-GC system is shown in Figure 3.41.
Hydrodistillation was used as a control method for the quantitative determination of carvone and
limonene in caraway fruit samples. A typical chromatogram is shown in Figure 3.42.
The GC method separated the volatiles of caraway well. The results proved that the volatile components
of caraway were quantitatively recovered by the cryogenic focusing at the head of the analytical GC
column. The carvone and limonene content of four caraway fruit samples is presented in Table 3.39.
The concentrations determined by both methods are markedly different, and the RSD for limonene is
considerably higher than that of carvone. These discrepancies may be because of the effect of the
splitter.
Both fresh and dried fruits of the genus Capsicum are frequently used as spices and as coloring agents
in foods. Because of the considerable commercial importance, much effort has been devoted to the
separation and quantitative
TABLE 3.37. Quantitative Results for Curcuminoids in Turmeric Powder by HPLC.

Powder 1 Powder 2 Powder 3 Powder 4 Powder 5
Curcuminoid (wt%) (wt%) (wt%) (wt%) (wt%)
Bisdemethoxycurcumin 0.49 0.60 0.55 0.50 0.90
Demethoxycurcumin 0.60 0.72 0.66 0.58 1.10
Curcumin 1.68 1.70 0.94 1.63 3.18
Column, Supelcosil LC-18, d p = 5 µm, 250 × 0.46 I.D.; mobile phase A, 1% citric acid
(pH 3.0); mobile phase B, acetonitrile; gradient, initial 50% B (hold 10 min), 80% B at 30
min (hold 10 min).
TABLE 3.38. Semi-Quantitative Results for Some Major and Minor Components in Turmeric Powders.
Retention Kovats Powder 1 Powder 2 Powder 3 Powder 4 Powder 5
Time (min) Index Assignment (ppm) (ppm) (ppm) (ppm)
15.71 747.85 3,4,5-Trimethyl-2-cyclopentene-1-one 1000 674 519 478
17.28 779.74 Internal standard 4000 4660 4720 3290
18.04 794.44 Coumaran 532 327 129 98
18.96 809.00 2-Hyroxy-5-methyl-acetophenone 2590 1150 1183 848
20.23 826.54 Vanillin 411 148 229 272
22.25 852.29 Curcumene 544 601 895 370
22.55 855.92 Compound 1 537 295 292 315
22.83 859.26 Zingiberene 107 137 173 98
23.17 863.26 β-Bisabolene 632 495 679 435
24.00 872.78 Dehydrocurcumene (proposed) 1710 647 690 707
24.46 877.92 Compound 2 1440 611 754 924
25.05 884.37 Ar-Tumerol 1510 327 822 533
25.28 886.84 Compound 3 3490 1340 1230 1590
25.68 891.09 Ar-Tumerone 59500 25700 25450 29200
25.89 893.29 Tumerone 45700 13900 6420 20500
26.59 900.64 Curlone 48000 15900 13100 23800
27.47 911.74 Compound 4 3470 906 1250 1480
27.72 914.83 Compound 5 5950 1620 963 1200
28.05 918.86 Compound 6 11400 3620 3510 4250
28.40 923.09 Dehydrozingerone 10700 2640 3890 3930
28.94 929.51 Compound 7 3320 632 467 1370
29.95 941.20 Compound 8 1470 432 301 978
30.447 946.73 Compound 9 2370 780 1740 946
30.98 952.72 Compound 10 2310 464 878 1480
Column DB -1 capillary, 60 m × 320 µm I.D., d f = 0.25 µm; temperature program, –20–150°C at 20 °C/min; 150–280°C at 5°
carrier, He at 1 mL/min; injection temperature, 220 °C; FID temperature, 325°C.
Page 377
Figure 3.41.
Schematic diagram of the on-line SFE-GC system with cryogenic
collection trap (F: frits; P: pump; V: valve; E-O: extraction oven;
C: cartridge: S: stream splitter; E: exit; MC: measuring cylinder;
I: injector; Ctr : trapping column; C an : analytical column;
GC-O: gas chromatograph oven).
publisher and the corresponding author, copyright
determination of the composition. Gas chromatography, combined with sniffing port analysis, was
employed for the evaluation of commercially dried bell peppers (Capsicum annuum) after rehydration
[489]. Rehydration was carried out by heating 3 g bell pepper cuttings and 35 mL of water in a water
bath at 100°C for 10 min. After cooling to 25°C, the cuttings were separated and 25 mL of artificial
saliva were added [5.208 g NaHCO 3 , 2.160 g mucin, 1.369 g K2HPO4 , 0.877 g NaCl, 0.500 g NaN 3,
0.447 g KCl, 0.441 g CaCl2, and 200, 000 units of α -amylase in 1 L of water (pH adjusted to 7)].
Nitrogen flow (20 mL/min) passed through the mixtures at 37°C for 2 h, and the volatiles were trapped
in 0.10 g Tenax (35/60 mesh). Thermal desorption was performed at 200°C for 10 min, and the volatiles
were collected in a cold trap (-100°C). GC separation of the volatiles was performed on a Supelcowax
10 capillary column (60 m × 0.25 mm I.D.). Initial oven temperature was 40°C for 4 min, then 2°C/min
to 92°C, and 6°C/min to 272°C. Compounds were detected by FID (275°C) or by 12 assessors at the
sniffing port. Identification was carried out by GC-MS. A typical chromatogram is shown in Figure
3.43. It was found that the same peaks were present in the samples originating from Chile, Hungary,
and Turkey; however, their quantity and ratio differed considerably. The results of the GC-MS and
sensory evaluation are given in Table 3.40. The data indicate
Page 378
Figure 3.42.
GC -FID chromatogram of the supercritical carbon dioxide extract
of caraway fruits: 1 = 2,4-hexadienal; 2 = α -pinene; 3 = sabinene;
4 = β-myrcene; 5 = limonene; 6 = (Z) -β-ocimene; 7 = (E)-β-ocimene;
8 = 2-methylbenzaldehyde; 9 = 4-methylbenzaldehyde; 10 = linalool;
11 = (E)-p-mentha-2,8-dien-1-ol; 12 = (Z)-p-mentha-2,8-dien-1-ol;
13 = α-terpineol; 14 = (Z)-dihydrocarvone; 15 = (E) -dihydrocarvone;
16 = decanal; 17 = (E)-carveol; 18 = carvone; 19 = geranial;
20 = perillaldehyde; 21 = methyl 3,7-dimethyl-2,6-octadienoate;
22 = β-caryophyllene; 23 = τ-elemene.
publisher and the corresponding author, copyright
that the composition of volatile odor compounds of bell pepper is very complicated, and a wide variety
of volatiles influence the characteristic odor of bell pepper.
Capsaicin (8-methyl-N-vanillin-6-nonenamide), the principle of hot flavor in the fruits of the genus
Capsicum, can be determined by both HPLC [ 490] and TLC methods [ 491]. Samples for TLC analysis
were dried at 65°C and ground, and 10.0 g of powder were refluxed for 4 h with 200 mL of ethyl
acetate. The extract was filtered, evaporated to dryness, and redissolved in 1 mL of ethyl acetate.
HPTLC plates were developed with toluene:acetone: chloroform (40:35:25 v/v). After development, the
spots were detected with 2,6-dichloroquinone-4-chloroimide. Capsaicin was extracted from the spots
Page 379
TABLE 3.39. Carvone and Limonene Content of Four Caraway Fruit Samples of Various
Origin Isolated by Hydrodistillation and Supercritical Fluid Extraction.*
Hydrodistillation Supercritical Fluid Extraction
Carvone Limonene C/L Carvone Limonene C/L

Origin (RSD) (RSD) Ratio (RSD) (RSD) Ratio
Cv. Polaris 2.7 (2.4) 1.8 (3.1) 1.5 2.7 (6.7) 1.3 (13.4) 2.0
Puumala ** 3.0 (7.3) 1.3 (10.1) 2.4 2.9 (8.8) 1.7 (18.6) 1.7
Vöyri** 2.8 (2.4) 1.6 (7.3) 1.8 2.7 (9.5) 1.1 (21.6) 2.4
Hämeenkyrö ** 2.6 (3.0) 1.5 (10.6) 1.8 2.2 (2.4) 1.0 (17.6) 2.2
* Results presented are the means of three replicative isolations calculated as g/100 g of
fruits and their relative standard deviations (%).
** Hydrodistillation of whole fruits.
Reprinted with permission from Reference [ 488], copyright © 1994, American Chemical
Society.
and detected at 595 nm. The results are shown in Table 3.41. The data prove that the capsaicin content
of paprika powders differs considerably and can be easily determined by both HPTLC or overpressured
TLC methods. The thermal decomposition rate and the thermal decomposition products of capsaicin
were studied by a GC -MS method [492]. Samples were treated at low (100 Torr) and atmospheric
pressure in a dry air flow and in a sealed tube at 200°C for 2 h. After heat treatment, the samples were
dissolved in dichloromethane, and the internal standard (methyl eicosanoate) was added.
Figure 3.43.
Gas chromatograms of volatile components of rehydrated Hungarian bell pepper
cuttings: (a) FID chromatogram and (b) chromatogram of the components
detected at the sniffing port. Numbers refer to compounds in Table 3.40.
TABLE 3.40. Volatile Components of Rehydrated Capsicum Cuttings of Three Different Origins:Their Retention Times, Odor
Descriptions and Average Peak Areas.
Peak Area (mV s)
Peak No. Retention Time Component Odor Description Chile Hungary

(min)
1 6.27 Dimethylsulphide 1840878 935803
2 7.30 Propanal 1026618 3348740
3 7.83 2-Methylpropanal Chocolate 12936741 26973318
4 8.11 Methyl acetate 88457 77390
5 9.50 2-Methylfuran 756175 3096111
6 9.62 Butanal 783889 1279480
7 9.99 Diethoxyethane 163819 1171945
8 10.33 2-Butanone 1772454 1688028
9 10.79 2-Methylbutanal Chocolate 21058713 54528985

10 11.24 3-Methylbutanal Chocolate 44997203 75734382
11 12.69 3-Buten-2-one 1612069 525114
12 13.95 2-Pentanone 107232 <10000
13 14.05 Pentanal 5005615 25434392
14 14.26 2,3-Butadione Caramel; butter 597711 2958329
15 16.18 1-Penten-3-one Plastic/chemical 688652 3939290
16 19.53 2,3-Pentadione 123015 1779999
17 20.10 Dimethyldisulphide 1242240 97820
18 20.55 Hexanal Grassy/green 16992232 212369451
19 20.95 3-Penten-2-one 62132 41490
20 23.42 1-Methyl -1H -pyrrole 3820132 7882184
21 23.72 1-Butanol 25171 1389427
22 25.42 1-Penten-3-ol 644311 3096888
23 27.06 2-Heptanone 419162 7850928
24 27.86 Heptanal Lemon/orange 2306133 81323827

Peak Area (mV s)

Retention Time
Peak No. (min) Component Odor Description Chile Hungary
25 28.62 Limonene 96643 14213807 282703
26 29.76 3-Methyl-1-butanol 1278667 2678512 987219
27 31.02 trans-2-Hexenal 81841 880410 52383
28 31.12 β -Ocimene Fish; rotten; sickly 958810 406779 311249
29 31.33 4-Methyl-2-hexanone 186340 1489611 151439
30 31.74 1-Pentanol 270199 2330559 203288
31 31.93 2,3-Hexadione 179561 81517 84299
32 34.01 5-Methyl-2-hexanone 144178 659495 113354
33 34.26 Octanal 583280 1688011 474113
34 34.97 trans-3-Hepten-2-one Mushrooms 689214 1104159 1188447
35 36.28 cis-2-Heptenal 1258834 3450004 524969
36 36.83 6-Methyl-5-hepten-2-one 1397735 3299050 1444512
37 37.37 1-Hexanol 542270 666915 360496
38 38.40 Dimethyltrisulphide Rotten: onion/leek 478317 128618 525891
39 39.43 Nonanal 198179 23728350 198948
40 40.53 tert-Dodecanethiol 106942 998882 73005
41 41.04 1-Octen-3-ol 1069494 5370009 521912
42 41.77 Acetic acid 841632 145456 199007
43 43.46 Decanal 1451095 4608510 159129
44 43.94 2-Methoxy-3- Bell pepper 193529 375886 85183

isobutylpyrazine
45 44.57 Benzaldehyde 617389 6990902 608350
46 47.81 β -Cyclocitral Fruity 21081 440575
Reprinted from Reference [ 489], copyright © 1994. With kind permission from Elsevier Science—NL, Sara Burgerhartstrant 25, 1055 KV
Page 382
TABLE 3.41. Capsaicin Content of Ground Red Pepper of Different Quality.

Quality Capsaicin Content (mg %) Coefficient of Variation (%)
Super 5.900 ± 0.098 2.960
Delicacy 8.600 ± 0.176 1.820
Delicacy hot 33.700 ± 0.281 1.090
Sweet-noble 16.200 ± 0.107 3.090
Half-sweet 26.800 ± 0.044 2.960
Rose 41.100 ± 0.211 1.860
Hot 50.080 ± 0.189 3.130
The composition of samples is given in Table 3.42. The decomposition products were separated on
fused-silica capillary columns and identified with MS. The thermal decomposition products of oleic
acid were hexanoic, heptanoic, octanoic, and nonanoic acids, and heptanal, octanal, and nonanal. The
thermal decomposition products of capsaicin are listed in Table 3.43. The thermal decomposition of
capsaicin results in many compounds. Cleavage, hydrogenation, and rearrangement equally play a
considerable role in the formation of decomposition products. Mixtures of capsaicin and oleic acid
produce a different decomposition pattern, as demonstrated in Table 3.44. The oxidation of oleic acid
decreases in the presence of capsaicin; that is, capsaicin serves as an antioxidant in this interactive
system. The amides formed during the thermal decomposition may influence the flavor of the
foodstuffs.
Phenolic compounds in green pepper berries were also analyzed by both TLC [ 493] and HPLC methods
[494]. For HPLC analysis, 50 g of green pepper was extracted with 7 × 100 mL of 80% aqueous
methanol. The extracts were dried under vacuum and redissolved in distilled water (0.1% w/v).
TABLE 3.42. Sample Composition for Oleic Acid—Capsaicin (CAP) Studies.

Sample Oleic Acid (mL) Capsaicin (mg)
Low-pressure oleic 2.0 0
Low-pressure 10% CAP 0.20 20
Low-pressure 50% CAP 0.20 200
Low-pressure CAP 0 100
Atm. pres. oleic 0.20 0
Atm. pres. 50% CAP 0.20 200
Atm. pres. CAP 0 100
Sealed tube 30% CAP 0.010 3.55
Sealed tube 50% CAP 0.010 10.34
Reprinted with permission from Reference [ 492], copyright © 1992, American Chemical Society.
Page 383
TABLE 3.43. Products of Thermal Decomposition of Capsaicin.

Compound MW Method of Identification Rel% *
Heptene 98 NBS 1213 <1
2-Methoxyphenol 124 authentic cmpd <1
2-Methoxy-4-methylphenol 138 authentic cmpd 1–10
Pentanamide 101 authentic cmpd <1
Nonanoic acid 158 authentic cmpd 1–10
8-Methyl -6-nonenoic acid 170 interpretation 1–10
Isononanamide 157 interpretation 1–10
Nonanamide 157 authentic cmpd 1–10
Methylvanillin 166 interpretation 1–10
8-Methyl -6-nonenamide 169 interpretation >10
Vanillin 152 authentic cmpd >10
N-Vanillin-di (8-methyl-6-nonen) imide 457 interpretation <1

* Quantitation of peak areas relative to internal standard.
TABLE 3.44. Products of Thermal Decomposition of Capsaicin (CAP) with Oleic Acid (auth.cmpd =
authentic compound).
Kovats Index
Compound IdentificationMethod RT 200 Stab-DA Oleic * 50 -50 * CAP*

x
2-heptene NBS 1213 1149 0.0 0.1 0.5

Heptanal Auth.cmpd 1087 1195 7.6 0.3 0.3
Octanal Auth.cmpd 1188 1299 14.9 0.1 0.0
Nonanal Auth.cmpd 1285 1401 28.8 0.3 0.1
Pentanoic acid Auth.cmpd ** 1747 1.4 0.0 0.0
2-methoxyphenol Auth.cmpd 1275 1860 0.0 0.5 0.7
2-methoxy -4-methyl- Auth.cmpd 1387 1956 0.0 0.4 1.0

phenol
Heptanoic acid Auth.cmpd ** 1963 10.0 0.1 0.0
Pentanamide Auth.cmpd 1356 2006 0.0 0.1 0.4
Octanoic acid Auth.cmpd ** 2076 17.5 0.2 0.1
Nonanoic acid Auth.cmpd ** 2188 17.8 1.1 2.9
8-methyl-6-nonenoic acid Interpret. 2259
** 0.5 44.5 4.0
Isonanamide Interpret. 1721 2397 0.0 0.7 2.0
Methylvanillin Interpret. 2409 0.0 0.3 0.9
Nonanamide Auth.cmpd 1742 2451 0.0 0.4 1.2
8-methyl-6-nonenamide Interpret. 1857 2529 3.0 41.7 84.5
Vanillin Auth.cmpd 1753 2556 0.3 7.2 10.6
9-octadecenamide NBS 31474 2776 3240 0.0 47.5 963.3
N-vanillin-9-octa- Interpret. 4062 0.0 tr 0.0

decenamide
* Percent normalized peak areas relative to internal standard.
** Retention varies with amount of material.
Reprinted with permission from Reference [ 492], copyright ©; 1992, American Chemical Society.
Page 384
Aliquots of extracts (200 mg) were hydrolyzed with 6 mL of 1 M HCl (100°C, 1 h), evaporated to
dryness, and redissolved in methanol:water (1:1) to give a concentration of 0.1% w/v. Separation was
performed on a C18 column (200 mm × 4.6 mm I.D.) using gradient elution: from 0% to 100% B in 30
min (solvent A and B being 2% aqueous acetic acid and methanol, respectively). The flow rate was 1.2
mL/min, and phenolic compounds were detected at 292 nm. Representative chromatograms of
hydrolyzed and unhydrolyzed phenolic extracts are shown in Figure 3.44. The lower number of peaks
in the
Figure 3.44.
Chromatogram of phenolic extract from Karimunda
green peppers. × chromatogram of unhydrolyzed
sample. Peaks: A = 3,4-dihydroxyphenylethanol
glucoside; B = 3,4-dohydroxy-6-(N-ethylamino)benzamide.
Y chromatogram of hydrolyzed sample.
Peaks: A' = 3,4-dihydroxyphenylethanol;
B = 3,4-dihydroxy-6-(N-ethylamino)benzamide;
b = gentisic acid + p-hydroxy benzoic acid;
c = vanillic acid and caffeic acid; d = syringic acid;
e = ferulic acid + synapic acid; f = salicylic acid.
Page 385
chromatogram of the unhydrolyzed sample indicates that phenol glycosides were not separated with this
method, and the chromatogram of solutes from the hydrolyzed extract also contained overlapping
peaks. The concentration of microcomponents of green pepper berries determined with this method is
given in Table 3.45. It has been stated that this HPLC method is rapid and can be used for the grading
of commercial black pepper.
Some Mexican spices such as Origanum vulgare and Pimpinella anisum were also studied by steam
distillation and SFE followed by GC [ 495].
Because of their volatility, the separation and identification of a sulphur compound from garlic extracts
was performed by GC-MS methods [ 496,497]. GC was performed on a fused-silica Carbowax 20 M
capillary column (50 m × 0.25 mm I.D.) using p-cymene as the internal standard. The carrier gas was
nitrogen (1.5 mL/min). Injector and detector temperatures were 200°C. The initial oven temperature
was 50°C, increased to 150°C at 1°C/min. A characteristic chromatogram is shown in Figure 3.45.
Volatiles from garlic extract are well separated on the capillary column, and the baseline separation
allows the exact quantitation of the fractions. The mean values of five parallel extractions are compiled
in Table 3.46. It was stated that compounds 16 and 18 are artifact formed during the separation process.
The recovery of the method varied between 74% and 90%, and the detection limit (ng/0.2 µL) was
1.76-3.00. Since the method is suitable for the separation and quantitative determination of the main
components in garlic, it was proposed for the analysis of garlic species and products containing garlic.
TABLE 3.45. Composition (area %) of Phenolic and Other Constituents in Total Hydrolyzed Samples of Six
Varieties of Fresh Green Pepper Berries by HPLC.
Enzyme Active Enzyme Inactive Phenolics

Phenolics
Gentisic
Acid +
p- Vanillic
Hydroxy- Acid + Ferulic Acid
Portocate benzoic Caffeic Syringic + Synapic Salicylic
Varieties A' B chuic Acid Acid Acid Acid Acid Acid
1 1.2 31.6 Trace 4.4 1.8 6.2 2.4 0.29
2 2.0 44.0 Trace 4.6 1.8 6.6 2.9 Trace
3 1.5 27.6 Trace 5.5 0.5 12.5 2.3 Trace
4 6.4 24.4 Trace 6.4 1.7 6.4 2.8 0.21
5 8.0 27.5 Trace 4.8 1.6 11.2 4.0 Trace
6 7.7 25.1 Trace 1.5 1.2 4.6 2.5 Trace
1 = Wayanadan; 2 = Karimunda; 3 = Geerakamundi; 4 = Nadesan; 5 = Balankotta; 6 = Panniyur-1. Each

value is the average of two determinations. A' = 3,4-dihydroxy phenyl ethanol (retention 8 min); B = 3,4-
dihydroxy-6-(N-ethylamino)benzamide (retention 3.0 min).
Page 386
Figure 3.45.
Capillary gas chromatogram of volatile components of garlic extract:
1 = ethyl acetate; 2 = allyl methyl sulphide; 3 = dimethyl disulphide;
4 = 2 -propen-1-ol; 5 = diallyl sulphide; 6 = allyl methyl disulphide;
7 = unknown; 8 = dimethyl trisulphide; 9 = unknown; 10 = diallyl disulphide;
11-13 = unknown; 14 = allyl methyl trisulphide; 15 = unknown;
16 = 3-vinyl -4(H)-1,2-dithiin; 17 = diallyl trisulphide;
18 = 2 -vinyl -4(H)-1,3-dithiin; STD = internal standard.
The flavor substances in wine vinegar have also been studied by various chromatographic techniques.
The volatiles in wine vinegar were determined by GC-MS, and the data were used as indicators of
quality [ 498]. RP-HPLC was used for the analysis of phenolic acids in wine vinegar [ 499]. Vinegars
were concentrated and extracted with diethyl ether, and the residue was dissolved in methanol:water
(2:1 v/v). Separation was performed on a C18 column (250 mm × 4 mm I.D.) thermostatted at 25°C. The
eluent was acetic acid:2-propanol:methanol:water (2:2:9:87 v/v). A linear gradient flow was employed
from 1 mL/min to 1.5 mL/min in 40 min. The solutes were detected at 280 nm. Typical chromatograms
of `Jerez' wine vinegar and a conventional wine vinegar are shown in Figure 3.46. The method allows
the separation of phenolic acids and aldehydes of vinegars using isocratic elution mode. The
composition of phenolics depends considerably on the quality of the vinegar. The quantitative data are
compiled in Table 3.47. The method was proposed for the comparison of vinegars of different types and
origins and to follow the acetification process.
Page 387
TABLE 3.46. Volatile Sulphur Compounds in Garlic (mg per 100 g fresh tissue ± SD).
Peak No. Sulphur Compound Mg/100 g Fresh Tissue ± SD
2 Allyl methyl sulphide 0.167 ± 0.015
3 Dimethyl disulphide 0.204 ± 0.026
5 Diallyl sulphide 0.122 ± 0.014
6 Allyl methyl disulphide 1.193 ± 0.068
8 Dimethyl trisulphide 0.075 ± 0.011
10 Diallyl disulphide 5.297 ± 0.846
14 Allyl methyl trisulphide 0.562 ± 0.078
16 3-Vinyl-4(H)-1,2-dithiin 4.088 ± 2.266
17 Diallyl trisulphide 1.288 ± 0.183
18 2-Vinyl-4(H)-1,3-dithiin 830.069 ± 1.757
3.2.1.2—
Wines
Phenolic compounds in wines influence not only the browning and aging processes, but also exert a
marked impact on the organoleptic characteristics of wines. HPLC has been used for the quantitative
determination of free phenolic acids in wines [ 500], and the phenolic content of wines was employed for
taxonomic studies [501]. Reversed-phase ion-pair chromatography was employed for the determination
of mono-, poly-, and hydroxycarboxylic acids in wines and other beverages [ 502]. Derivatization was
performed by the following manner: 50 µL of wine, 25 µL of fruit juice, and 50 µL of Japanese sake
were diluted by water to 100 µL (100 µL of beer was further used without dilution). The samples were
mixed with 200 µL of ethanol containing 400 nmol of 3-methylglutaric acid (internal standard), 200 µL
of 2-NPH.HCl solution, and 200 µL of 1-EDC.HCl solution. [A solution of 0.02 M 2-NPH.HCl was
prepared by dissolving 2-nitrophenylhydrazine hydrochloride in 0.1 M HCl:ethanol (1:1 v/v); 0.25 M of
1-EDC.HCl solution was prepared by dissolving 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide
hydrochloride in pyridine:ethanol (3:97 w/v)]. The mixture was heated at 80°C for 5 min; then 200 µL
of 10% w/v KOH solution was added, heated further at 80°C for 5 min, and then cooled. Separation
was performed on a C18 column (250 mm × 6 mm I.D., particle size 4 µm) at 35°C using 0.005 M
phosphate buffer:acetonitrile:methanol (80:10:10 v/v) containing 0.005 M tetraethylammonium
bromide. The flow rate was 2 mL/min and the carboxylic acids were detected at 400 nm. Typical
chromatograms are shown in Figure 3.47. Citric and malic acid hydrazides eluted in two peaks because
of the stereochemical isomers of the derivatives. The correlation coefficients of the relationship peak
height ratio versus the amount of acid (nmol) were, in each instance, over
Page 388
Figure 3.46.
Chromatograms obtained from a (A) `Jerez' wine
vinegar and from (B) a vinegar by quick acetification.
Peaks: 1 = gallic acid; 2 = pyrogallic acid;
3 = protocatechuic acid; 4 = catechin; 5 = gentisic acid;
6 = p-hydroxybenzoic acid; 7 = vanillic acid;
8 = caffeic acid; 9 = syringic acid; 10 = vanillin;
11 = p-coumaric acid; A-E = unidentified.
The Netherlands.)
Page 389
TABLE 3.47. Relative Percentages of Phenolic Acids in the Vinegars.

Relative Percentages
Jerez Vinegars Conventional
Vinegars
Number Substances 1 2 3 4
1 Gallic acid 48.0 57.0 21.2 50.9
2 Pyrogallic acid — — 3.0 2.1
3 Protocatechuic acid 4.9 4.8 3.4 4.9
4 Catechin 16.9 — 14.7 6.0
5 Gentisic acid — 3.8 — 2.8
6 p-Hydroxybenzoic 3.3 4.2 4.9 3.7

acid
7 Vanillic acid 5.0 3.1 1.9 1.4
8 Caffeic acid 8.3 11.8 28.6 14.1
9 Syringic acid 2.9 2.3 — 1.8

10 Vanillin 3.1 2.5 10.8 3.2
11 p-Coumaric acid 7.6 10.5 11.5 9.1
12 Veratric acid — — — —
13 m-Coumaric acid — — — —
Netherlands.
0.999, showing the good linearity of the correlation. The recovery varied between 97.5% and 102.8%,
depending on the type of carboxylic acid. The intra-assay and interassay precision of the method was
between 0.5% and 3.7%. The quantitative data are compiled in Table 3.48. It was established that the
method is suitable for the separation and quantitative determination of the major carboxylic acids in
beverages. The advantage of the method is that the chromatograms are very clean because of the visible
detection wavelength. It was proposed as a tool for routine analysis of carboxylic acids in beverages.
The volatile aroma components of wine are mainly analyzed by various gas chromatographic methods.
Because the concentration of the majority of volatile aroma components in wine are low, extraction and
preconcentration methods are necessary before they can be analyzed by GC. Thus, the aroma
composition of Galician (northwestern Spain) white wines was determined by a purge and cold trapping
injection—capillary gas chromatographic method [ 503,504]. The volatile compounds were purged from
wine and carried to a capillary trap held at -120°C. The volatiles were cryofocused into the column
head. The schematic diagram of the purge and cold trap injector system is shown in Figure 3.48.
Separation was performed on a capillary column (30 m × 0.22 mm I.D., film thickness 0.25 µm). The
temperature gradient was 5 min at 40°C, to 150°C at 5°C and final hold for 3 min. The carrier gas was
nitrogen (FID) or helium (MS). The concentration of volatiles determined with a
Page 390
Figure 3.47.
Carboxylic acid profile of (A) orange juice, (B) white wine, and (C) beer. Peaks:
1 = citric; 2 = tartaric; 3 = malic; 4 = succinic; 5 = fumalic; 6 = internal
standard; 7 = glycolic; 8 = L-pyroglutamic; 9 = lactic; 10 = acetic acid hydrazide.
slightly modified method is given in Table 3.49. The data represent the average of 14 Rias Baixas and
eight non-Rias Baixas wines.
Various multivariate mathematical statistical methods such as principal component analysis and
hierarchical cluster analysis indicated that the two types of wines can be differentiated by the
composition of the volatile compounds; however, some outliers were found in both groups.
In order to avoid the use of complicated sample preparation processes, SPME has also been employed
for the fast screening of wines [ 505]. A 100-µm polydimethylsiloxane fiber was immersed in the
headspace of 125 mL of wine for 10 min at room temperature and then desorbed in the injector port of a
gas chromatograph for 5 min. Separation was performed on a PAG fusedsilica capillary column (30 m
× 0.25 mm I.D., 0.25 µm film thickness). The initial oven temperature was 35°C for 5 min, to 100°C at
5°C/min, to 200°C at 3°C/min, 1 min hold, to 220°C at 10°C/min. The detector (FID) and injector
temperatures were 250°C. Characteristic chromatograms of Hungarian and
TABLE 3.48. Determination of Major Carboxylic Acids in Beverages.
Carboxylic Acid g/L
Beverage Citric Tartaric Malic Succinic L-Pyroglutaric Lactic
Apple juice 0.52 ± 0.01 ND 2.37 ± 0.04 ND ND ND
Orange juice 5.35 ± 0.14 ND 1.34 ± 0.03 ND ND ND
White wine 0.41 ± 0.01 1.28 ± 0.01 1.59 ± 0.02 0.39 ± 0.01 ND 0.91 ± 0.03
Red wine 0.20 ± 0.01 1.95 ± 0.02 0.29 ± 0.004 0.66 ± 0.02 ND 2.99 ± 0.05
Beer 0.16 ± 0.01 ND 0.05 ± 0.001 0.04 ± 0.001 0.18 ± 0.004 0.08 ± 0.001
Japanese sake 0.14 ± 0.004 ND 0.07 ± 0.002 0.17 ± 0.003 0.27 ± 0.01 1.19 ± 0.03
Data are expressed as the mean ± SD (n = 3).
Page 392
Figure 3.48.
Schematic view of the purge and cold trap injector system showing the two main
modifications introduced: (A) sample refrigerator device; (B) stopped purge flow valve.
corresponding author, copyright © 1995, American Chemical Society.)
Italian muscat wines are shown in Figure 3.49. It was stated that the method allows the detection of the
addition of 1 ppm cold-pressed coriander seed oil and is suitable for the differentiation between wines
of different origin. Sorbent extraction has also been used for the separation of aroma components in
wine, and the results were compared with those of liquid-liquid extraction (LLE) [ 506]. For the sorbent
extraction, a column (30 cm × 6 cm I.D.) was filled with 150 g of an inert, high surface area
diatomaceous earth. Samples of Chardonnay wine mixed with internal standards were poured into the
column. After 15 min, 200 mL of pentane-methylene chloride (2:1 v/v) were added to the column, and
after a further 10 min; 100 mL of eluate were collected. The eluate was dried with sodium sulfate,
reduced to 1 mL, and used for GC analysis. LLE was carried out by extracting 500 mL of wine with 70
mL of extraction solvent for 8 h. The extract was treated as the sorbent extraction. Separation was
performed on a fused-silica capillary column (50 m × 0.2 mm I.D., coated with Carbowax 20M).
Injector and detector (FID) temperatures were 240°C and 260°C, respectively. Oven temperature was
60°C for 3 min, to 180°C at 3.5°C/min, and 60 min hold. Helium was the carrier gas. The quantitative
results are presented in Table 3.50. The concentration of aroma
Page 393
TABLE 3.49. Volatile Compounds Determined in Galician White Wines (as % area
relative to the internal standard).
Rias Baixes Wines Non-Rias Baixas Wines
Compound Mean SD Mean SD
Ethyl butyrate 0.33 0.089 0.2 0.22
Isobutanol 3 1.2 3 1.8
Isoamyl acetate 3 1.5 2 3.5
Unknown 0.08 0.03 0.04 0.028
Ethyl 2-butenoate 0.2 0.071 0.1 0.089
3-methylbutanol + 2- 78 12 64 20
methylbutanol
Ethyl hexanoate 1.4 0.4 2 1.9
Hexyl acetate 0.02 0.004 0.01 0.012
4-Methyl -1-pentanol 0.15 0.061 0.07 0.05
Ethyl lactate 24 16 11 11
trans-3-Hexen-1-ol 0.09 0.047 0.1 0.14
3-Ethoxypropanol 0.1 0.15 0.02 0.025
cis-3-Hexen-1-ol 0.2 0.05 0.2 0.13
2-Butoxyethanol 0.02 0.007 0.01 0.014
Ethyl octanoate 2.1 0.61 1.1 0.86
Furfural 0.2 0.12 0.01 0.028
Ethyl-3-hydroxybutyrate 0.1 0.03 0.09 0.054
2-Methylthioethanol 0.02 0.022 0.05 0.058
Linalool 0.11 0.045 0.07 0.035
Isobutyric acid 0.11 0.053 0.13 0.053
Butyric acid + Hotrienol 0.7 0.24 0.4 0.41
Ethyl decanoate 0.29 0.086 0.23 0.07
3-Hydroxybutyric acid 0.6 0.28 0.4 0.18
Diethyl succinate 1.4 0.8 1 2.2
Isovaleric acid 0.3 0.12 0.3 0.16
3-Methylthiopropanol 0.14 0.074 0.2 0.23
Terpineol 0.19 0.063 0.04 0.045
2-Phenylethyl acetate 0.3 0.12 0.3 0.39

Hexanoic acid 6 1.8 4 2.1
Citronellol 0.02 0.015 0.01 0.012
Nerol 0.27 0.052 0.2 0.1
Benzyl alcohol 0.12 0.058 0.06 0.097
2-Phenylethanol 26 6.1 36 39
Diendiol 1 0.4 0.13 0.4 1.2
Geraniol 0.01 0.012 0.04 0.06
Diethyl malate 2 1.4 0.3 0.25
Octanoic acid 11 4.3 8 5
Decanoic acid 3 1.3 3 2.4
Reprinted from Reference [ 504] with permission

Page 394
Figure 3.49.
Comparison of muscat wines by SPME -GS. Peaks: 1 = isobutanol; 2 = isoamyl
acetate; 3 = 3-methyl-1-butanol; 4 = ethyl hexanoate; 5 = hexanol;
6 = 3-hexen-1-ol(Z); 7 = ethyl octanoate; 8 = linalool; 9 = linalyl acetate;
10 = ethyl decanoate; 11 = terpineol; 12 = citronellol; 13 = geraniol;
14 = phenethyl alcohol.
(Reprinted from Reference [ 505] with permission of Supelco, Bellefonte, PA.)
compounds determined by both methods showed good agreement in the majority of cases. This finding
indicates that sorbent extraction can be for the determination of aroma components in wines. It was
stated that sorbent extraction is simple, accurate, and precise and less time-consuming than the
traditional LLE technique.
The highly apolar solvent Freon 113 has also been used for the extraction of the aroma components
from wine [ 507] and from wine distillate [ 508]. The adsorption-thermal desorption-GC has also been
applied for the determination of wine aromas [ 509]. Volatiles were concentrated by bubbling dry
nitrogen (flow rate 67 mL/min) over 50 mL of wine at 30°C and by adsorbing in a metal tube (88.9 mm
× 4.50 mm I.D., filled with 0.2 ± 0.01 g Tenax GC, 60 mesh). After adsorption, the remaining ethanol
and water were removed from the tube by nitrogen flow. The scheme of the adsorption is shown in
Figure 3.50. Thermal desorption of the aroma compounds from the tube was performed at 300°C for 5
min. The low and high temperatures of the cold trap were -30°C and 350°C, respectively; injection time
was 40 s. Separation was carried out on a fused-silica capillary column (50 m × 0.22 mm I.D., 0.25 µm
of BP-21). The initial temperature was 50°C, up to 180°C at 2.5°C/min, and final hold 8 min. A typical
chromatogram is shown in Figure 3.51. The regression coefficient was over 0.96 for each compound,
and the variation coefficient was between 3% and 5%. Because of its reliability and simple sample
preparation step, this method was proposed for the determination of the volatile components in wines.
TABLE 3.50. Concentrations (c), 95% Confidence Interval Limits, Bandwidths, and Percentage Overlap (O) of the Confidence Intervals
Obtained Determining the Added Compounds and Those Naturally Occuring with Continuous Liquid-Liquid Extraction and Sorbent
Extraction.
Liquid-Liquid Extraction Sorbent Extraction
Confidence Confidence
Compound Added c/µg.L-1 Limits/µg.L-1 Bandwidth (%) O (%) c/ µg.L-1 Limits/µg.L-1
Methyl decanoate (63 µg/L) 54 36–69 28 c* 54 39 –71
9-Decen-1-ol (32 µg/L) 35 25–45 29 58 30 19 –40
Methyl tridecanoate (61 µg/L) 23 0–44 95 c 32 0–79
trans-Hex-2-enoic acid (249 250 210–280 16 c 230 150–300

µg/L)
Naturally occurring
Ethyl propanoate 145 117–169 18 30 111 69–147
Ethyl 2-methylpropanoate 150 65–210 49 c 150 98–200
Ethyl butanoate 112 84–137 24 c 110 94–125
Ethyl 2-methylbutanoate 12 15–27 27 c 11 8–14
Ethyl 3-methylbutanoate 27 21–32 19 64 25 18 –30
Ethyl hexanoate 210 180–250 16 72 200 150–250
Ethyl octanoate 330 260–380 19 82 350 300–400
Ehtyl 3-hydroxybutanoate 370 290–450 21 26 460 390–520
Ethyl decanoate 116 57–166 47 76 133 62–194
Diethyl succinate 660 560–750 15 25 790 670–900
2-Methylpropyl ethanoate 68 44 –Ft91 35 c 84 17–138
3-Methylbutyl ethanoate 1300 1000–1500 17 44 1100 840–1300
4-Ethoxycarbonyl-4-
hydroxybutanoic acid τ-lactone 13200 10500–15000 18 c 15300 7780–19800
2-Phenylethyl ethanoate 340 300–450 19 c 330 190–460
Butan-1-ol 1200 850–1500 26 c 1200 830–1500

TABLE 3.50.
Liquid -Liquid Extraction Sorbent Extraction
Confidence Confidence
Compound Added c/ µg.L-1 Limits/µg.L-1 Bandwidth (%) O (%) c/ µg.L-1 Limits/µg.L-1 Bandwith (%)
4-Methyl pentan-1-ol 92 80–103 12 c 91 72–110
3-Methyl pentan-1-ol 190 150–220 19 86 180 150–210
Hexan-1-ol 1740 1440–2010 16 c 1660 1460–1850
Trans-Hexen-3-ol 124 102–144 17 58 113 96–130
3-Ethoxypropane-1-ol 110 87–130 19 c 110 65–140
Cis-Hexen-3-ol 75 62 –88 17 c 76 59–96
2-Methylpropanoic acid 2600 2100–3000 17 c 2300 1400–3100
Butanoic acid 910 770–1000 14 39 750 510–970
3-Methylbutanoic acid 2900 2100–3600 25 53 2600 2100–3000
Hexanoic acid 1160 1040–1280 10 c 1130 981–1300
Octanoic acid 2160 1580–2710 26 77 2000 1500–2500
Decanoic acid 870 640–1100 26 c 830 550-1100
* c = Complete overlapping.
Reprinted from Reference [ 506] by permision of The Royal Society of Chemistry.

Page 397
Figure 3.50.
Adsorption scheme: (1), (2), (4), (5), (8),
and (9) stop valve; (3) 50 mL of sample
in a 30°C bath; (6) metal tube containing 0.2
g of Tenax at 25°C; (7) flow control.
Line A: volatile adsorption,
1—3—5—6—7. Line B: ethanol
and water elimination, 1—8—9—6—7.
corresponding author, copyright
Figure 3.51.
Chromatogram for a wine whose compounds
and their concentrations are (in ppm) as follows:
1, isoamyl acetate (1.82); 2 = limonene (0.39);
3 = trans-2-hexenal (5.02); 4 = ethyl hexanoate
(0.55); 5 = pentanol (0.13); 6 = ethyl pyruvate (0.14);
7 = hexyl acetate (1.27); 8 = ethyl heptanoate (2.24);
9 = hexanol (0.72); 10 = ethyl acetate (0.96);
11 = methyl octanoate (IS);
12 = trans-2-hexen-1-ol (12.00);
13 = benzaldehyde (0.37); 14 = linalool (1.56);
15 = octanol (0.13); 16 = cis-3-hexen-1-ol (0.15);
17 = τ-butyrolactone (1.70); 18 = diethyl
succinate (2.30); 19 = cironellol (1.73);
20 = α-terpineol (0.24); 21 = ethyl decanoate
(0.12); 22 = 2-phenethyl acetate;
23 = ethyl laurate; 24 = hexanoic acid (0.96);
25 = benzyl alcohol (3.59); 26 = heptanoic acid (1.40).
(Reprinted from Reference [ 509 ] with permission
from the publisher and the corresponding author,
Page 398
Off-line and on-line SFE combined with GC has also found application in the analysis of wine aroma
[ 510]. Three different SFE-GC methods were compared. Samples were retained on a solid support for
SPE. Method A uses SFE, and the extract is collected at -5°C in a solid-phase trap containing
octadecyl-silica. After extraction, the aroma compounds are eluted with dichloromethane and used for
GC analysis. Method B uses the quartz liner of the programmed temperature vaporizer (PTV) to collect
the extract. After extraction, the liner is placed in the injector body of GC. Method C directly connects
the SFE apparatus to the GC via PTV. The schematic diagram of on-line SFE-GC is shown in Figure
3.52. It was established that filter paper was the best sorbent for wines. Some chromatograms of aroma
compounds in wines are shown in Figure 3.53. The relative standard deviation of methods A and B was
markedly lower than that of the on-line SFE-GC (method C); however, the recovery of method C was
higher than those of methods A and B. The detection limit of method C varied between 0.08 ng and
0.34 ng. Because of its simplicity, method C was proposed for the analysis of aroma composition of
wines.
Amberlite XAD -2 resin has also been used for preconcentration of volatile compounds of white wines
before capillary GC analysis [ 511]. The results of direct injection (DI) of wines have been compared
with the results of liquid-liquid extraction with external concentration (LLE-EC), liquid-liquid
extraction with internal concentration (LLE-IC), simultaneous
Figure 3.52.
Schematic diagram off on-line SFE-GC coupling using a programmed
temperature vaporizer as interface. The extraction, collection,
and chromatographic analysis can be accomplished in a single step.
(Reprinted from Reference [ 510 ] with permission from the publisher and the
Page 399
Figure 3.53.
Chromatograms obtained from (a) and (b), two white wines, (c and d), two rosè wines,
and (e and f) two red wines using the on -line SFE-GC via PTV approach.
(Reprinted from Reference [ 510] with permission from the publisher and the corresponding author,
Page 400
distillation-extraction with concentration (SDEC), and simultaneous distillation-extraction without

concentration (SDENC) [ 512]. LLE was performed by extracting 250 mL of wine with 150 mL of
Freon 11 for 24 h. LLE-EC was carried out by concentrating the Freon extract at 32°C. The PTV was
operated in the cold-split mode, and the body injector was held at 40°C during sampling and then
increased to 375°C at 14°C/min and held for 5 min. For LLE-IC, a 2-cm plug of Tenax TA was packed
in the vaporization insert. SDEC was carried by concentrating the extract of simultaneous distillation-
extraction procedure under a stream of inert gas. SDENC was performed by using the extract without
preconcentration using PTV. DI was performed by using a modified PTV device. The vaporization inlet
of PTV was packed with GasChrom 220:Tenax TA (55:45). GC analyses were carried out on a fused-
silica capillary column (50 m × 0.22 mm I.D., coating 0.25 µm of cross-linked BP-21). The initial oven
temperature was 35°C for 8 min and then increased to 180°C at 2°C/min. Gas chromatograms of a wine
sample obtained by various preparation methods are shown in Figure 3.54. The chromatograms indicate
that the simultaneous distillation-extraction methods result in a considerable loss of aroma compounds
while the efficiency of LLE is similar to that of the direction injection method. The RSDs were also the
lowest for the direct injection method, indicating that this procedure is superior to the other methods
using various sample preconcentration steps. Because the method is simple, rapid, and does not require
solvent, it can be used for the study of the aroma composition of wines. GC has also been used for the
study of the aroma compounds formed from must during yeast fermentation [ 513]. SPE, followed by
GC-MS, has been employed for the analysis of the aroma components in cider [514]. The internal
standard 2-ethyl-1-hexanol (16.69 mg/L) was added to 20 mL of cider. The sample was acidified with
HCl (pH 3.00), degassed, and percolated through a kieselguhr cartridge. After 30 min equilibration, the
cartridge was washed with dichloromethane. The organic fraction was dried over anhydrous sodium
sulphate, evaporated to 0.5 mL, and used for GCMS. GC separation was performed on a FFAP column
(50 m × 0.22 mm I.D., film thickness 0.33 µm). The injector and detector temperatures were 250°C.
The column temperature varied from 40°C to 220°C at 3°C/min. The total ion chromatograms of
volatile compounds in fruity and sharp cider are shown in Figure 3.55. The aroma compounds were
well separated under these experimental conditions; however, organic acids with low molecular mass
were not extracted, and the short-chain esters were eluted in the solvent front. The quantitative results
of aroma components of fruity and sharp ciders are given in Table 3.51. The concentration of the aroma
compounds considerably differed in fruity and sharp cider, indicating that these compounds exert a
marked impact on the aroma of cider.
Page 401
Figure 3.54.
Chromatograms from the aroma of a wine of the Verdejo variety obtained
by (a) liquid-liquid extraction with external concentration, (b) liquid-liquid
extraction with internal concentration, (c) simultaneous distillation-extraction
with concentration, (d) simultaneous ditillation-extraction without
concentration, and (e) direct injection: 1 = ethyl lactate + cis-3-hexen-1-ol;
2 = acetic acid; 3 = propanoic acid; 4 = linalool; 5 = τ-butyrolactone;
6 = butanoic acid; 7 = α-terpineol; 8 = hexanoix acid; 9 = benzyl alcohol;
10 = 2 -phenylethanol; 11 = octanoic acid;
12 = ethyl hexadecanoate; 13 = decanoic acid.
and the corresponding author, copyright © 1995, American Chemical Society.)
Page 402
Figure 3.55.
Total ion chromatograms of volatile compounds in (A) fruity and (B) sharp cider:
1 = isobutanol; 2 = isoamyl acetate; 3 = butanol; 4 = amyl alcohols; 5 = acetoin;
6 = ethyl lactate; 7 = ethyl caprylate; 8 = 2,3(R, R and/or S, S)-butanediol;
9 = 2,3-butanediol (meso form); 10 = ethyl caprylate; 11 = dihydro2(3H)-furanone;
12 = diethyl succinate; 13 = 3-methylthio)-1-propanol; 14 = 2 -phenylethyl acetate;
15 = ethyl laurate; 16 = benzyl alcohol; 17 = 2-phenylethanol; 18 = caprylic acid;
19 = 4-ethylphenol; 20 = ethyl palmitate; 21 = capric acid; 22 = lauric acid; 23 = myristic acid.
(Reprinted from Reference [ 514] with permission from Chromatographia and the corresponding author.)
Page 403
TABLE 3.51. Content (mg L-1 ± RSD) of Volatile Compoenents of Two Ciders
Determined by Solvent Extraction and Extrelut Resin.
Component Rt (min) Fruity Cider Sharp Cider
1-Butanol 14.65 2.24 ± 4.09 3.88 ± 4.60
Amyl alcohols 17.43 134.29 ± 3.46 170.85 ± 5.50
Isobutanol 12.59 7.73 ± 3.37 21.92 ± 7.55
2-Phenylethanol 47.95 185.24 ± 4.24 57.30 ± 5.90
Benzyl alcohol 46.25 0.53 ± 5.60 1.54 ± 8.83
Ethyl lactate 23.68 11.86 ± 4.80 100.27 ± 3.87
Diethyl succinate 38.42 2.27 ± 4.80 2.32 ± 3.28
Ethyl caprylate 27.88 2.22 ± 3.50 1.92 ± 6.43
Ethyl caprate 36.73 3.39 ± 7.80 2.15 ± 5.89
Ethyl laurate 44.92 0.68 ± 7.00 ND
Ethyl palmitate 58.74 0.38 ± 3.66 0.66 ± 4.14
i-Amyl acetate 13.98 0.28 ± 6.30 0.58 ± 9.50
2-Phenylethyl acetate 44.01 1.21 ± 5.15 ND
Dihydro-2(3H)-furanone 37.21 3.50 ± 5.45 7.90 ± 2.94
Acetoin 21.20 ND 1.92 ± 4.88
2,3(R, R -S, S)-Butanediol 32.44 37.36 ± 6.14 90.89 ± 4.84
2,3-Butanediol(meso form) 33.98 22.10 ± 6.30 75.34 ± 7392
3-(Methylthio)-1-propanol 40.00 1.41 ± 8.88 1.80 ± 6017
4-Ethylphenol 56.49 1.37 ± 8.92 3.33 ± 5.56
Caprylic acid 52.23 1.22 ± 8.03 3.51 ± 4.39
Capric acid 59.13 1.25 ± 8.10 2.20 ± 2.42
Lauric acid 66.16 4.88 ± 5.00 0.77 ± 8.18
Myristic acid 76.40 1.91 ± 4.80 ND
3.2.1.3—
Beer, Malt, and Hop
Because the concentration of 2-furaldehyde (F) and 5-hydroxymethyl-2-furaldehyde (HMF)

considerably influence the taste and browning of beer, an RP-HPLC method was developed for their
separation and simultaneous determination in beer [ 515]. Beer was degassed and filtered prior to
derivatization. A 5-mL aliquot was mixed with 4 mL of 2,4-dinitrophenylhydrazine (DNPH) in
acetonitrile; 0.4 mL of 70% perchloric acid was added, and the volume was adjusted to 10 mL with
DNPH solution. The pH was adjusted to 1, and the mixture was stirred at room temperature for at least
25 min. After derivatization, samples were immediately injected into the HPLC system. Separation was
performed on a C18 column (250 × 4.6 mm I.D., particle size 5 µm). Eluent was acetonitrile:water
(55:45 v/v). Flow rate was 1 mL/min, and the detection wavelength was set to 385 nm. The RP-HPLC
method separated the DNPH derivatives of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde well; as
demonstrated in Figure 3.56. The method showed good validation
Page 404
Figure 3.56.
Liquid chromatographic separation of the
2,4-dinitrophenylhydrazones of carbonyl
compounds from a commercial sample
of beer. Peak identification: (1)
2,4-dinitrophenylhydrazine;
(2) 2,4 -dinitrophenylhydrazone of
5-hydroxymethyl -2-furaldehyde;
(3) 2,4 -dinitrophenylhydrazone of 2-furaldehyde.
(Reprinted from Reference [ 515],
copyright © 1995. With kind permission
from Elsevier Science—NL, Sara Burgerhartstraat
parameters: the coefficient of regressions between the peak area and solute concentration were over
0.9999, and the recovery was between 91% and 98%. The detection limits were 2.2 10-8 mol/L and 2.4
10-8 mol/L for F and HMF, respectively. The RSDs were 5% for F and 3% for HMF. The
concentration of F and HMF in commercial beer samples is given in Table 3.52. It was established that
beers stored at elevated temperatures have a higher F and HMF content, indicating that their
concentrations can be used as an indicator of the quality of beer.
Because purine nucleosides and free bases also contribute to the taste of beer, an HPSEC method was
developed for their separation and quantitative
Page 405
TABLE 3.52. Concentrations of 2 -Furaldehyde and 5 -Hydroxymethyl-2-Furaldehyde Found in Some

Commercial Beers. *
Concentration of 5-Hydroxymethyl 2 - Concentration of 2-Furaldehyde
Sample No. Furaldehyde (mol/L) (mol/L)
1 (7.30 ± 0.21) 10 -5 (2.32 ± 0.11) 10 -6
2 (5.40 ± 0.11) 10 -5 (2.45 ± 0.09) 10 -6
3 (6.80 ± 0.21) 10 -5 (1.01 ± 0.03) 10 -6
4 (2.62 ± 0.06) 10 -5 (9.36 ± 0.28) 10 -7
5 (3.24 ± 0.07) 10 -5 (3.60 ± 0.12) 10 -6
6 (6.31 ± 0.20) 10 -5 (3.13 ± 0.11) 10 -6
7 (5.84 ± 0.12) 10 -5 (1.32 ± 0.05) 10 -6
8 (3.24 ± 0.05) 10 -5 (8.41 ± 0.29) 10 -7
* Mean of six determinations ± SD
Sara Burgerharstraat 25, 1055 KV Amsterdam, The Netherlands.
determination [ 516]. Beer was applied to the chromatographic columns (300 × 10 mm I.D.) after
degassing and filtering. Columns were filled with HPSEC supports (Superose 12 = cross-linked agarose
and Superdex 75 = composite of cross-linked agarose and dextran). Eluent was 100 mM NaCl, and the
flow rate was 0.25 mL/min. Solutes were detected at 260 nm and 280 nm. Purine nucleosides were well
separated on both columns (Figures 3.57 and 3.58), the elution order being identical. The concentrations
of purine nucleosides and free bases in a variety of commercial beers determined by column
chromatography using the HPSEC matrix Superdex 75 are presented in Table 3.53. The quantitative
results were also similar on both columns, indicating that both of them can be used for the separation
and quantitative determination of purine nucleosides and free bases in beer and wort. Since the method
is rapid and allows the analysis of purine nuleosides without any pretreatment of the sample, it was
proposed for routine quality control and for research concerning the fate of nucleosides during the
brewing process.
GC has also found application in the analysis of the volatile compounds in beer. Headspace GC was
employed for the determination of vicinal diketones in beer [ 517]. The beer samples were degassed,
saturated with ammonium sulphate, 2.3-hexanedione internal standard added, and the mixture was held
at 35°C for 40 min, then the concentration of vicinal diketones in the headspace was measured by GC.
Both packed (2 m × 3.125 mm I.D., packed with 0.2% Carbowax 1500 on 80/100 mesh Cabopack B)
and fused-silica capillary (60 m × 0.25 mm I.D., film thickness of CP-WAX 57 CB 0.4 µm) columns
were included in the experiment. Nitrogen carrier gas and an electron capture detector were used for
both columns. The chromatographic conditions for a packed column were: injector, column, and
detector temperatures 200°C, 120°C, and 150°C, respectively; for capillary column the same
temperatures were 105°C,
Page 406
Figure 3.57.
Fractionation of a sample of commercial beer on a column of
Superose 12 (F: phenylalanine; Y: tyrosine; G: guanosine; dG:
deoxyguanosine; W: tryptophan; A: adenosine; dA: deoxyadenosine;
x: xanthine; Gn: guanine; Ad: adenine).
Figure 3.58.
Fractionation of a sample of commercial beer on a column
of Superdex 75 (F: phenylalanine; Y: tyrosine; G: guanosine;
dG: deoxyguanosine; W: tryptophan; A: adenosine; dA:
deoxyadenosine; x: xanthine; Gn: guanine; Ad: adenine).
Page 407
TABLE 3.53. Analysis of Purine Nucleosides and Free Bases in a Variety of Commercial Beers by Column
Chromatography Using the High-Performance, Size Exclusion Chromatography Matrix Superdex 75.
Beer Origin ABV % G + dG A + dA X Gn Ad
Lager Germany 4.8 109 18 21 6 7
Lager Spain 5.4 39 7 24 3 2
Lager Canada 5.0 55 19 5 3 2
Lager Great Britain 3.5 55 19 9 3 2
Lager Czechoslovakia 5.0 41 11 30 11 1
Lager Belgium 4.0 57 13 38 3 5
Wheat beer Germany 5.5 48 15 29 4 4
Mild ale Great Britain 3.5 73 40 7 2 1
Ale Great Britain 3.0 39 23 6 2 1
Ale Great Britain 3.0 39 11 6 1 1
Light ale Great Britain 3.0 47 26 7 1 1
Ale Great Britain 3.5 72 43 <1 2 1
Low-alcohol beer Great Britain 1 19 16 5 3 4
Stout Great Britain 4.1 69 20 16 2 <1>
ABV % = ethanol content percent by volume, G = guanosine, dG = deoxyguanosine, A = adenosine, dA =
deoxyadenosine, X = xanthine, Gn = guanine, Ad = adenine).
55°C, and 110°C. The results of the statistical evaluation of the data are presented in Tables 3.54 and
3.55. The low values of repeatability and reproducibility indicate that both packed column and capillary
column can be successfully used for the separation and quantitative determination of diacetyl and 2,3-
pentanediol in beer.
GC has also been used for the determination of the ethanol content of beer [ 518]. Beers were degassed,
2-propanol internal standard added, and directly injected to six different GC apparatuses. The
chromatographic conditions were the same in each instance. Column (2 m × 2 mm I.D., packed with
Chromasorb 102, 80-100 mesh) temperature was 170°C. The injector and FID temperatures were 225°C
and 250°C, respectively, and the carrier gas was nitrogen. The retention times of ethanol and 2-propanol
were 1.75 min and 2.75 min, respectively. The ethanol concentrations determined by GC and by the
traditional distillation method are given in Table 3.56. The concentrations determined by GC are in
good agreement with the results of the official distillation method. Because the GC method is rapid and
the repeatability is similar to that of the official method, it is an acceptable alternative for the
determination of the ethanol content in beer.
Headspace gas chromatography was employed for the determination of S-methylmethionine (SMM) in
malt [ 519]. Because SMM is a precursor of
Page 408
TABLE 3.54. Summary of Precision Data for Diacetyl (concentrations in mg/L).

Sample No. Capillary Column Packed Column Combined Results
Mean 0.048 0.056 0.051
r95 0.014 0.011 0.014
1 sr 0.005 0.004 0.005
R95 0.039 0.040 0.038
SR 0.014 0.014 0.014
No. of labs 6 3 9
Mean 0.150 0.169 0.155
r95 0.068 0.019 0.049
2 sr 0.024 0.007 0.017
R95 0.128 0.079 0.104
SR 0.049 0.028 0.037
No. of labs 6 4 10
Mean 0.165 0.187 0.173
r95 0.021 0.034 0.027
3 SR 0.007 0.012 0.010
R95 0.135 0.106 0.122
SR 0.048 0.038 0.044
No. of labs 6 3 9
Mean 0.037 0.043 0.040
r95 0.010 0.017 0.013
4 Sr 0.004 0.006 0.005
R95 0.036 0.029 0.040
SR 0.013 0.010 0.012
No. of labs 6 4 10
r95 = repeatability; S r = standard deviation of repeatability; R 95 = reproducibility; S R =

standard deviation of reproducibility.
Reprinted from Reference [ 517] with permission from the Brewing Institute, London.
dimethyl sulphide (DMS), its concentration in malt has a considerable impact on the quality of malt.
The free DMS is generally determined from the cold water extract of malt. Total DMS was measured
after the alkaline hydrolysate of the cold water extract. The concentration of SMM was calculated from
the difference between the total and free DMS content of the malt. The laboratories in the collaborative
trial used either packed or capillary columns. DSM was detected with a flame ionization detector, flame
photometric detector, or photoionization detector, depending on the laboratories. The internal standards
were diisopropyl sulphide, ethyl methyl sulphide, butanol, or propanol. The concentrations of SMM and
DMS determined by the different laboratories are presented in Table 3.57. The repeatability and
reproducibility of the results were good, and no stragglers were found for the determination of free
DMS and SMM. These data indicate that each headspace GC method can be used for the determination
of SMM in malt.
Page 409
TABLE 3.55. Summary of Precision Data for 2,3-Pentanedione (concentrations in mg/L).

Sample No. Capillary Column Packed Column Combined Results
Mean 0.027 Insufficient data 0.029
r95 0.017 0.015
1 sr 0.006 0.005
R95 0.064 0.057
SR 0.023 0.020
No. of labs 6 8
Mean 0.015 0.019 0.017
r95 0.004 0.002 0.003
2 Sr 0.001 0.0006 0.001
R95 0.015 0.029 0.020
SR 0.005 0.010 0.007
No. of labs 6 3 9
Mean 0.064 0.071 0.067
r95 0.014 0.012 0.013
3 Sr 0.005 0.004 0.005
R95 0.049 0.060 0.050
SR 0.018 0.021 0.018
No. of labs 6 3 9
Mean 0.018 0.027 0.021
r95 0.001 0.012 0.007
4 Sr 0.0004 0.004 0.002
R95 0.018 0.035 0.026
SR 0.007 0.123 0.009
No. of labs 6 3 9
r95 = repeatability; S r = standard deviation of repeatability; R 95 = reproducibility; S R =

standard deviation of reproducibility.
The bitter acids in hop (Homulus lupulus L.) exert a marked influence on the taste and character of beer.
Because of their considerable importance, many chromatographic methods were developed for their
separation and quantitative determination, such as microcolumn analysis [ 520], are centrifugal partition
chromatography [ 521]. RP-HPLC has also been employed for the separation and characterization of
bitter acids in hop [522]. The supercritical CO 2 hop extract was added to the same volume of methanol
and stored at 5-6°C for 24 h. The mixture was centrifuged, and the methanol was evaporated. The
residue was dissolved again in methanol and used for HPLC analysis. Separation was carried out on a
C18 column (250 × 4 mm I.D., particle size 5 µm) with a corresponding precolumn. Gradient elution
was run from 60% to 80% A in 40 min (A = acetonitrile - 0.05% H3 PO4 85%; B = H2 O - 0.05% H3 PO4
85%). The flow rate was 1 mL/min. The detector wavelength was 315 nm. When the system was
coupled to NMR, C18 columns were coupled,
Page 410
TABLE 3.56. R95 Data for Individual Beers Analyzed by GC and Ethanol by Distillation Results.
Sample Ethanol by GC (% v/v) Ethanol by Distillation (% v/v) R95 for Individual Beers by
Average of Six Systems Average of Four GC
Beer 1 0.95 0.91 0.128
Beer 2 1.06 1.03 0.043
Beer 3 3.04 3.01 0.059
Beer 4 3.51 3.40 0.058
Beer 5 3.56 3.46 0.060
Beer 6 3.54 3.45 0.053
Beer 7 3.78 3.70 0.068
Beer 8 3.40 3.30 0.103
Beer 9 3.75 3.72 0.047
Beer 10 4.05 3.98 0.088
Beer 11 4.58 4.57 0.081
Beer 12 5.02 5.01 0.107
Beer 13 4.51 4.47 0.157
Beer 14 5.21 5.19 0.075
Beer 15 5.41 5.34 0.061
Beer 16 5.23 5.14 0.124
Beer 17 6.01 5.95 0.071
Beer 18 6.32 6.33 0.107
Beer 19 9.48 9.25 0.256
Beer 20 11.15 11.04 0.197
Reprinted from reference [ 518] with permission from the Institute of Brewing, London.
TABLE 3.57. Concentrations of S-Methylmethionine and Free Dimethylsulphide in Malts

Determined by Different Laboratories.
S-Methylmethionine (mg/kg dry) Free DMS (mg/kg dry)
Pair 1 Pair 2 Pair 1 Pair 2
Lab A B C D A B C D
A 7.079 8.981 12.918 9.584 1.512 1.698 6.127 4.794
B 2.335 4.562 10.221 6.846 4.353 3.819 5.164 5.162
C 3.708 5.058 7.493 5.192 — — — —
D 3.791 6.098 8.081 5.311 0.760 1.769 7.783 6.059
E 3.228 4.057 6.937 2.663 2.335 3.497 9.918 10.576
F 4.100 5.200 7.800 5.200 2.300 3.800 8.500 8.300
G 2.354 3.753 5.621 3.449 1.576 1.839 6.550 5.078
H 4.797 7.495 10.443 4.388 — — — —
I 5.522 8.072 10.134 7.178 0.531 0.637 1.689 1.689

J 4.611 6.181 9.113 7.630 1.560 1.918 4.874 4.556
K 3.719 5.311 8.971 4.868 1.382 2.018 6.227 4.550
Mean 4.113 5.888 8.885 5.664 1.812 2.333 6.315 5.640
Page 411
and the gradient was 80 min. The HPLC chromatogram of supercritical CO2 hop extract is shown in
Figure 3.59. This HPLC system allowed the separation of humulone-adhumulone and lupulone-
adlupulone pairs, which are not separated by using methanol as the organic modifier. The stopped flow
1H NMR spectra of bitter acids are in agreement with the spetra conventionally recorded.
Microemulsion electrokinetic chromatography (MEEKC) also separated the main bitter acids in hop,
and the results were compared with those of RP -HPLC [ 523]. Samples were prepared by extracting 2 g
of hop cones with 150 mL of isooctane under reflux in nitrogen atmosphere for 60 min. Extraction was
repeated twice; the pooled extracts were filtered, evaporated under vacuum, redissolved in methanol,
and centrifuged. RP-HPLC used C18 columns, separation was performed at 35°C, and the bitter acids
were detected at 280 nm. The performance of two different methods was compared. Method I: 250 ×
4.6 mm I.D. analytical and 40 × 4.6 mm I.D. precolumn packed with ODS (10 µm); flow rate: 2.5
mL/min; eluent A: 5% formic acid in water; eluent B: methanol; gradient: 0-2 min 25% B, 2-12 min to
55% B, 12-37 min to 95% B, 37-42 min to 25% B, 42-45 min 25% B. Method II: 250 × 4.0 mm I.D.
analytical and 11 × 4.0 mm I.D. precolumn packed with ODS (5 µm);
Figure 3.59.
HPLC chromatogram of supercritical CO2 hop extract. Peaks: 1 = cohumulone,
2 = humulone, 3 = adhumulone, 4 = colupulone, 5 = lupulone, 6 = adlupulone.
Page 412
Figure 3.60.
The MEEKC analysis of isooctane extracts of different hop
cultivars: (a) Saaz, (b) Nugget, (c) Wye Target; 1 = colupulone,
2 = cohumulone, 3 = lupulone, 4 = adlupulone, 5 = adhumulone,
6 = humulone. (Reprinted from Reference [ 523] with
permission from the Institute of Brewing, London.)
flow rate: 0.9 mL/min; eluent A: 5% formic acid in water; eluent B: 5% acetonitrile in methanol;
gradient: 0-2 min 60% B, 2-32 min to 95% B, 32-37 min 95% B, 37-45 min to 60% B. MEEKC was
performed on a fused-silica capillary (60 cm × 50 µm I.D., detection window at 52.8 cm). Buffer was
10 mM borax (pH 9.2), 40 mM SDS, 3% v/v n-butanol, and 0.3% (v/v) n-heptane. The separation
voltage was 25 kV, and bitter acids were detected at 214 nm. A typical electropherogram is shown in
Figure 3.60. The six main bitter acids are well separated in a very short analysis time (10 min). The hop
acid ratios determined by two RP -HPLC methods and by MEEKC are given in Table 3.58. The data
indicate that the composition of bitter acids in hop cultivars is different and markedly depends on the
type of cultivar. The hop acid ratios determined by MEEKC and HPLC are similar, suggesting that
MEEKC can be used as a substitute of HPLC for the analysis of hop bitter acids.
A collaborative trial was carried out for the RP-HPLC determination of iso- α-, α-, and β-acids in hop
and isomerized hop extracts [524]. It was estab-
Page 413
TABLE 3.58. Hop Acid Ratios for Three Different Hop Cultivars(three extracts each).
Ratio Adlupulone
Cohumulone Ratio Colupulone Ratio Adhumulone Ratio
Hop Cultivar (a) (b) (c) (a) (b) (c) (c) (c)
Saaz I 24.1 24.6 24.1 48.0 42.8 44.0 12.5 12.1
Saaz II 24.0 24.3 25.7 45.0 43.5 42.7 12.2 12.8
Saaz III 24.1 25.2 24.4 46.6 42.8 43.1 11.9 12.4
Nugget I 36.4 35.8 35.8 60.9 60.5 59.9 12.8 12.2
Nugget II 34.9 34.6 37.3 60.6 60.0 59.7 12.4 14.5
Nugget III 36.0 35.2 36.5 61.7 60.4 59.2 13.1 13.6
Wye Target I 40.8 40.6 41.6 62.8 60.2 60.7 12.8 11.2
Wye Target 39.6 39.5 41.9 62.4 60.3 59.7 12.7 12.0
II
Wye Target 39.7 39.8 41.2 62.5 61.2 60.0 12.8 11.5
III
(a) HPLC, 10 µm, single analysis; (b) HPLC, 5 µm, single analysis; (c) MEEKC, average of three
measurements.
lished that the repeatability and reproducibility of the method comply with the requirements; that is, this
method can be used for the quantitation of these components of hop and hop extract. GC, coupled with
MS, has also found application in the analysis of hop. The humulene diepoxides influencing the aroma
of beer were separated and quantitatively determined by a capillary GC-MS system [ 525].
3.2.1.4—
Coffee and Dairy Products
A hyphenated method (purge and trap + GC + MS + FID) has been developed for the analysis of
volatile components in various food products [ 526]. Aqueous extracts were prepared by stirring the
sample with warm water; then the mixture was filtered and heated. Separation of volatiles was
performed on a fused-silica capillary column (30 m × 0.32 mm I.D., film thickness 1 µm). Initial
column temperature was 5°C, increased to 47°C at 3°C/min, and to 220°C at 10°C. The column flow
was divided and directed to both MS and FID. A characteristic FID headspace profile is shown in
Figure 3.61. It was established that many heated food products (green coffee beans, green tea, popcorn,
rice, potatoes, etc.) show a similar FID profile (Figure 3.62); however, the concentration and relative
ratio of the components are characteristic for the products, as demonstrated in Table 3.59. Owing to its
high sensitivity and identification capacity, this method has been proposed for the analysis of the
composition of volatile components in natural products. The same method has been used for the study
of the influence of ion concentration and pH on the
Page 414
Figure 3.61.
FID headspace profile of roasted peanut extract. Peaks: (A) furan;
(B) propanal; (C) acetone; (D) 2-methyl-propanal; (E) 2-methyl-furan;
(F) butanone; (G) 2,3-butanedione; (H) 3-methyl butanal;
(I) 2-methylbutanal; (J) 2,3 -pentenedione; (K) 1 -hydroxy -2-propanone;
(L) pyrazine; (M) 3-hydroxy-2-butanone; (N) hexanal;
(O) methyl pyrazine; (P) furfural; (Q) dimethylpyrazine;
(R) acetylfuran; (S) benzaldehide; (T) methyl furfural;
(U) benzeneacetaldehide; (V) C3 pyrazine; (W) decanal; (X) phenylfuran.
Publications, A Division of Preston Industries, Inc.)
release of volatile compounds [ 527]. Both the concentration of sodium chloride and the pH exerted a
considerable influence on the quantity of volatile compounds released in the headspace. The data
concerning the impact of NaCl and pH are compiled in Tables 3.60 and 3.61, respectively. The marked
increase in the quantity of volatiles at higher NaCl concentrations suggests that the modification of the
amount of NaCl in the products may change their organoleptic characteristics. Interestingly, the
addition of NaClO 4 was not effective, and the influence of CaCl2 ad MgSO4 was lower than that of
NaCl. Similar to the effect of salts, pH also influenced the quantity of volatile compounds in the
headspace. It was proposed that the method can be used for the design and formulation of new aroma
mixtures.
GC-MS has also been used for the separation and identification of headspace volatiles from brewed
coffee [ 528]. Gas chromatography-olfactometry of headspace (GCO-H) sample was frequently applied
for the analysis of coffee [529]. The difference between the odor characteristics of Arabica (Coffea
arabica) and Robusta (Coffea canephora var. Robusta) coffees was also studied by GCO-H [ 530].
Coffee brews were prepared by pouring 1.1 L hot water on 54 g coffee powder, and 10 mL of brew
were used for GC analysis. Sepa-
Page 415
Figure 3.62.
FID traces of selected heat-treated extracts.
A Division of Preston Industries, Inc.)
ration was performed on a fused-silica capillary column (30 m × 0.52 mm I.D., film thickness 1.5 µm).
The effluent was split for FID and sniffing port. The initial oven temperature was 0°C for 2 min,
increased to 50°C at 6°C/min, held for 2 min, and increased to 250°C at 8°C min. The same temperature
program was also used for GC-MS. The results are shown in Table 3.62. It was
TABLE 3.59. Percent Contribution and Concentration of Selected Components in

Heat-Treated Aqueous Extracts.
Coffee Peanut Tea
Component % Conc. * % Conc. * % Conc. *

Acetone 30.01 10.84 17.1 7.16 14.76 4.58
2-Methylpropanal 13.46 4.86 10.69 4.44 2.86 0.88
Furfural 5.81 2.1 5.9 2.4 55.16 17.12
Dimethlypyrazines 2.28 0.82 3.47 1.46 0.15 0.04
* Concentration in µg/mL.
Reprinted from Reference [ 526] with permission from Preston Publications, A Division
of Preston Industries, Inc.
Page 416
TABLE 3.60. Concentration of Volatile Material for Heat-Treated Aqueous Extracts.

Molarity NaCL Tea Leaves (µg/mL) Coffee Beans (µg/mL) Peanuts ( µg/mL)
0 20.75 39.96 34.85
0.86 20.75 37.25 31.73
1.72 19.22 37.15 31.12
2.59 23.18 37.35 32.94
3.45 23.62 37.81 31.87
4.31 26.61 38.67 32.94
5.17 26.03 37.11
Reprinted from Reference [ 527] with permission from Preston Publications, A Division of
established that both Arabica and Robusta coffees and the brews prepared differ considerably in the
composition of volatile aroma components.
The aroma-forming chlorogenic acid (quinic acid esters) in green coffee was previously determined by
high-performance gel filtration chromatography (HPGFC) [ 531], and the influence of the low- and high-
molecular mass fractions isolated of green Arabica coffee on the volatile profile of roasted coffee beans
was studied by a combined method (HPGFC and GC) [ 532]. Lipids were removed from ground green
coffee by extraction with petroleum ether, and 100 g of defatted sample were extracted with 4 L of hot
bidistilled water (80°C) for 15 min by shaking in a water bath. After extraction, the slurry was filtered
and freeze-dried. An aliquot of 20 g was redissolved in 25 mL of water (50°C), and 100 mL of ethanol
were added. After 1 h of shaking, the sample was centrifuged and the supernatant removed. The
extraction was repeated
TABLE 3.61. Effects of pH on Extract FID Area Counts.

FID Area Counts
Sample pH Total IS Volatile Conc. ( µg/mL)

Peanut 4.3 7852.78 2949.01 4903.77 35.25
6.8 7938.06 2955.32 4982.74 35.74
8.3 8897.19 3029.45 5867.74 41.06
Coffee 4.7 9655.99 2994.58 6661.41 47.16

5.9 7740.76 2710.43 5030.33 39.35
7.6 8106.95 2920.46 5186.49 37.65
Tea 4.4 6328.25 3104.66 3223.59 22.01

6.4 6602.13 3049.15 3552.98 24.70
7.7 7071.89 3237.38 3834.51 25.11
Reprinted from Reference [ 527] with permission from Preston Publications, A

Division of Preston Industries, Inc.
Page 417
TABLE 3.62. CO—H of Roasted Coffee Powders and Brews.

FD-Factor*** Powder
No. of Compound* Odor Description** RI on RTX-5 Arabica Robusta
1 Acetaldehyde**** Fruity, pungent <400 25 25
2 Methanethiol**** Putrid, cabbage–like, <400 5 12.5
Sulphurous
3 Propanal Fruity ≈450 5 5
4 Methylpropanal**** Fruity, malty ≈550 5 5
5 2,3–Butenedione **** Buttery 580 62.5 125
6 3–Methylbutanal**** Malty 653 12.5 25
7 2–Methylbutanal**** Malty 662 5 12.5
8 3,3–Pentanedione **** Buttery 697 125 125
9 3–Methyl –2–butenthiol§ Foxy, skunky 822 62.5 62.5
10 Unknown Meaty, roasty 839 ND 5
11 2–Methyl –3–furanthiol § Boiled meat-like 870 25 125
12 Methional§ Boiled potato-like 906 62.5 62.5
13 2-Furfurylthiol§ Roasty 911 62.5 125
14 Unknown Roasty 939 ND 5
15 Dimethyltrisulphide§ Cabbage -like 970 ND 1
16 1–Octen–3–one§ Mushroom-like 978 ND ND
17 Unknown Roasty 986 1 25
18 Unknown Musty, earthy 1002 ND ND
19 Unknown Earthy, roasty 1012 ND ND
20 3–Mercapto–3–methylbutylformate § Catty, roasty 1022 62.5 62.5
21 2–Ethyl–3,5–dimethylpyrazine§ Earthy, roasty 1086 25 62.5
22 Guaiacol§ Phenolic, burnt 1092 12.5 25
23 Unknown Earthy, roasty 1107 25 125
24 2,3–Diethyl–5–methylpyrazine§ Earthy 1155 25 125
25 Unknown Earthy 1182 12.5 62.5
26 2–Isobutyl–3–methoxypyrazine§ Green, earthy 1186 25 1
27 Unknown Roasty, sulphurous 1225 1 5

28 4-Ethylguaiacol§ Phenolic, spicy 1282 ND ND
29 4–Vinylguaiacol § Phenolic, spicy 1317 ND ND
30 (E)-β–Damascenone § Honey-like, fruity 1400 5 5
FD-Factor*** Brew
No. of Compound Arabica Robusta Soluble Coffee
1 Acetaldehyde**** 125 62.5 12.5
2 Methanethiol**** 1 1 ND
3 Propanal 25 25 25
4 Methylpropanal**** 25 25 25
5 2,3-Butenedione **** 125 125 62.5
6 3-Methylbutanal**** 62.5 62.5 25
7 2-Methylbutanal**** 25 25 12.5
8 3,3-Pentanedione **** 125 62.5 62.5
9 3-Methyl -2-butenthiol§ 5 5 1

Page 418
TABLE 3.62.
FD-Factor***Brew
No. of Compound Arabica Robusta Soluble Coffee
10 Unknown ND ND ND
11 2–Methyl –3–furanthiol § 5 5 ND
12 Methional§ 25 25 5
13 2–Furfurylthiol§ 12.5 12.5 ND
14 Unknown ND ND ND
15 Dimethyltrisulphide§ 12.5 25 ND
16 1–Octen–3–one§ 5 1 ND
17 Unknown ND 1 ND
18 Unknown 5 5 ND
19 Unknown 5 5 ND
20 3–Mercapto–3–methylbutylformate § 1 1 ND
21 2–Ethyl–3,5–dimethylpyrazine§ 25 62.5 25
22 Guaiacol§ 25 12.5 5
23 Unknown 25 25 5
24 2,3–Diethyl-5–methylpyrazine§ 25 125 12.5
25 Unknown 1 25 12.5
26 2–Isobutyl–3–methoxypyrazine§ 125 1 ND
27 Unknown 1 5 ND
28 4-Ethylguaiacol§ ND 5 ND
29 4-Vinylguaiacol § 5 25 ND
30 (E)–β–Damascenone § ND ND ND
* The compounds that appeared in one of the coffee samples with an FD-factor of at least
five are compared.
** Odor description assigned during GCO—H.
*** The relationship between FD -factor and headspace volume.
**** The compound was identified by comparing it with the reference substance using the
following criteria: retention index (RI) on the capillary RTX–5, the mass spectrum and the
odor quality perceived at the sniffing port.
§ The compound was identified by comparing it with the reference substance on the basis of
the RI on capillary RTX –5 and the odor quality perceived during GCO—H.
ND: not detectable in the highest headspace volume of 25 mL.
FD: flavor dilution.
Page 419
Figure 3.63.
Gel filtration chromatography of (1) green
coffee and fractions (2) B and (3) C, using the
TSK-G 3000 SW column. Mobile phase
was bidistilled water containing 0.05% (w/v)
of sodium azide at a flow rate of 0.5
mL/min. Detection at 280 nm.
0.25 mm I.D.). Injector and FID temperatures were 100°C and 280°C, respectively. The initial oven
temperature was 40°C for 6 min and increased to 190°C at 3°C/min. The high-performance gel
chromatograms of green coffee and fractions B and C showed marked differences, as shown in Figure
3.63. The composition of fractions determined by other methods are goven in Table 3.63. The
headspace profiles of roasted green coffee and fractions B and C showed marked differences. It was
stated that the different fractions behave differently as aroma precursors during the roasting process.
HPGFC has also been employed for the simultaneous determination of total chlorogenic acid,
trigonelline, and caffeine in green coffee [ 533]. Defatted coffee samples (0.5 g) were extracted with 30
mL of hot distilled water (80°C) for 15 min with continuous shaking. The slurry was filtered, and the
volume was adjusted to 50 mL with distilled water. This filtrate was used for analysis. The
Reprinted from Reference [ 530] with permission from Academic Press Ltd., a Subsidiary of
Harcourt Brace & Company, Ltd.
four times, and the collected supernatants (fraction B) and pellets (fraction C) were freeze-dried again.
Sugars were determined by GC as alditol acetate derivatives. HPGFC was performed on a TSK-G 3000
SW (300 × 8 mm I.D.) column using bidistilled water with 0.05% (w/v) sodium azide as eluent
(detection wavelength 280 nm, flow rate 0.5 mL/min). Defatted coffee and low- and high-molecular
mass fractions were roasted at 220 ± 2°C for 14 min in a closed tube. The headspace of tubes was used
for GC analysis. Separation was carried out on a fused-silica Supelcowax 10 capillary column (30 m ×
Page 420
Figure 3.64.
Chromatograms of (a) standard and green coffee
(b) using the TSK G-3000 SW HPGF chromatographic
column: (1) CGA, (2) trigonelline, (3) caffeine. Mobile
phase was bidistilled water at flow rate of 0.5 mL/min.
Detection at 272 nm.
With kind permission from Elsevier Science—NL
The Netherlands.)
same column was used as in the previous study, but the eluent did not contain sodium azide and the
detection wavelength was 272 nm. The results were compared with those of various RP -HPLC methods
chlorogenic acid (CGA) [ 534], trigonelline [535], and caffeine [ 536]). The method successfully separated
CGA, trigonelline, and caffeine from each other, and no interfering impurities were found (Figure 3.64).
Good linear relationships were found between the peak height and the concentration of solutes, the
correlation coefficients being over 0.9996. The recoveries of CGA, trigonelline, and caffeine were 97%,
96%, and 96%, respectively. The quantitative results are presented in Table 3.64. The results of the
proposed method did not deviate significantly from those obtained by RP-HPLC. Because the method is
inexpensive (uses only distilled water as eluent) and simultaneously separates CGA, trigonelline, and
caffeine in one isocratic step, it was proposed for industrial quality control and research purposes.
RP-HPLC was also used for the determination of the caffeine content in coffee, tea, and other beverages
[537]. Beverages were degassed and then were injected directly into the column. In the case of low
caffeine concentrations, 25 mL of beverage were applied to a C 18 cartridge and then eluted with
methanol. Coffee and tea were filtered and injected directly. Separation was performed on a C 18 column
(250 × 4 mm I.D.) using methanol:water eluent
Page 421
TABLE 3.63. Distribution of Carbohydrates, Trigonelline, Total Chlorogenic Acid

(CGA) and Protein in Fractions Obtained from Green Coffee.
Fractionsn * (g %)
80% Ethanol-Soluble (B) 80% Ethanol-Insoluble (C)
Galactose tr 21.2
Arabinose tr 18.1
Mannose tr 6.9
Glucose tr 1.9
Xylose tr 1.9
Sucrose 17.4 –
Trigonelline 5.3 –
Total CGA 33.2 –
Protein (N × 6.25) 7.5 40.0
* Results are average of duplicate determinations; g % dry basis.
Netherlands.
(40:60 v/v) at 0.7 mL/min flow rate. Caffeine was detected at 273 nm. Good linear correlation was
found between the peak area and the concentration of caffeine (10-50 µg/mL). The detection limit was
1 ng/mL, and the precision was under 3.2%. Caffeine levels were 2-41 mg/can in Brazilian cola
beverages, 2-40 mg/150 mL in tea, and 0.2-109 mg/150 mL in coffee. Since the method does not need
complicated sample preparation steps or derivatization, it was proposed for food control laboratories.
HPLC has also been used for the determination of theobromine, theophylline, and caffeine in various
food products [ 538]. Not only HPLC, but also MECC, has been used for the determination of caffeine in
coffee, and the results were compared with those of RP -HPLC [ 539]. Samples were prepared by
extracting caffeine with boiling distilled water, filtering, and diluting. Separation was carried out on a
fused-silica capillary column (55 cm × 75 µm I.D.) at 20 kV voltage. Caffeine was detected at 214 nm.
RP-HPLC separations were performed on a C18 column (150 × 4.6 mm I.D.). The optimal MECC
method consisted of prerinsing the column with 0.1 N HC1 for 30 min and with the mobile phase for 2
min. Buffer was 50 mM cetyltrimethylammonium chloride, 0.5 mM Na 2HPO4, and 0.5 mM Na3PO4.
The mean and RSD of the migration time, peak, and peak height were 5.522 (0.74%), 5058.15 (1.04%),
and 3579.49 (2.07%), respectively. These data prove the good reproducibility of the method. The data
obtained by MECC and RP-HPLC techniques are compared in Table 3.65. The differences in the
caffeine content of coffees determined by MECC and RP-HPLC are negligible, except in decaffeinated
coffees. This difference may be because of the different method of sample preparation. Nevertheless, it
was assumed that this MECC method can
Page 422
TABLE 3.64. Average of Results of CGA, Trigonelline, and Caffeine in Different Green
Coffee Samples Obtained by Different Methods.
CGA Trigonelline Methods Caffeine
Samples Proposed RP–HPLC Proposed RP–HPLC Proposed RP –HPLC

A 8.0 9.0 1.1 1.1 1.1 1.2
SD 0.32 0.31 0.05 0.04 0.03 0.04
CV% 4.0 3.4 4.5 3.6 2.7 3.3
B 6.7 7.7 1.2 1.2 1.1 1.3
C 7.4 8.1 1.0 1.1 1.1 1.1
D 7.2 8.0 1.6 1.6 0.7 0.7
E 6.8 7.1 1.5 1.4 1.1 1.3
F 6.8 7.0 1.2 1.2 1.2 1.3
A: Results are average of 12 replicates originating the respective SD and CV (%); B -F:
results are average of duplicate determinations.
TABLE 3.65. Determination of Caffeine in Coffee Samples by MECC and

HPLC.
Caffeine %
Sample No. Coffee Type MECC HPLC Difference*

1 Instant 3.57 3.36 0.21
2 Instant 2.75 2.75 0.00
3 Instant 3.82 3.74 0.08
4 Instant 3.95 3.66 0.29
5 Instant 3.94 3.66 0.28
6 Decaf. instant 0.10 0.12 –0.02
7 Decaf. instant 0.12 0.14 –0.02
8 Decaf. instant 0.13 0.14 –0.01
9 Regular 1.46 1.24 0.22
10 Regular 1.73 1.49 0.24
11 Regular 1.40 1.07 0.33
12 Regular 1.68 1.45 0.23
13 Regular 1.41 1.19 0.22
14 Decaf. R&G 0.09 0.06 0.03 **
15 Decaf. R&G 0.10 0.05 0.05 **

16 Decaf. green 0.11 0.04 0.07 **
17 Decaf. green 0.07 0.05 0.02
18 Decaf. green 0.09 0.05 0.04 **
19 Decaf. green 0.08 0.04 0.04 **
20 Decaf. green 0.07 0.04 0.03 **

* (% caffeine determined by MECC) - (% caffeine determined by HPLC).
**Out of limit of reproducibility for decaffeinated roasted coffee beans
under ISO/DIS #10095.
Reprinted from Reference [ 539] with permission from John Wiley & Sons,
Inc., copyright © 1994, John Wiley & Sons.
Page 423
be a valuable substitute of HPLC in the determination of the caffeine content of coffees.
The quality and quantity of volatile aroma compounds in various dairy products have also been
frequently investigated [ 540,541,542,543]. Because headspace gas chromatography is a method of
preference for the study of aroma volatiles [ 544], this method was employed for the monitoring of the
oxidative deterioration of milk powder [545]. Aroma volatiles were steam distilled; then 2 mL of
distillate and 1.0 g of NaCl were added to a headspace vial and thermostatted at 60°C. GC separation
was performed on a fused-silica capillary column (25 m × 0.32 mm I.D., SE-54 film thickness 1.0 µm).
Initial oven temperature was 38°C for 3 min and then increased to 170°C at 6°C/min. Carrier gas was
hydrogen. The gas chromatograms of a whole milk powder and a milk-based bay formula are shown in
Figure 3.65. The data indicate that this headspace
Figure 3.65.
Headspace volatiles of (A) whole milk powder stored in air at 40°C
(peroxide value = 1.01) and (B) a milk-based baby formula also stored in air at
40°C (peroxide value = 1.23): 1 = Pentanone, 2 = pentanal, 3 = methyl
butyrate (internal standard), 4 = hexanal, 5 = heptanone, 6 = heptanal,
7 = octanal, 8 = octanol, 9 = nonanal.
Page 424
TABLE 3.66. Comparison of Regression Statistics of Artificial Neural

Networks (ANN) and Principal Components Regression (PCR) for Prediction
of Milk Shelf Life*.
All Samples (n = 134) Cross-Validation (n = 21)
Method r2 SEE r2 SEE
ANN 0.92 2.25 0.50 (0.82)** 5.34 (3.0) **

PCR 0.62 4.21 — —
* Squared correlation coefficient (r2) and standard error of the estimate were
calculated for the predicted versus the experimental shelf lives. ANN and PCR
were computed using 46 variables (GC areas) with shelf life as the response.
ANN consisted of 46 input neurons, eight hidden neurons, and shelf life as the
output neuron. PCR consisted of the first 30 principal components as the
independent variables and shelf life as the dependent variable in forward
stepwise regression.
**Squared correlation coefficient (r2 ) and standard error of the estimate
calculated after elimination of five outlying observations.
SEE = standard error of the estimate.
GC method separates the straight-chain aldehydes well; therefore, it can be used for the rapid quality
grading of dried milk products. The combination of headspace GC retention data with various
multivariate mathematical-statistical methods such as factor optimization [ 546], principal component
regression (PCR) [ 547], and linear discriminant analysis [ 548] was successfully used in the analysis of
milk aroma compounds. Artificial neural network (ANN) has also been used for the prediction of milk
shelf life based on headspace gas chromatographic data, and the results were compared with those of
PCR [ 549]. The results of calculation are shown in Tables 3.66 and 3.67. The data clearly prove that
ANN shows a better predicting power than PCR does.
TABLE 3.67. Comparison of the Standard Error of Prediction of

Artifical Neural Networks (ANN) and Principal Component
Regression (PCR) for Shelf Life Prediction of Milk Samples
Assigned to Different Quality Groups.*
Standard Error of Prediction (%)
Method Total Good Marginal Poor

ANN 15.0 10.4 22.1 32.0
PCR 28.0 17.0 38.4 76.0
* Milk samples with different shelf life ratings were assigned to
three quality groups: ''good" (shelf life of 15 days or more, n = 60);
"marginal" (shelf life of 8 -14 days, n = 42; and "poor" (shelf life of
1-7 days, n = 32).

Page 425
This finding indicates that this new method can be successfully used for the prediction of the shelf life
of milk based on headspace GC data.
GC has also been applied to study the flavor components of cheese [ 550] and the stereoisomeric flavor
compounds in dairy products [ 551]. Enantiomeric separation of C8-C18 δ (τ)-lactones was motivated by
the fact that their odor thresholds differ considerably. Isolation of lactones was carried out by steam
distillation under reduced pressure at 50°C. Distillate was extracted three times with dichloromethane,
dried with anhydrous sodium sulfate, and concentrated. Enantioselective multidimensional GC was
performed on two capillary columns. The precolumn (30 m × 0.32 mm I.D.) was coated with OV 1701
(df = 0.25 µm). For C13-C18 lactones, the initial temperature was 45°C for 2 min, increased to 180°C at
20°C/min, and then further increased to 260°C at 3°C; for C8-C12 lactones, the initial temperature was
45°C for 2 min, increased to 120°C at 20°C/min, and then further increased to 260°C at 5°C. The main
column for lactones C13-C18 (23 m × 0.23 mm I.D.) was coated with 50% heptakis (2,3-di-O-acetyl-6-O-
TBDMS)- β-cyclodextrin in PS 255 (df ≈ 0.06 µm); the initial temperature was 45°C for 2 min,
increased to 120°C at 20°C/min, held for 35 min, and then increased to 160°C at 2°C and to 200°C at 1°
C/min. The main column for lactones C8-C12 (30 m × 0.25 mm I.D.) was coated with 40% heptakis (2,3-
di-O-acetyl-6-O-TBDMS)-β-cyclodextrin in OV 1701 (d f ≈ 0.25 µm); the initial temperature was 45°C
for 2 min, increased to 120°C at 20°C/min, held for 15 min, and then increased to 125°C at 3°C and to
175°C at 1.5°C/min and to 200°C at 5°C/min. Enantiomeric multidimensional gas chromatograms of
cheeses and butter are shown in Figure 3.66. The two columns separate the lactones well; both
according to the carbon number and stereoisometry. The stereodifferentiation of δ- and τ-lactones in
dairy products, margarine, and coconut are listed in Tables 3.68 and 3.69. Although the origin of the
dairy products was different, their lactone profile was surprisingly similar. Even the heating did not
change the ratio of stereoisomers considerably. Racemic lactones as additives can be detected in
margarine. The total concentration of lactones determined by one-dimensional GC are compiled in
Tables 3.70 and 3.71. The concentration of δ-lactones is higher in each food product than that of τ-
lactones, and the quantity considerably depends on the type of the sample. It was assumed that the
determination of the amount and enantiomeric ratio of lactones may contribute to the study of the
organoleptic properties of various foodstuffs.
GC and profile-rank method has been used for the evaluation of the relationship between sensory and
GC characteristics of Dutch cheese [ 552].
3.2.1.5—
Meats and Oils
GC, combined with MS [ 553] and olfactometry [ 554], was employed for the detection of odorants in
boiled trout (Salmo fario) and cod (Gadus morhua).
Page 426
Figure 3.66.
Enantiomer multidimensional gas chromatograms
of δ- and τ-lactones from cheese and butter.
Page 427
TABLE 3.68. Enantiomeric Ratios for δ- and τ-Lactones (C 8–C18 ) from Dairy Products.
(R):(S) (%) in Lactones

Food τ-C10 τ-C12 δ -C8 δ-C10 δ -C12 δ -C14 δ-C
Country butter Trace * 78:22 15:85 82:18 92:8 85:15 90:10
Best quality butter (sweet

cream) 78:22 80:20 15:85 82:18 92:8 83:17 89:11
cream) after heating to
200ºC for 15 min 78:22 76:24 15:85 82:18 92:8 83:17 89:11
Whipped cream
(pastuerized) Trace 78:22 13:87 81:19 92:8 85:15 88:12
Evaporated milk (7.5% fat) Trace R>74 Trace 82:18 89:11 Trace Trace
Full -cream milk Trace Trace 13:87 82:18 92:8 81:19 Trace
(pasteurized)
Parmesan cheese R>82 R>82 R>87 86:14 Trace
Cheddar cheese Trace R>75 17:83 81:19 90:10 85:15 Trace

Limburger cheese Trace 85:15 78:22 78:22 R>87 83:17 Trace
Emmental cheese Trace 85:15 85:15 90:10 83:17
Blue cheese Trace 83:17 Trace 80:20 89:11 87:13 Trace

* Trace—stereodifferentiation impossible.
Separation of volatile aroma compounds was performed on a fused-silica capillary column (30 m × 0.52 mm
I.D., df = 1.5 µm) coated with SE-54 (RTX 5) for simultaneous FID and olfactometry. The initial oven
temperature was 5°C and was raised to 230°C at 6°C/min. The gas chromatograms of boiled cod samples
prepared from raw material stored under different conditions are
TABLE 3.69. Enantiomeric Ratios for δ-Lactones (C 6–C18 ) from Margarine and Coconut.
(R):(S) (%) in Lactones
Food δ–C6 δ–C8 δ–C10 δ–C12 δ–C14 δ–C16
Margarine (from sunflower oil) 50:50 50:50
Coconut juice R>85* R>70** 84:16 60:40 na na

Coconut flesh 87:13 90:10 85:15 62:38 23:77
* Approximate value (overlap occurred).
** Trace: stereodifferentiation impossible.
na = Not analyzed.
Page 428
TABLE 3.70. Concentration of τ- and δ-Lactones in Dairy Products.

Concentration (µg/kg) of Lactones
C10 C12
Food δ-C8 τ- δ- τ- δ- δ-C14 δ-C16
Country butter 1984 46 3859 323 1906 223

Best quality butter (sweet 446 46 3390 136 1197 147
cream)
cream)
After heating to 200ºC for 15 2898 320 4873 699 1856 213
min
Whipped cream (pasteurized) 293 Trace * 742 45 298 49
Evaporated milk (7.5% fat) Trace Trace 33 30 43 Trace Trace

Full-cream milk (pasteurized) 46 Trace 277 Trace 108 21 Trace
Parmesan cheese Trace Trace 858 264 480 18 Trace
Cheddar cheese 292 Trace 1406 535 823 64
Limburger cheese 319 Trace 319 2446 4810 400 Trace
Emmental cheese 202 Trace 1396 65 726 106
Blue cheese Trace Trace 2315 187 1603 95 Trace
* Trace: concentrations <10 µg/kg.
shown in Figure 3.67. The chromatograms show marked differences, indicating that this GC method can be
used for the detection of the differences in the storage conditions of raw material after boiling the sample. The
lowest headspace volume required to perceive the odorant of boiled cod and trout at the sniffing port is given
in Tables 3.72 and 3.73, respectively. Marked differences were observed in the lowest headspace volumes.
These differences indicate that the method is suitable for the objective evaluation of odor profile of trout and
cod and can be used for the assesment of inappropriate storage conditions.
TABLE 3.71. Concentrations of τ- and δ-lactones in Margarine, Coconut, and Soya Milk.
Concentration (µg/kg) of Lactones
C6 C8 C10
Food τ– δ– τ– δ– τ– δ– δ –C12 δ–C
Margarine (sunflower 4303 331

oil)
Coconut (flesh) Trace * 579 Trace 9613 Trace 3513 372 89
Soya milk Trace Trace Trace

* Trace: concentrations <10 µg/kg.

Page 429
Figure 3.67.
Capillary gas chromatograms obtained by GCO—H of cod samples
A and B. A headspace volume of 10 mL was drawn from boiled cod mince
of which the raw material had been stored for 26 weeks at -60° (sample A)
and at -13°C (sample B). Peaks: 1 = acetaldehyde; 2 = methanethiol;
3 = trimethylamine; 4 = dimethyl sulfide; 5 = 2-methylpropanal; 6 = butane-2,3-dione;
7 = 3-methylbutanal; 8 = 2-methylbutanal; 9 = unknown; 10 = methional;
11 = dimethyl trisulfide; 12 = 1 -octen-3-on; 13 = (Z)-1,5-octen-3-on;
14 = unknown; 15 = dimethyl tetrasulfide.
Page 430
TABLE 3.72. Lowest Headspace Volumes Required to Perceive the Odorant at the Sniffing Port in GCO—H
of Boiled Cod Minces after Storage of the Raw Material at Different Temperatures.*
Volume*** (mL)
Compound Rl on Odor Description ** RTX 5 A B
Acetaldehyde**** <400 sweet 5 2.5
Methanethiol**** <400 Sulfurous 10 20
Trimethylamine **** <500 amine-like >25 5
Dimethyl sulfide**** 505 cabbage-like 5 10
2-Methylpropanal**** ≈550 malty >20 5
Butane -2,3-dione **** 595 buttery-like 10 2.5
3-Methylbutanal**** 653 malty 20 0.5
2-Methylbutanal**** 663 malty >20 5
Unknown 785 vegetable-like, pungent >20 10

Methional§ 908 boiled potato-like 10 5
Dimethyl trisulfide **** 977 cabbage-like, putrid 1 1
1-Octen-3-on § 980 mushroom-like 10 5
(Z)-1,5-Octen-3-on § 983 geranium-like 5 5

Unknown 1077 mushroom-like >20 10
Dimethyl tetrasulfide § 1232 cabbage -like, putrid 10 10
*The cod minces were stored for 26 weeks at (A) -60ºC, and (B) -13ºC before boiling. Samples A and B
were thermostatted at 40ºC during GCO—H.
*** The lowest headspace volume required to perceive the odorant at the sniffing port.
**** The compound was identified by comparing it with the reference substance on the basis of the following
criteria: retention time on the capillary RTX 5, mass spectra obtained by MS–EI and MS–chemical
ionization (CI), and odor quality perceived at the sniffing port.
§ The MS signals were too weak for an interpretation: the compound was identified by comparing it with the
reference substance on the basis of the remaining criteria reported in footnote "*".
RI = retention index.
GC has been used, not only for the study of the odorants of fish, but also for the study of the aroma
compounds in chicken [555,556]. Dynamic headspace GC was applied for the study of the influence of
various endpoint temperatures (EPTs) on the volatile compounds in chicken breast [ 557]. The EPT of
meat varied between 60°C and 80°C; 0.5 g sample was purged with helium for 10 min. The
conditioning and purge temperatures were both 50°C. Volatiles were collected on a Tenax trap at
ambient temperature; the trap was heated to 175°C, and desorption took place at 180°C for 4 min.
Separation was performed on a fused-silica capillary column (50 m × 0.32 mm I.D., film thickness of
cross-linked 5% phenyl methyl silicone 1.05 µm). The oven temperature was 0°C during desorption and
then increased to 4°C at 1°C/min, to 190°C at 6°C/min, and to 250°C at 20°C/min and held for 8 min.
Injector and
Page 431
TABLE 3.73. Lowest Headspace Volumes Required to Perceive the Odorant at the Sniffing Port in GCO—H
of Boiled Trouts before and after Storage of the Raw Material.*
Volume*** (mL)
Compound Rl on Odor Description ** RTX 5 C D
Acetaldehyde**** <400 sweet 5 1
Propionaldehyde**** ≈450 sweet 2.5 0.5
2-Methylpropanal**** ≈550 malty >30 5
Butane -2,3-dione **** 595 buttery-like 10 2.5
Unknown 645 vegetable-like, pungent 30 2.5

3-Methylbutanal**** 653 malty >30 5
Unknown 685 pungent, ethereal >30 10

Pentane-2,3-dione **** 700 buttery-like 10 2.5
Unknown 748 vegetable-like 30 2.5

hexanal****
(Z)-3-Hexenal**** 800 green 10 1
(Z)-4-Heptenal**** 900 biscuit-like 30 5
Methional§ 908 boiled potato-like 5 5
1-Octen-3-on § 980 mushroom-like 5 2.5
(Z)-1,5-Octen-3-on § 983 geranium-like 2.5 0.5
Octanal§ 1006 citrus-like 30 10

Unknown 1077 mushroom-like, earthy 30 10
(Z, Z)-3,6-Nonadienal § 1100 fatty, green 30 10
(E, Z)-2,6-Nonadienal § 1149 cucumber-like 30 5

*The trouts were (C) freshly harvested and (D) stored for 1 year at –13 ºC before boiling. Samples C and D
were thermostatted at 40ºC during GCO—H.
*** The lowest headspace volume required to perceive the odorant at the sniffing port.
**** The compound was identified by comparing with the reference substance on the basis of the following
criteria: retention time on the capillary RTX 5, mass spectra obtained by MS–EI and MS–CI, and odor
quality perceived at the sniffing port.
§ The MS signals were too weak for an interpretation: the compound was identified by comparing it with
reference substance on the basis of the remaining criteria reported in footnote "*"
RI = retention index.
FID temperatures were 110°C and 290°C, respectively. Carrier gas was helium. A typical
chromatogram is shown in Figure 3.68. More than 50 compounds were detected by the method,
indicating the complexity of the aroma profile of boiled chicken breast meat. The majority of peaks
were identified either with the use of authentic standard compounds or by MS. The concentration and
the coefficient of variation of some volatiles are presented in Table 3.74. The data prove that the
quantity of volatiles increases with increasing EPT, mainly between 60°C and 70°C. Because the
method is complicated
Page 432
Figure 3.68.
Dynamic headspace GC chromatograms of volatiles from chicken breast
meat previously heated to endpoint temperatures of 70°C: 1 = H 2S, CO 2:
2 = methanol; 3 = propanal and acetone; 4 = 2 -propanol and thiobismethane;
5 = 2 -methylpropanal; 6 = 1-propanol; 7 = 2,3-butanedione; 8 = hexane;
9 = 2 -butanone; 11 = 3-methylbutanal; 13 = 2-methylbutanal; 14 = 1-penten-3-ol;
15 = pentanal; 18 = dimethyl disulfide; 19 = hexanal; 21 = 1-hexanol;
22 = 2-heptanone; 23 = heptanal; 26 = 1-heptanol; 27 = benzaldehyde;
28 = 7 -octen-4-ol; 33 = octanal; 38 = dlimonene;
43 = undecane; 44 = nonanal; 52 = dodecane.
and needs trained personnel, it was not proposed for routine analysis in the industry, but only for
research purposes.
Because androsterone, skatole, and indole unfavorably influence the odor and flavor characteristics of
meat, the determination is of considerable importance. Because of the low volatility of the compounds,
HPLC is the method of preference for the determination of their concentrations in adipose tissues of
pigs [ 558,559]. A rapid RP-HPLC method was developed for the simultaneous determination of
androsterone, skatole, and indole in back fat of pigs [ 560]. Samples were prepared by homogenizing 0.5
g of back fat with 3.0 mL of methanol containing androstanone and 2-methylindole internal standards.
After homogenization, the sample was cooled in an ice bath for 15 min and centrifuged. An aliquot of
140 µL of supernatant was mixed with 30 µL of 2% dansylhydrazine in methanol, 4.4 µL of water, and
10 µL of 20% BF3 in methanol. After 5 min at room temperature, the samples were injected.
Page 433
TABLE 3.74. Reproducibility of Volatile Compound Data from Chicken Breast Muscle Heated to Three Endpoint Temperatures as
Analyzed by Dynamic Headspace Method (n = 4).
65ºC 70 ºC 75 ºC
Peak No. Retention Time (min) X CV (%) X CV (%) X CV (%) av CV (%)

14 18.270 21.6 12.6 64.2 10.8 56.9 4.23 9.21
15 18.794 43.9 6.25 141 9.13 155 3.59 6.32
18 21.765 79.1 8.89 194 11.8 153 8.62 9.77
19 22.961 540 13.5 150 × 10 8.13 134 × 10 3.07 8.23
22 26.250 2.76 1.68 5.27 15.0 4.47 1.04 5.91
23 26.630 29.9 7.59 62.6 6.85 56.2 7.16 7.20
28 29.196 48.3 10.9 125 10.2 116 9.66 10.2
33 29.981 17.5 6.68 44.1 4.82 43.5 4.87 5.46
44 33.058 6.26 9.58 115 4.62 111 6.86 7.02
X: mean. 14 = 1-penten-3-ol; 15 = pentanal; 18 = dimethyl disulfide; 19 = hexanal; 22 = 2 -heptanone; 23 = heptanal; 28 = 7 -
octen-4-ol; 33 = octanal; 44 = nonanal.
Page 434
Separation was performed on a C18 column (60 × 4.6 mm I.D., particle size 3 µm) at 40°C. The
components of the mobile phase were: (A) THF:25 mM potassium phosphate buffer pH 6.0:acetic acid
(31:67.6:1.4, v/v); (B) THF:acetonitrile:25 mM potassium phosphate buffer pH 6.0:acetic acid
(34:23.8:41.1:0.8, v/v); and (C) THF:water (90:10, v/v). The gradient was 0-4.5 min, 100% A; 4.5-5.0
min, 100% A 100% B; 5.0-10.5 min, 100% B; 10.5-10.6 min, 100% B-90%C + 10% A (2.0 mL/min);
12.0-12.5 min, 90% C + 10% A-100% A; 12.5-14.0 min, 100% A (2 mL/min - 1.5 mL/min). The
excitation and emission wavelengths were 285 nm and 340 nm, respectively, for the first 5 min
detection (indole and skatol) and then were changed to 346 nm and 521 nm for androsterone and
androstanone. The selectivity of the chromatographic system is demonstrated with the chromatograms
in Figure 3.69. The chromatograms prove that solutes and internal standards are well separated from
each other, and no interference can be observed from the other
Figure 3.69.
Selectivity of chromatographic system: (A) standard solution containing:
33 ng/mL of indole, skatol, and 2-methylindole; 400 ng/mL of androsterone;
and 330 ng/mL of androstanone. (B) Back fat sample spiked with internal
standards (2-methylindole and androstanone, 200 and 2000 ng/g,
respectively. Androsterone, skatol, and indole content 1010, 190,
and 110 ng/g, respectively. (C) as (B) but without internal standards.
The Netherlands.)
Page 435
components of back fat samples. The limit of detection and the limit of quantitation were <3 ng/mL and
30 ng/g for indole and skatol, respectively, and 20 ng/mL and 200 ng/g for androsterone. The recoveries
were also good, being 100.7 ± 5, 96.9 ± 6, and 104.8 ± 9% for indole, skatol, and androsterone,
respectively. The data of the within-day and between-day reproducibility of the method are given in
Table 3.75. The CV values of both within-day and between-day determinations were low, indicating the
stability and reliability of the method. The concentrations determined by HPLC agreed well with those
of the traditional colorimetric method, suggesting that it can be used as an alternative procedure for the
measurement of boar taint compounds. The method is rapid and does not employ time-consuming
sample preparation steps, and the solvent consumption is low.
A similar RP -HPLC method was developed for the determination of indole and skatol in adipode tissue
of pig [ 561]. Fat samples with the 2-methylindole internal standard were extracted with methanol, and
the lipids were removed from the extract by freezing the sample. Separation was performed on a
precolumn (4 × 4 mm I.D., particle size 5 µm) and on an analytical column (75 × 4 mm I.D., particle
size 4 µm), both filled with octadecylsilica. Eluents were: 0.02 mol acetic acid:acetonitrile:isopropanol
(65:20:15, v/v) (A) and acetonitrile:isopropanol (70:30, v/v) (B). The gradient program was 0-5 min,
100% A; 5-6 min, 100% B; 6-7 min, 100% B; 7-8 min, 100% A; 8-10 min, 100% A. The column
temperature was 40°C and the flow rate was 1 mL/min. The excitation and emission wavelengths of the
fluorescence detector were 270 nm and 370 nm, respectively. The retention times of indole, 2-
methylindole, and skatol were 2.9, 4.3, and 5.0 min, and no interference from the other components of
the extract was observed. The recovery of both indole and skatol varied from 92-100%, the interassay
coefficient of variation was 10%. Owing to its rapidity, this method also has been proposed for routine
control of the indole and skatol content of pig fat.
Low-molecular-mass aldehydes having an unpleasant, pungent odor were determined as thiazolidine

derivatives by GC [ 562]. Fat and oil samples (each of 0.1 g) were extracted twice with 0.5 mL of
acetonitrile, and the combined extract was used for derivatization. Solid samples (ca. 1 g) were
homogenized with 4 mL of acetonitrile and centrifuged. The supernatant was decanted, and the pellet
was extracted again. The combined supernatants were used for the derivatization, which was carried out
by mixing 0.1 mL of extract with 0.2 mL of 0.2 mg/mL cysteamine and 0.1 mL of 0.1 M NaOH, and
the volume of the mixture was adjusted to 1 mL with water. After 10 min shaking at room temperature,
0.5 mL of water was added; then it was saturated with NaC1 and extracted with 0.2 mL of ethyl acetate
containing 1 µg/mL phenyl sulphide internal standard. A fused-silica capillary column DB-17 (15 m ×
0.53 mm I.D., df 1.0 µm) connected with a DB -210 column of the same dimensions was used for the
separation. Column temperature was raised from 100°C to 240°C
TABLE 3.75. Intra and Inter-Assay Variability.
Indole Skatol Androsterone
CV (%) CV (%) CV (%)
Concentration Within Between Concentration Within Between Concentration Within Between

(mean ± SD) Day Day (mean ± SD) Day Day (mean ± SD) Day Day
(ng/g) (n = 7) (n = 5) (ng/g) (n = 7) (n = 5) (ng/g) (n = 7) (n
1 74 ± 4 5.5 8.2 209 ± 8 3.9 7.0 2940 ± 88 3.0
2 90 ± 2 2.2 5.1 173 ± 2 1.4 4.0 870 ± 34 3.9
3 46 ± 3 5.4 9.6 228 ± 9 3.7 6.7 1970 ± 114 5.8
4 32 ± 1 3.9 6.2 46 ± 2 3.3 4.4 610 ± 24 3.9
5 44 ± 2 5.2 7.4 114 ± 2 1.1 5.6 1310 ± 61 4.7
6 36 ± 2 4.3 8.9 36 ± 2 4.9 7.9 250 ± 20 8.2
7 41 ± 2 4.2 12.6 126 ± 7 5.3 13.3 390 ± 9 2.2
8 103 ± 7 6.6 4.5 314 ± 20 6.2 5.3 490 ± 55 11.1
9 54 ± 4 7.2 7.3 171 ± 7 3.8 5.4 790 ± 57 7.2
10 69 ± 2 3.4 5.0 136 ± 4 2.6 5.9 670 ± 17 2.6
KV Amsterdam, The Netherlands.
Page 437
at 6° C/min. Injector and detector temperatures were 250° C, and nitrogen was the carrier gas. The
thiazolidine derivatives of aldehydes were well separated under these GC conditions, as demonstrated
in Figure 3.70. The minimum detectable amount depended considerably on the length of the alkyl
chain; it was 4 pg injected for C3 and 100 pg injected for C6-1. The coefficients of variation of within-run
and between-run determinations varied from 1.3-7.1% and 2.6-15.2%, respectively. The coefficients of
regression between peak height ratio and amount of aldehyde were in each instance over r = 0.992, and
the recoveries were 82-111%. The aldehyde content of some food products is given in Table 3.76. The
data prove that the aldehyde content of foods differs
Figure 3.70.
Typical gas chromatogram obtained from standard and
food sample: (A) standard (containing 100 ng of C 2 - C8
and 500 ng of C9, C 10, C 6-1 and C 6-2), (B) sesame oil (10 mL).
Peaks: 1 = acetaldehyde (C2 ), 2 = propanal (C3), 3 = isobutanal
(i -C4), 4 = butanal (C 4), 5 = isopentanal (i-C5), 6 = pentanal (C 5),
7 = hexanal (C6 ), 8 = trans-2-hexenal (C6-1), 9 = heptanal (C 7),
10 = 2,4-hexadienal (C 6-2), 11 = octanal (C 8), 12 = nonanal (C 9),
13 = phenyl sulphide (IS), 14 = decanal (C10).
Page 438
TABLE 3.76. Aliphatic Aldehyde Contents in Various Foods from Commercial Sources.
Aldehyde * Content ( µg/mL or g) (mean ± SD, n = 4)
Sample C3 C4 i-C5 C6 C7 C9> C6-1
Sesame oil 2.4 ± 0.1 6.9 ± 0.3 6.7 ± 0.1 4.0 ± 0.1 ND 11.9 ± 0.7 ND
Soybean oil 1.6 ± 0.1 5.4 ± 0.3 5.1 ± 0.3 1.8 ± 0.1 ND 8.1 ± 0.5 ND
Rapeseed oil 2.5 ± 0.1 5.3 ± 0.3 4.9 ± 0.3 3.6 ± 0.2 ND 10.6 ± 0.6 ND
Cottonseed oil 1.6 ± 0.1 5.3 ± 0.3 5.1 ± 0.3 2.3 ± 0.02 ND 10.0 ± 0.6 ND
Corn oil 1.3 ± 0.1 5.0 ± 0.2 4.0 ± 0.4 2.2 ± 0.1 ND 9.1 ± 0.2 ND
Olive oil 1.8 ± 0.1 5.5 ± 0.2 4.8 ± 0.3 3.5 ± 0.2 ND 12.6 ± 1.0 ND
Sardine oil 5.9 ± 0.1 6.2 ± 0.2 3.3 ± 0.2 6.5 ± 0.2 5.3 ± 0.2 23.7 ± 1.2 ND
Beef fat 2.7 ± 0.3 5.1 ± 0.3 5.4 ± 0.2 2.4 ± 0.2 ND 12.6 ± 1.7 ND
Lard 3.8 ± 0.2 5.5 ± 0.3 4.7 ± 0.3 2.6 ± 0.1 ND 9.0 ± 0.9 ND
Butter 2.1 ± 0.1 4.9 ± 0.2 5.1 ± 0.3 2.0 ± 0.2 ND 13.0 ± 0.8 ND
Margarine 1.9 ± 0.2 5.1 ± 0.4 4.8 ± 0.3 1.2 ± 0.1 ND 8.6 ± 0.8 ND
Mayonnaise 2.1 ± 0.3 3.8 ± 0.2 3.8 ± 0.4 2.2 ± 0.2 ND 10.6 ± 0.5 ND
Cheese 2.4 ± 0.1 4.6 ± 0.2 3.4 ± 0.1 3.5 ± 0.1 ND 22.8 ± 1.3 ND
Egg yolk 2.7 ± 0.3 4.0 ± 0.2 3.5 ± 0.2 1.5 ± 0.1 ND 9.0 ± 0.2 ND
Fresh milk 3.0 ± 0.5 3.7 ± 0.4 5.1 ± 0.1 1.5 ± 0.1 ND 9.4 ± 0.5 17.8 ± 2.1
Chocolate 1.9 ± 0.2 4.4 ± 0.3 3.5 ± 0.4 5.1 ± 0.2 3.4 ± 0.1 19.1 ± 0.5 18.7 ± 0.8
Potato chips 2.0 ± 0.1 3.2 ± 0.2 4.1 ± 0.2 1.8 ± 0.1 ND 9.9 ± 0.9 ND
*C3 = propanal; C 4 = butanal; i–C5 = isopentanal; C 6 = hexanal; C 7 = heptanal; C 9 = nonanal; C6–1 = trans–2–
hexenal.
Page 439
considerably according to the type of the product. It was further established that heat treatment and
exposure to UV radiation markedly increase the aldehyde content of food products, suggesting quality
deterioration.
Thermal desorption-GC-MS has been used for the analysis of volatile aroma compounds in cooking oils
[ 563]. One milliliter of the oil sample was heated to 80°C for 30 min and purged with high-purity
helium. Volatiles were trapped in a tube containing 100 mg of Tenax TA. Desorption was performed at
220°C for 5 min. The inlet of the capillary GC column (30 m × 0.25 mm I.D., DB-5MS, df = 0.25 µm)
was held at -70°C and then heated to 200° C. Initial oven temperature was 35° C for 5 min, to 80°C at
10° C/min, to 200°C at 4°C/min, and to 260°C at 10°C/min. More than 30 volatile compounds were
identified in the cooking oils (Figure 3.71.), and their concentration markedly depended on the origin
and type of oil. The relative amounts of aroma compounds are compiled in Table 3.77. As the GC -MS
method found marked differences between the composition of volatile aroma compounds in cooking
oils, it was proposed that this method can be used for quality control and comparison of cooking oils
and for the determination of the origin.
Figure 3.71.
Chromatogram of extra virgin olive oil, brand A. Peaks: 2 = hexane;
3 = ethyl acetate; 7 = heptane; 9 = toluene-d8 internal standard;
11 = 1-octene; 13 = octane; 17 = (E)-2-hexenal; 18 = (Z)-3-hexenal;
23 = styrene; 24 = heptanal; 25 = (E)-2-heptenal; 29 = (Z)-3-hexen-1-ol;
33 = cymene-d 14 internal standard; 35 = (E)-2-octenal; 36 = nonanal;
37 = naphthalene-d 8 internal standard; 39 = (Z)-2-decanal.
and the corresponding author, copyright © 1995,
American Chemical Society.)
TABLE 3.77. Relative Amounts (nanograms per milliliter) of Volatile Organics in Cooking Oils.
Brand A Brand B Brand C Brand D
Extra Extra Extra Extra Extra
Peak No. Virgin Virgin Regular Light Virgin Regular Virgin Regular
1 Butanal 3.3 3.9 15.5 2.0
2 Hexane 53.0 18.4 95.3

3 Ethyl acetate 87.4 165.1 15.9 1.3 82.6 6.3 133.8 15.4
4 3-Methylbutanal 11.8 21.7 18.6 3.1
5 2-Methylbutanal 11.2 22.7 20.0

6 3-Pentanone 62.5 5.0
7 Heptane 144.0 390.3
8 Pentanal 48.7 50.8 68.1 295.1 52.4
9 Toluene -d 8 internal standard
10 Toluene 115.1 28.3 19.1 9.1 22.7 8.8 60.7 11.3

11 1-Octene 50.5 13.8 14.5 24.8 10.6 63.2 13.0
12 2-Octene 17.8
13 Octane 2310.1 228.1 121.4 3107.8
14 Hexanal 208.6 516.4 319.5 1903.0 24.2 255.9

15 (E)-2-octene 147.5
16 (Z)-2-octene 79.3
17 (E)-2-hexenal 72.6 119.6 8.8 689.4 153.3 254.4
18 (Z)-3-hexenal 32.6 869.1 1330.9

19 (Z)-3-hexen-1-ol 88.2
20 (E)-2-hexen-1-ol 463.2 8.9 183.7 249.1 36.2
21 4-Hexen-1-ol 117.6 26.0
22 2-Heptanone 22.2 48.0 17.5 17.6 16.7 62.0 21.7

Brand A Brand B Brand C Brand D

Extra Extra Extra Extra Extra
Peak No. Virgin Virgin Regular Light Virgin Regular Virgin Regular
23 Styrene 113.8 175.4
24 Heptanal 25.8 46.1 15.9 58.4 227.1 13.6 128.9 18.5

25 (E)-2-heptenal 210.7 23.1 20.9 8.5 200.6
26 (Z)-2-heptenal 124.3
27 2-Pentylfuran 35.4 149.5 12.0 38.1

28 Octanal 43.5 174.2 30.3
29 (Z)-3-hexen-1-ol 324.8 227.4
30 (Z)-3-hexen-1-ol acetate 246.6
31 (Z)-4-hexen-1-ol 18.0 23.3 22.9

32 Acetic acid, hexyl ester 45.6 80.7 14.9 6.7 64.3 4.4
33 Cymene-d.14 internal standard
34 Limonene 7.6 11.8 10.7 2.0 286.4 418.4
35 (E)-2-octenal 40.3 9.2 138.1 49.8
36 Nonanal 274.6 84.7 18.9 68.6 334.6 19.2 307.0 186.0

37 Naphthalene-d8. internal standard
38 Methylcyclo heptane 38.8 17.8
39 (Z)-2-decanal 42.2 7.6 1.7
40 2-Cyclohexen-1-ol 19.9 28.8 34.8

41 2-Undecenal 8.2 16.8 11.4

Page 442
3.2.1.6—
Other Food Products
Because of their considerable commercial importance, the separation, identification, and quantitation of
flavor compounds in juices and fruits has been extensively studied. Thus, SPME [ 564], dynamic
headspace sampling [ 565], various other extraction methods [ 566], and simultaneous distillation-
extraction [ 567] equally found application in the flavor analysis of juices and fruits. Similar to other
foodstuffs, GC [568], HPLC [ 569] and ion-exclusion chromatography with UV detection [ 570] were
frequently used for these purposes. Much effort was devoted to the elucidation of the composition of
flavor compounds in orange juices. The performance of static and dynamic headspace GC [ 571] method
and the volatile profile of fresh and processed orange juices were compared [ 572]. The flavor profile of
various orange juices has been proposed for the identification of orange varieties [ 573] and commercial
orange juices [ 574]. Dynamic headspace GC was applied for the determination of the volatile
constituents in various cultivars [575]. Samples were prepared by both hand-extracted and mechanically
extracted orange juices. Analyses were performed with dynamic headspace GC using a cold capillary
trap and an HP -5 capillary column (30 m × 0.53 mm I.D., film thickness 2.65 µm). FID detector and
injector temperatures were 250°C. Original oven temperature was 40°C for 6 min and increased to 200°
C at 6°C/min. It was established that the composition of volatile constituents markedly depends on the
mode of preparation.
To facilitate the detection of adulteration, the chiral separation and isotopic analysis of the most
important aroma compounds of some raspberry cultivars were performed [ 576]. Samples were prepared
by grounding 500 g of raspberries with 300 mL of distilled water and 200 mL of acetone. The slurry
was filtered through sand, and the acetone was removed under vacuum. The aqueous phase was
extracted with 250 mL of pentane:diethyl ether (50:50). The organic fraction was dried over anhydrous
sodium sulfate, concentrated at 40°C to 25 mL, and used for GC-MS. The sample solution was further
concentrated for 2 mL before multidimensional GC (MDGC) and isotopic ratio mass spectrometry (GC -
IRMS). GC-MS was carried out on an HP1 capillary column (25 m × 0.25 mm I.D., film thickness 0.25
µm). Initial column temperature was 50°C for 5 min, increased to 200°C at 5°C/min, to 295°C at 25°
C/min, and final hold 10 min. Helium was used as the carrier gas. Chiral separation was performed on a
double oven system. The first column was of 25 m × 0.32 mm I.D. coated with 0.5 µm of BP 20. The
injector and FID detector temperatures were 250°C. Oven temperature program was 120°C for 5 min
and increased to 220°C at 10°C/min. The second capillary column was of 25 m × 0.22 mm I.D. and
coated with 10% octakis-2,6-dimethyl-3-trifluoracetyl-τ-cyclodextrin in OV-1701. Initial oven
temperature was 110°C for 10 min and then increased to 180°C at 1°C/min. For the determination of
the isotope ratios 13C/ 12C, the same column was used as for GC-MS. The column temperature
was 50°C for 15 min, increased to 140°C at 5°C/min, to 250°C at 2°C/min, and held for 15 min. The results of
chiral separation and isotope measurements are presented in Tables 3.78 and 3.79. It was concluded from the
data that this combined method is suitable for the discrimination of cultivars and offers information about the
taste quality of products. It was further established that
TABLE 3.78. Chiral and Isotope 13C/ 12C Measurement with Quantitative Ratios of Natural Raspberry Samples.
δ –Decalactone δ–Decalactone α–lonone
Cultivar and Enantiomeric Isotope Ratio Enantiomeric
Characterization Excess (%) 13C/ 12 C (0/00) Excess (%)
Natural Mecker July (1993) 98 to 100 (S) –34 to –39 98 to 100 (R)
August (1993) 98 to 99 (S) –36 to –39.1 98 (R)
Natural Heritage August (1993) 100 (S) –33.3 to –35.9 98 (R)

X 99 (S) –40.2 98 (R)
Natural wild (1993) 100 (S) –37.2 99 (R)

Natural Williamette (1992) 99 (S) –35.0 99 (R)
Natural Malling (1992) 99 (S) –34.0 100 (R)
Promise
α –lonone Raspberry Ketone cis-3-Hexen–1–

Cultivar and Isotope Ratio Isotope Ratio Isotope Ratio
13C/ 12C (0/00) 13C/ 12 C (0/00) 13C/ 12 C (0/00)*
Characterization
Natural Mecker July (1993) 33 to –35 –28.9 to 31.9 –39.1
August (1993) –33 to –36 –30.6 to –32 –39.1
Natural Heritage August (1993) –36.5 to –37.4 –28.8 to –30.2 –35.2

X –36 –30.1 ND
Natural wild (1993) –33.6 –35.1 –34.6

Natural Williamatte (1992) –34.3 –30.0 –35.4
Natural Malling (1992) –32.5 –30.0 –33
Promise
Quantitative Quantitative β-lonone Isotope

Cultivar and Ratio R1 = Ratio R2 = Ratio
Characterization δC10/α–lonone α–lonone/β–lonone 13C/ 12 C (0/00)*
Natural Mecker July (1993) 20 to 130 0.4 to 2.4 –32.2 to –

August (1993) 20 to 31 0.9 to 1.0 –32.3 to –
Natural Heritage August (1993) 7 to 20 0.6 to 1.5 –32.2 to 34.1
X 55 1.0 –33.7
Natural wild (1993) 8 0.9 –33.0

Natural Williamette (1992) 50 0.6 –34.4
Natural Malling (1992) 16 0.7 ND
Promise
* Measurements made on a mixture of different samples.
X is a Mecker's sample supplied under the name Heritage.
KV Amsterdam, The Netherlands.
Page 444
TABLE 3.79. Chiral and Isotope 13C/ 12 C Measurement with Quantitative Ratios of Raspberry-Flavored
Products.
δ -Decalactone δ-Decalactone α -lonone
Commercial Enantiomeric Isotope Ratio Enantiomeric
Samples Excess (%) 13C/ 12C (0/00) Excess (%)
Syrups SA 100 (S) –34 100 (R)
SB 2 (S) –26.3 4 (R)
Heavy BA 100 (S) –28.3 100 (R)
Juice BB 100 (S) –29.5 4 (R)
Tea 0 –27.6 8 (R)
α -lonone Raspberry Ketone cis-3-Hexen-1-ol
Commercial Isotope Ratio Isotope Ratio Isotope Ratio
13C/ 12C (0/00) 13C/ 12C (0/00) 13C/ 12C (0/00)*
Samples
Syrups SA –35.8 –31.9 nd
SB –27.8 –24.0 nd
Heavy BA –34.3 –29.1 nd
Juice BB –26.0 –29.3 nd
Tea nm –25.2 nd
Quantitative Quantitative β-lonone Isotope
Commercial Ratio R1 = Ratio R2 = Ratio
Samples δ C10/α-lonone α-lonone/β-lonone 13C/ 12C (0/00)*
Syrups SA 11 50 –29.5
SB 0.5 9 –26.3
Heavy BA 23 3 –33.1
Juice BB 4 2.1 –26.0
Tea 0.2 100* nd
* R2 theoretically equal + ∞ .
nm = Detected but not measurable.
nd = Not detected.
chiral chromatography and isotopic ratio mass spectrometry can be successfully used for the detection
of adulteration.
GC-MS and organoleptic analysis have been applied for the determination of the aromatic qualities of
ready-made tomato sauces [ 577]. Volatiles were distilled, collected in a cold trap, and extracted with n-
pentane. GC-MS was performed on an OV-1 capillary column (25 m × 0.25 mm I.D., film thickness 0.1
µm). Helium was used as the carrier gas. Initial oven temperature was 30°C for 1 min, to 40°C at 10°
C/min, 5 min hold, to 280°C at 10°C/min, and 15 min final hold. The chromatograms of freshly
prepared and stored sauce (tomato from northern Italy, fresh onion, and fresh greenhouse-grown basil)
are shown in Figure 3.72. The chromatograms clearly demonstrate that the
Page 445
Figure 3.72.
Comparison between the plot of the volatile compounds in a ready-made tomato sauce (left) and the same product
after storage (right). Peaks: 1 = 6-methyl-5-hepten-2-one (tomato); 2 = cineole (basil); 3 = di -2-propenyl
sulphide (garlic); 4 = octanol (internal standard); 5 = 3,7-dimethyl -1,6-octadien-3-ol (tomato); 6 = unknown (garlic);
7 = unknown (garlic); 8 = 2-dodecenal (olive oil); 9 = unknown; 10 = 2,4-decadienal (olive oil); 11 = 2 -methoxy -4-(1-
propenyl)
phenol (basil); 12 = 6,10-dimethyl-5,9-undecadien-2-one (tomato); 13 = 6-allyl -4-methoxy -1,3-
benzodioxole (myristicin) (parsley);
14 = 6,10-dimethyl-3,5,9-undecatrien-2-one (tomato); 15 = 5-allyl-4,7-dimethoxy-1,3-benzodioxole (apiole) (parsley);
16 = 2,4-dimethyl-5,6-dithio-2,7-nonadienal (onion); 17 = palmitic acid (tomato).
(Reprinted from Reference [ 577], copyright © 1994. With kind permission from Elsevier Science—NL,
Page 446
GC/MS method can be used for the discrimination of volatiles originating from different components of
the ready-made tomato sauce, facilitating in this manner the quality control of the products.
Organoleptic analysis indicated that aromatizing processed products can be employed in the preparation
of ready-made tomato sauce without significant loss of quality.
HPLC, combined with high-resolution GC (HRGC), has also found application in the analysis of
various essential oils such as citrus [ 578], orange [ 579], bergamot [ 580], and other essential oils [ 581]. A
combined HPLC-HRGC -MS method was employed for the detection of the authenticity of bitter
orange, sweet orange, lemon, and mandarin leaf oils by analyzing the composition of hydrocarbon and
oxygenated fractions [ 582]. Samples of essential oils were dissolved in pentane and separated on a silica
HPLC column (100 × 2 mm I.D., particle size 5 µm). Hydrocarbon fraction was eluted with pentane,
and the oxygenated compounds were backflushed with 1 mL of diethyl ether. The flow rate was 180
µL/min, and the detection wavelength was set to 220 nm. Fractions were on-line transferred to GC.
Deactivated fused-silica precolumn (10 m × 0.53 mm I.D.) was used as a retention gap. The retaining
percolumn (4 m × 0.32 mm I.D.) was coated with SE-52 (film thickness 0.40-0.45 µm). Separation was
performed on a fused-silica SE-52 column (30 m × 0.32 mm I.D., film thickness 0.40-0.45 µm). The
temperature was 45°C during the transfer of HPLC fractions (6 min) and then increased to 240°C at 3°
C/min. Helium was used as the carrier gas. Typical HPLC and GC chromatograms of bitter orange
petitgrain oil are shown in Figures 3.73 and 3.74. The chromatograms indicate that the preseparation of
hydrocarbon and
Figure 3.73.
LC chromatogram of bitter orange petitgrain oil.
A Division of Preston Industries, Inc., and the corresponding author.)
Page 447
Figure 3.74.
Total ion chromatogram of (A) a bitter orange petitgrain
oil and of the (B) F1 (hydrocarbons), and (C) F2 (oxygenated
compounds fractions from its LC separation. Peaks:
1 = tricyclene; 2 = α-thujene; 3 = α-pinene; 4 = camphene;
5 = sabinene; 6 = β-pinene; 7 = 6-methyl-5-hepten-2-one;
8 = myrcene; 9 = α-phellandrene; 10 = δ -3-carene;
11 = α-terpinene; 12 = p-cymene; 13 = limonene;
14 = 1,8-cineole = 15 = (Z)-β-ocimene;
16 = (E)- β-ocimene = 17 = τ-terpinene; 18 = cis-linalool oxide;
19 = terpinolene; 20 = trans-linalool oxide; 21 = linalool;
22 = citronellal; 23 = terpinen-4-ol; 24 = α -terpineol;
25 = nerol; 26 = neral; 27 = linalyl acetate;
28 = geranial; 29 = δ-elemenene; 30 = α -cubebene;
31 = α-terpinyl acetate, 32 = citronellil acetate;
33 = α-copaene; 34 = neryl acetate; 35 = geranyl acetate;
36 = β -elemenene; 37 = N-methyl methyl anthranylate;
38 = (E)-caryophyllene; 39 = trans-α-bergamotene;
40 = α -humulene; 41 = (Z)- β-franesene = 42 = bicyclogeracrene;
43 = α -farnesne; 44 = δ -cadinene; 45 = (E)-nerolidol;
46 = sphatulenol; 47 = caryophyllene oxide.
Publications, A Division of Preston Industries,
Page 448
oxygenated fractions of essential oils facilitate the GC separation and quantitation of each volatile
fraction. The composition of essential oils is given in Table 3.80. It was stated that this combined
HPLC-HRGC -MS method is suitable for the separation and quantitative determination of volatile
constituents in oils. Moreover, the method was proposed for the authenticity control of oils and for the
detection of added contaminants.
The relationship between the sensory characteristics and GC-MS profile of four brands of chocolate
flakes was also determined [ 583]. Dynamic headspace chromatography using simulated mouth
conditions and oral vapor GC indicated that 2-methylbutanal was one of the constituents responsible for
chocolate flavor (Figure 3.75).
The aroma composition and the change in time of aroma constituents in a corn-based snack were
studied by sensory analysis, GC, and MS [ 584]. Samples were prepared by mixing 500 g of finely
ground snake with 50 mL of distilled water and allowing to stand for 1 h at room temperature. The
mixture was extracted with 2 × 500 mL of methanol, filtered, mixed with 800 mL of distilled water, and
extracted three times with a total of 500 mL of dichloromethane, and the organic phase was
concentrated to 2 mL. GC separation was performed on a Supelcowax 10 fused-silica capillary column
(30 m × 0.32 mm I.D., film thickness 0.50 µm). The injector and detector temperatures were 150°C and
240°C, respectively. The initial oven temperature was 50°C for 6.0 min, increased to 240°C at 5.0°
C/min, and final hold 30 min. The gas chromatogram and Osmegram of the extract for the 0-month
storage snack are shown in Figure 3.76. The flavor extract contained about 150 volatile compounds;
according to the sensorial analysis, only 49 volatiles were odor-active compounds. The results of the
qualitative GC and Osmegram analyses are compiled in Table 3.81. The combined method established
that the quality and quantity of aroma compounds of snacks markedly change during storage. It was
suggested that this procedure may promote the better understanding of the flavor properties and stability
of snacks.
The aroma profiles of many other foodstuffs and food products were also studied. Thus, the aroma
profile of unifloral honeys [ 585], mineral waters packed in polyethylene laminated packages [586],
spirits [587], green and black tea powders [ 588], and peanuts and cashews [ 589] was determined.
3.2.2—
Color Pigments
One of the main parameters used for commercial evaluation of many food products is the color. A
bright and characteristic color is an important requirement of marketability. Becuase of the considerable
commercial importance of pigment composition and the inherent low volatility of pigments, much
effort has been devoted to the development of liquid chromatographic methods suitable for the
separation of and quantitative determination of pigments in
Page 449
TABLE 3.80. Components Identified by HPLC-HRGC-MS and Quantitative Composition

for Four Oils Analyzed.
Bitter Sweet Mandarin
Orange Orange Orange Lemon
Compounds (g/100 g) (g/100 g) (g/100 g) (g/100 g)
Tricyclene TRNS TRNS 0.01 TRNS
α–Thujene 0.02 0.39 0.98 0.07
α–Pinene 0.17 1.51 2.16 0.87
α–Fenchene — 0.01 0.01 0.01

Camphene TRNS 0.04 0.02 0.05
Sabinene 0.45 38.26 0.30 3.01
β–Pinene 2.20 2.62 2.31 12.57

6-Methyl –5–hepten–2–one 0.08 0.05 0.01 1.06
Myrcene 2.31 2.88 0.79 0.83
Octanal — 0.01 — —
α–Phellandrene 0.05 0.23 0.04 0.03
δ –3–Carene 1.15 3.46 0.02 0.75
α–Terpinene 0.03 0.65 0.24 0.04

o–Cymene — 0.05 — 0.01
p–Cymene 0.12 2.89 5.19 0.51
Limonene 2.02 5.85 12.59 30.66
1,8–Cineole 0.06 0.03 0.02 1.34
(Z)–β–Ocimene 0.89 0.21 0.18 0.30
(E)– β–Ocimene 3.64 4.49 0.59 1.50
τ–Terpinene 0.18 2.62 27.64 0.42

cis–Sabinene hydrate — 0.23 0.01 0.03
cis–Linalool oxide 0.05 — TRNS 0.01
Octanol — — TRNS 0.01
Terpinolene 0.59 0.98 0.97 0.19
trans–Linalool oxide 0.03 — — —
Linalool 29.80 4.34 0.93 3.09
Nonanal — 0.04 0.01 0.08
cis–Limonene oxide — — TRNS TRNS
cis–p–Menth–2–en–1–ol — 0.09 — 0.02
trans–p–Menth–2–en -1–ol — 0.07 0.02 —
Isopulegol — — — TRNS
Citronellal 0.05 0.43 — 0.78
Terpinen–4–ol 0.12 2.36 0.24 0.51
p–Cymen-8–ol — — 0.02 —
α–Terpineol 5.39 0.21 0.26 0.96

Decanal — 0.03 0.01 —
Cironellol — 0.03 0.01 —
Nerol 1.28 0.26 0.10 2.66
Neral 0.40 0.28 0.06 8.13
Linalyl acetate 39.75 0.40 0.96 5.44
Geranial 0.67 0.59 0.10 11.67
Undecanal — — — 0.04
Thymol — 0.05 0.11 —

Page 450

Bitter Sweet Mandarin
Orange Orange Orange Lemon
Compounds (g/100 g) (g/100 g) (g/100 g) (g/100 g)
δ –Elemenene 0.02 — — —
α–Cubebene 0.02 — — —
Methyl anthranilate — — 0.01 —
α–Terpinyl acetate 0.06 — 0.01 —

Citronellil acetate 0.11 0.25 — 0.21
α–Copaene 0.01 0.01 — —
Neryl acetate 2.27 0.38 0.03 5.89
β–Cubebene — 0.10 — —
Geranyl acetate 4.22 0.28 0.05 2.92
β–Elemenene 0.04 3.80 — 0.03
cis–α –Bergamotene — TRNS — —

N–methyl methyl anthranylate 0.17 10.29 41.61 0.24
(E)–Caryophyllene 0.71 2.47 0.92 0.96
trans–α–Bergamotene 0.01 — — 0.06

α–Humulene 0.07 0.60 0.07 0.09
(Z)– β–Farnesene 0.07 0.58 — 0.02

N–Dimethyl methyl anthranylate — — 0.03 —
Bicyclogeracrene 0.28 — — 0.17
α–Selinene — 0.04 0.02 —
Valencene — 0.02 — —
α–Farnesene 0.05 0.12 TRNS 0.14
δ–Cadinene 0.04 — TRNS 0.02

(E)–Nerolidol 0.06 0.05 — 0.01
Sphatulenol 0.03 — — 0.04
Caryophyllene oxide 0.02 0.06 0.02 0.05
2,3–Dimethyl–3– (4–methyl–3–
pentenyl–bis)–2– norbornanol — — — 0.02
Campherenol — — — 0.01
α–Bisabolol — — — 0.01
β–Sinensal — 1.25 — —
δ–Sinensal — 0.24 — —
TRNS = transferred.
Reprinted from Reference [ 582] with permission from Preston Publications, A Division of
various food products. Thus, RP-HPLC has been successfully used for the analysis of black tea liquors
with special reference to the thearubigins [ 590] and for the separation of curcuminoid pigments in
turmeric (Curcuma domestica Val.) [ 591].
Caramel colors as food additives have been simultaneously separated by HPLC and CE [592]. The
HPLC system consisted of an ODS 2 column
Page 451
Figure 3.75.
Dynamic headspace gas chromatogram from plain chocolate flakes. C1, C2, and C3
are peaks with a chocolate -like smell; C1 = 2-methylpropanal, C2 = 3-methylbutanal,
C3 = 2-methylbutanal.
(250 cm × 4.6 mm I.D.) and a water-methanol gradient elution containing 5 mM pentanesulfonic acid.
Fractions were detected at 275 nm. CE used a capillary column (64 cm × 75 µm I.D.), 30 mM
phosphate buffer (pH = 1.9), 20 kV, and 35°C. The HPLC chromatograms of caramels produced with
sodium hydroxide (class I), ammonia (class III), and ammonium sulphite (class IV) are shown in Figure
3.77. The peak characterizing class III caramel eluted at 3.2 min. It was reported that this peak was
present in each class III caramel sample, and it was absent in other (class I and class IV) caramel
samples; that is, this fraction can be used for the identification of class III caramels. The results of CE
entirely supported the conclusions drawn from the HPLC analysis, as demonstrated in Figure 3.78. It
was stated that both HPLC and CE are suitable for the detection and identification of class III caramel
in foods and can be used for caramel analysis in various food products.
The two major anthocyanins were isolated from champagne vintage by-products by gradient elution
centrifugal partition chromatography [ 593].
Page 452
Figure 3.76.
Gas chromatogram and Osmegram of the extract for the 0 -month storage extract.
Anthocyanins were extracted from the by-products of champagne vintage (1.015 kg) by macerating it
with 1.45 L of methanol:acetic acid (99:1 v/v) for 5 days. After extraction, the slurry was filtered, and
the liquid phase was concentrated to 0.25 L under vacuum at 28°C. This solution was extracted with 4 ×
0.2 L of n-butanol saturated with water. The organic phase was evaporated at 65°C, and the residue was
lyophilized (14.29 g, 1.4%). The initial eluent for the high-performance centrifugal partition
chromatography was ethyl
Page 453
TABLE 3.81. Peak Number, Kovats' Index (KI20M ), Odor Descriptors, Associated Odorant, Maximum
Odor Intensity, (I max ) and Area Under the Odor Peak for Odorants Showing High and Intermediate Odor
Intensity (I max ) in the 0 Month Stored Snack as Perceived by the GCO Panel (means across panelists and
replications).
Osme****
Identified (GC-MS)***
Peak* KI 20M** Compound Imax Area
Descriptors
2 1342 Toasted corn 2–Acetyl –1–pyrroline pi 3.8 0.3
4 1383 Garlic/onion Dimethyltrisulfide pi 4.2 0.4
9 1442 Floral/plastic/oxidized 8–Nonen–onepi 3.7 0.3
10 1463 Cooked or baked potato Methionalpi 8.8 1.7
19 1596 Garlic/onion Nethyl allyl trisulfide pi 6.1 0.8
21 1640 Romano cheese/butyric acid Butyric acid pi 8.5 2.0
28 1795 Garlic/onion Diallyl trisulfide pi 4.0 0.7
29 1818 Body odor/rotted onion and T, t -2,4–Decadienalnc 7.4 2.0

garlic
30 1849 Spicy/cooked onion/ celery 2-Vinyl –4H–1,3–dithiinpi 5.0 0.8
34 1967 Caramel+strawberry/cotton 3–Hydroxy –2–methylpyran– 4– 5.1 0.7

candy One (maltol)pi
36 2012 Musty/oxidized/vinyl/ 2–Methyl -phenol pi 4.2 0.7

metallic
37 2039 Caramel+strawberry/cotton 2,5–Dimethyl–4–hydroxy–3 7.6 1.6
candy (2H)–furanone(furaneol)pi
40 2204 Colve/eugenol/medicinal 2–Methoxy–4–vinylphenolpi 9.2 1.8
41 2225 Stale corn chip/sweet floral O-Amino–acetophenonepi 7.2 1.5
42 2291 Soapy/rubber/plastic/vinyl Decanoic acidpi 4.2 0.9
46 2491 Soapy/bad breath Skatolepi 4.8 0.8
48 2574 Vanilla Vanillinpi 7.5 1.5
49 ND Burnt caramel/musky floral 4,5–Dimethyl–3–hydroxy–2 4.8 2.8

(5H)–Furanonepi
MSD§ 2.56
0.84
* Peak number as shown in Figue 3.76.
** Kovats indices as detected by the GC -MS.
*** Odorant as identified by the GC-MS: pipositive identification, nc identification not 100% agreed to by
the GCO panel when pure standard was evaluated.
**** Sixteen-point intensity scale (0 = none, 15 = extreme).
§ Minimum standardized difference at p < 0.05.
ND = not detected by GC -MS.
Means with the same superscript within the same column are not significantly different from each other (p
< 0.05).
Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.
Page 454
Figure 3.77.
HPLC traces of caramel colors. (a) Class I caramel; (b) Class III caramel; (c) Class
IV caramel with low nitrogen content (1.48% wt); (d) Class IV
caramel with high nitrogen content (4.49% wt).
acetate:n-butanol:water (77:15:8 v/v/v), and it was modified to the final eluent ethyl acetate: n-
butanol:water (40:46:14 v/v/v). Each phase was acidified with 0.8% of trifluoroacetic acid. Fractions
were detected at 540 nm. The purity of fractions was checked by TLC [cellulose F plates, n-
butanol:acetic acid:water (61:10:5 v/v/v) eluent]. The authenticity of the fractions of anthocyanins
malvidin-3-glucoside and peonidin-3-glucoside was also controlled by 1H HMR. The method
effectively separated the two anthocyanins in pure form. Because these compounds show a neutralizing
effect against toxic free radicals, they
Page 455
Figure 3.78.
Electropherogram of the identifying peak at 275 nm.
can be used in food products as protecting agents, and their separation in large quantitities is of
considerable economical importance.
RP-HPLC has been used for the separation of pigment fractions of tomato fruit (Lycopersicum
esculentum cv. Venture and Bulka), carrot (Daucus carote cv. Nanti), and paprika powders [594]. The
extraction and sample purification steps are shown in Figure 3.79. Color pigments from paprika powder
were extracted with acetone. Some characteristic chromatograms are presented in Figure 3.80. The
chromatograms clearly show that the pigment profile of paprika is more complicated than those of red
tomato and carrot, and the RP-HPLC method is suitable for the separation of color pigments of carrot,
red tomato, and paprika powder. Many liquid chromatographic methods were developed and applied for
the separation and quantitative determination of color pigments of paprika powders. Adsorption [ 595]
and RP -TLC methods [ 596] separated the pigments well; however, because of the low reproducibility of
these methods, they found only limited applications in the chromatographic practice [ 597]. A wide
variety of adsorption and RP-HPLC methods were employed for the analysis of paprika pigments. The
number of fractions separated by the method depend considerably on both the characteristics of the
column support and those of the eluent system. The methods can separate the carotenoid esters or the
carotenoids after hydrolysis [ 598]. An adsorption HPLC method was used for the separation of
individual carotenoids after
Page 456
Figure 3.79.
Scheme illustrating the extraction and preparation steps of tomato fruit pigments for HPLC investigation.
(Reprinted from Reference [ 594] with permission from Akadémiai Kiadó, Hungary.)
Page 457
Figure 3.80.
HPLC chromatograms of (A) carrot, (B) ripe red tomato,
and (C) paprika extracts separated on a Chromsil C 18 column
with an acetone: isopropanol:water (39:57:4) mixture used as the
mobile phase. Peak identification: (A) 1 = lutein; 2 = τ-carotene.
(B) 1 = zeaxanthin; 2 = lutein; 3 = neoxanthin; 4 = lycoxanthin;
5 = lycopene; 6 = neurosporene; 7 = β -carotene. (C) 1 = capsorubin;
2 = capsanthin; 3 = zeaxanthin; 4 = capsanthin ester I;
5 = capsanthin ester II; 6 = zeaxanthin ester; 7 = β-carotene;
8 = capsorubin ester I; 9 = capsorubin ester II; 10 = capsanthin
ester III; 11 = capsanthin ester IV; 12 = oxidation product.
from Akadémiai Kiadó, Hungary.)
saponification [ 599]. Samples were prepared by triturating 25 g of paprika powder with methanol for 20
min. The mixture was filtered and the residue extracted with methanol:diethyl ether (90:10 v/v) until the
residue was colorless. The combined filtrates were filled up to 500 mL with the same solvent, and 50
mL were saponified with aqueous potassium hydroxide (60%). The nonsaponified carotenoid fraction
was extracted with diethyl ether, dried in a nitrogen stream, redissolved in acetone, and used for HPLC
analysis. Color pigments were separated on a silica column (250 × 4.6 mm I.D., particle size 5 µm).
The solvents were (A) light petroleum (b.p. 40-60°C) and (B) acetone. The initial mobile phase was
A:B (95:5 v/v). The ratio of B was increased to
Page 458
25% in 30 min using a linear gradient, a 5-min hold, and then an increase of A to 95% in 5 min with a
convex gradient curve. The flow rate was 1 mL/min, and the detection wavelength was set to 460 nm. A
typical chromatogram is shown in Figure 3.81. The majority of fractions was well separated under the
adsorption HPLC conditions, indicating that the method can be employed for the analysis of
carotenoids in Capsicum annuum. The concentrations of the individual color pigments are given in
Table 3.82. It was concluded from the data that this normal-phase HPLC method is suitable for the
separation and
Figure 3.81.
Normal-phase HPLC of Capsicum annuum cv. Belrubi carotenoids.
Peak numbers: 1 = β-carotene; 2 = cryptocapsin; 3 = cryptoflavin;
4 = β-cryptoxanthin; 5 = antheraxanthin; 7 = capsolutein;
8 = luteoxanthin; 9 = zeaxanthin; 10 = mutatoxanthin;
11 = capsanthin; 12 = capsanthin 5,6-epoxide;
13 = violaxanthin; 14 = capsorubin; 15 = capsorubin isomer;
16 = neoxanthin; × = unknown.
Page 459
quantitation of color pigments in the fresh fruit of Capsicum annuum cv. and its commercial products.
The same normal-phase HPLC method was used for the determination of the carotenoid composition of
various cultivars [ 600]. The concentration and percentage of carotenoids found in various cultivars are
given in Table 3.83. The data indicate that both the pigment content and pigment composition of the
various cultivars show marked differences. It was proposed that these differences can be used for the
identification and selection of cultivars for different purposes.
Becuase of its simplicity and good separation power, RP-HPLC has found frequent application in the
analysis of paprika pigments. An isocratic elution mode was employed for the study of the stability of
the individual pigment fractions [ 601]. Pigments were extracted with acetone and separated on a C 18
column (250 × 4.6 mm I.D., particle size 10 µm) using acetonitrile:2-propanol:water (39:57:4 v/v/v)
eluent. The flow rate was 1 mL/min, and the detection wavelength was set to 438 nm. To assess the
stability of various fractions, they were treated with lipoxigenase before analysis. An aliquot of
pigments was saponified by refluxing it at 60°C with 5 mL of 20% KOH in
TABLE 3.82. Quantitative Distribution of Capsicum annuum cv. Belrubi Carotenoids

Determined by Normal-Phase HPLC.
Concentration
Carotenoid (g/g fresh weight × 10 -4 ) Proportion %
β–Carotene 2.75 6.72
Cryptocapsin 8.14 19.90
Cryptoflavin 1.09 2.66
β–Cryptoxanthin 0.79 1.93

Antheraxanthin 0.38 0.93
Capsolutein 2.13 5.21
Luteoxanthin 0.85 2.07
Zeaxanthin 1.25 3.06
Mutatoxanthin 1.64 4.01
Capsanthin 12.07 29.51
Capsanthin 5,6–epoxide 2.16 5.28
Violaxanthin 0.98 2.40
Capsorubin 1.79 4.38
Capsorubin isomer 1.38 3.37
Neoxanthin 1.74 4.25
Unidentified pigments (as β–carotene) 1.77 4.32

Total 40.91 100.00
Red pigments 25.54 62.43
Yellow pigments 15.37 37.57
Page 460
TABLE 3.83. Carotenoid Composition of Different Red Pepper Cultivars. *

Belrubi Negral AmlerB51
Carotenoid mg/g % mg/g % mg/g %

β-Carotene 1.22 ± 0.02 7.79 1.09 ± 0.02 7.37 1.13 ± 0.02 8.48
Cryptocapsin 2.79 ± 0.14 17.80 0.10 ± 0.02 0.68 1.67 ± 0.08 12.54
Cryptoflavin 0.48 ± 0.03 3.06 1.04 ± 0.03 7.03 — —
β-Cryptoxanthin 0.35 ± 0.03 2.23 0.42 ± 0.03 2.84 0.41 ± 0.04 3.10
Antheraxanthin 0.16 ± 0.02 1.02 0.24 ± 0.02 1.62 0.13 ± 0.03 0.98
Lutein — — 0.17 ± 0.03 1.15 0.13 ± 0.03 0.95
Capsolutein 0.96 ± 0.01 6.13 0.84 ± 0.02 5.68 0.84 ± 0.03 6.31
Luteoxanthin 0.38 ± 0.04 2.43 0.44 ± 0.03 2.57 0.14 ± 0.02 3.30
Zeaxanthin 0.60 ± 0.05 3.83 0.43 ± 0.05 2.91 0.58 ± 0.04 4.35
Mutatoxanthin 0.73 ± 0.03 4.66 0.67 ± 0.04 4.53 0.36 ± 0.03 2.70
Capsanthin 4.14 ± 0.20 26.42 5.68 ± 0.18 38.40 4.69 ± 0.16 35.21
Capsanthin
5,6-epoxide 0.74 ± 0.07 4.72 0.37 ± 0.04 2.50 0.08 ± 0.05 0.60
Violaxanthin 0.44 ± 0.03 2.81 0.58 ± 0.04 3.92 0.08 ± 0.05 0.60
Capsorubin 0.61 ± 0.06 3.89 0.74 ± 0.07 5.00 0.94 ± 0.04 7.06
Capsorubin isomer 0.47 ± 0.03 3.00 0.80 ± 0.05 5.41 0.68 ± 0.03 5.11
Neoxanthin 0.78 ± 0.02 4.98 0.64 ± 0.01 4.33 0.34 ± 0.02 2.48
Unidentified pigments 0.82 ± 0.27 5.23 0.60 ± 0.12 4.06 0.34 ± 0.11 2.52
NAN AmlerB51E81 Albar × M.CA
Carotenoid mg/g % mg/g % mg/g %

β-Carotene 1.05 ± 0.01 8.37 1.56 ± 0.01 12.72 0.35 ± 0.01 3.53
Cryptocapsin 2.00 ± 0.07 15.95 2.42 ± 0.07 19.74 0.15 ± 0.01 1.51
Cryptoflavin 0.41 ± 0.04 3.27 0.39 ± 0.02 3.18 0.08 ± 0.01 0.82
β-Cryptoxanthin 0.30 ± 0.04 2.30 0.51 ± 0.02 4.16 0.16 ± 0.02 0.84
Antheraxanthin 0.67 ± 0.03 5.34 0.06 ± 0.02 0.49 0.31 ± 0.02 3.13
Lutein 0.35 ± 0.04 2.79 0.09 ± 0.03 0.73 0.07 ± 0.01 0.70
Capsolutein 0.53 ± 0.03 4.23 0.08 ± 0.01 0.65 0.07 ± 0.01 0.70
Luteoxanthin 0.20 ± 0.03 1.12 0.20 ± 0.03 1.63 0.09 ± 0.01 0.92
Zeaxanthin 0.26 ± 0.03 2.07 0.53 ± 0.02 4.32 0.27 ± 0.02 2.73
Mutatoxanthin 0.62 ± 0.02 4.94 0.51 ± 0.02 4.16 0.24 ± 0.02 2.43
Capsanthin 2.92 ± 0.15 23.27 4.80 ± 0.20 39.15 5.36 ± 0.22 53.98
Capsanthin 5,6- 0.34 ± 0.04 2.71 0.10 ± 0.02 0.82 0.21 ± 0.03 2.11
epoxide
Violaxanthin 0.27 ± 0.05 2.15 0.06 ± 0.02 0.49 0.04 ± 0.01 0.41
Capsorubin 0.25 ± 0.03 1.99 0.10 ± 0.02 0.82 1.33 ± 0.04 13.39
Capsorubin isomer 0.19 ± 0.02 1.52 0.03 ± 0.01 0.24 0.69 ± 0.03 9.95
Neoxanthin 0.51 ± 0.02 4.07 0.52 ± 0.01 4.24 0.13 ± 0.01 1.32
Unidentified pigments 1.74 ± 0.31 13.91 0.30 ± 0.15 2.46 0.45 ± 0.14 4.53

Page 461
TABLE 3.83.
Americano
Carotenoid mg/g %
β-Carotene 0.43 ± 0.01 5.38
Cryptocapsin 0.05 ± 0.01 0.63

Cryptoflavin 0.34 ± 0.02 4.26
β-Cryptoxanthin 0.14 ± 0.02 1.75
Antheraxanthin 0.07 ± 0.01 0.88

Lutein 0.03 ± 0.02 0.88
Capsolutein 0.47 ± 0.03 5.88
Luteoxanthin 0.18 ± 0.02 2.24
Zeaxanthin 0.34 ± 0.03 4.26

Mutatoxanthin 0.27 ± 0.02 3.38
Capsanthin 3.82 ± 0.16 47.81
Capsanthin 5,6- 0.29 ± 0.03 3.63

epoxide
Violaxanthin 0.04 ± 0.01 0.50
Capsorubin 0.53 ± 0.03 6.63
Capsorubin
isomer 0.49 ± 0.03 6.13
Neoxanthin 0.25 ± 0.01 3.13
Unidentified pigments 0.25 ± 0.10 3.13

* Presented data (milligrams per gram of dry weight) are means of three analyses ± standard error.
methanol, 30 mL of methanol, and 0.1 g of ascorbic acid for 40 min. After saponification, the mixture
was extracted with 2 × 30 mL of petroleum ether, and the organic phase was dried with Na2 SO4. The
fatty acid methyl esters were separated by isothermal GC (10% polyethylene glycol succinate on Chrom
W-80). The injector, column, and detector temperatures were 230°, 180°C, and 240°C, respectively. It
was established that carotenoids form esters mainly with saturated C 12, C14, and C16 fatty acids, and
esterified capsanthin consisted mainly of unsaturated fatty acids (C 18:2 ). It was established that the esters
of capsorubin were more sensitive to enzymatic oxidation than the esters of capsanthin.
Carotenoids and carotenoid esters were separated and quantitated with a different RP-HPLC method
[602]. Samples were prepared by homogenizing 2-3 g of fruits with 50 mL of methanol and 1 g of
MgCO 3 . The mixture was filtered, and the solid phase was extracted with 100 mL of acetone:hexane
(80:20 v/v). The combined filtrates were mixed with the same volume of water, and the organic phase
was used for further analysis. RP-HPLC separation was performed on a C18 column (150 × 3.9 mm I.D.)
using a linear gradient of methanol and ethyl acetate. Canthaxanthin was the internal standard.
Page 462
The flow rate was 1.8 mL/min, and the detection wavelength was set to 475 nm. The fatty acid
composition of the fractions was determined by packed column GC. A typical chromatogram is shown
in Figure 3.82. The RP -HPLC method separated the color pigments well, according to the character of
the pigment and the length of the fatty acid chain. The concentration and the retention time relative to
the internal standard of the pigment fractions are presented in Table 3.84. The RSD of the RP-HPLC
method was 5.1% and 2.7% for
Figure 3.82.
A typical HPLC chromatogram of red bell peppers. Peaks:
1, 4-10, 20-22, 24, 26 = unidentified; 2 = capsorubin; 3 = capsanthin;
11 = β-apo 8 ′-carotenal; 12 = capsorubin monoester;
13-15 = capsanthin monoester; 16 = capsanthin c 14:0 ;
17 = capsorubin monoester; 18 = capsanthin monoester;
19 = capsorubin monoester; 23 = β-carotene; 25, 27 = capsorubin
diester; 28 = capsanthin diester; 29 = capsorubin diester;
30 = capsanthin C 12:0 &C14:0 ; 31 = capsorubin diester;
32 = capsanthin C 14:0&14:0; 33 = capsorubin diester;
34 = capsanthin C 14:0 &C16:0 ; 35 = lutein diester;
36 = capsanthin diester; 37, 38 = lutein diester.
Page 463
TABLE 3.84. Carotenoid Composition of Red Bell Peppers. Retention Times are Given
Relative to Internal Standard (RRT).
Peak No. RRT Carotenoid Conc. (µg/g)
1 0.21 Unidentified —
2 0.29 Capsorubin 0.15 *
3 0.33 Capsanthin 1.96
4 0.37 Unidentified 2.55
9 0.80 Unidentified 3.73 **
11 1.00 β-apo 8'-carotenal IS
12 1.10 Capsorubin monoester —
13 1.17 Capsanthin monoester 6.63 ***
14 1.23 Capsanthin monoester 16.97
16 1.45 Capsanthin C 14:0 29.25
17 1.56 Capsorubin monoester 9.64
19 1.77 Capsorubin monoester 10.44
22 1.94 Unidentified 4.88 ****
23 2.00 β-Carotene 29.78
25 2.23 Capsorubin diester 6.81
28 2.46 Capsanthin diester 12.66
30 2.63 28.46
Capsanthin C 12:0 &C14:0
32 2.80 Capsanthin C 14:0&14:0 25.38
34 2.95 Capsanthin C 14:0 &C16:0 8.02
35 3.05 Lutein diester 2.20
36 3.10 Capsanthin diester 1.33

Total 280.47
* Includes peak 1.
** Includes peaks 5 to 8.
*** Includes peak 12.
**** Includes peak 21.

Page 464
β-carotene and total carotenoids, respectively. The majority of pigments were capsanthin esters
followed by capsorubins and β-carotene. It was stated that the method allows the rapid and direct
quantitative analysis of red bell pepper carotenoids and carotenoid esters.
The optimal conditions for the RP-HPLC separation of carotenoids have been extensively studied. It
was established that acetonitrile and tetrahydrofuran exert a considerable impact on the separation
efficiency [ 603], and both the characteristics of column [ 604] and the temperature of separation
influence the retention behavior of carotenoids [605]. The effect of column temperature has been studied
in more detail [ 606]. Carotenoids were extracted from a food mix containing sweet corn, tomato, and
carrot by a tetrahydrofuran-methanol mixture. Separations were performed on a 250 × 4.6 mm I.D.
column guarded with a 100 × 4.6. mm I.D. precolumn. The mobile phase consisted of methanol:
tetrahydrofuran (95:5 v/v). Fractions were detected at 450 nm. The effect of temperature on the
retention behavior of carotenoids is demonstrated on the chromatograms in Figure 3.83. The data prove
that the column temperature exerts a marked influence on the retention time and separation of
carotenoids. Column temperatures between 20°C and 22.5°C were proposed for optimal resolution.
Diode array detection, combined with RP -HPLC, has also been employed for the assessment of the
effect of the various processing steps on the stability of color pigments in paprika powders [ 607].
Separation of pigments was performed on a C18 column (250 × 4 mm I.D., particle size 5 µm). The flow
rate was 1 mL/min, and the detection wavelength range was between 340 nm and 560 nm. Eluents A
and B were bidistilled water and acetonitrile:methanol (2:8 v/v), respectively. The gradient program
began with eluent A:eluent B (5:95 v/v), 5 min hold, to 97% of eluent B in two min, final hold 53 min.
The RP-HPLC method separated more than 40 pigment fractions. The RSDs of the quantitation and
extraction were 3.55% and 5.19%, respectively. It was established that the inhomogeneity of the raw
material was very high (27.11%); therefore, the impact of the various technological steps on the
stability of the individual pigments cannot be determined beyond this limit. The data indicated that the
technological steps do not exert a marked influence on the stability of the pigments.
The separation capacity of adsorption and RP-TLC and HPLC was also compared [608]. Precoated
silica plates and various mixtures of n-hexane, acetone, tetrahydrofuran, and bidistilled water were used
as eluents for adsorption TLC. Precoated silica plates impregnated by overnight predevelopment in n-
hexane:paraffon oil (95:5 v/v) were used as RP -TLC supports. Bidistilled water, acetone, and
tetrahydrofuran were used in various concentrations as eluents. The use of uncommon
tetrahydrofuran:water mixtures was motivated by the finding that a strongly retained pigment fraction
remained on the start in each TLC and RP-TLC system, which can be removed only with
tetrahydrofuran:
Page 465
Figure 3.83.
The effect of three column temperatures on the separation of five
carotenoids plus echinenone (internal standard) in an unsaponified
''food mix" containing sweet corn, tomato, and carrot: 1 = lutein;
2 = zeaxanthin; 4 = echinenone; 5 = lycopene;
6 = α-carotene; 7 = β-carotene.
Page 466
water (9:1 v/v) eluent. Taking into consideration the results of TLC and RP-TLC as pilot methods, the
following HPLC systems were employed for the separation of pigments, including the strongly retained
fraction. Adsorption HPLC was performed on a silica column (250 × 4 mm I.D., particle size 5 µm) or
C18 column (250 × 4 mm. I.D., particle size 5 µm). The solvents were (A) n-hexane:carbon tetrachloride
(99:1 v/v); (B) acetone; and (C) tetrahydrofuran:water (98:2 v/v). The gradient was 100% A for 7 min;
to 50% of B in 50 min; 100% C in 3 min and final hold 15 min. RP-HPLC was performed on a C18
column (250 × 4 mm I.D., particle size 5 µm) or C18 column (250 × 4 mm. I.D., particle size 5 µm). The
components of the eluent were (A) bidistilled water, (B) acetone, and (C) tetrahydrofuran. The gradient
steps were: step 1, 15% of A and 85% of B for 10 min; 5% of A and 95% of B in 3 min; 10 min hold;
2% of A and 98% of C in 5 min; final hold 30 min. In both instances the flow rate was 1 mL/min, and
the detection wavelength range was between 340 nm and 460 nm. Color pigments were well separated
on both silica and octadecylsilica supports, as demonstrated in Figures 3.84 and 3.85.
Figure 3.84.
Separation of pigments of Capsicum annuum on a silica column.
Detection wavelength 440 nm, flow rate 1 mL/min. Gradient: A = n-hexane:carbon
tetrachloride (99:1 v/v); B = acetone and C = tetrahydrofurane/water
(98:2 v/v). Step 1: A during 7 min; Step 2: 50% of A and 50% of B in
50 min; Step 3: 100% C in 3 min; Step 4: 100% of C during 15 min.
Page 467
The last peak on both chromatograms contained the strongly retained pigment fraction. The fact that
this fraction eluted very slowly in adsorption and RP systems was tentatively explained by the
supposition that the molecular mass of this pigment fraction is relatively high, and it contains both
hydrophobic and hydrophilic substructures on the surface of the molecule facilitating its binding to
polar and apolar surfaces of the supports.
Figure 3.85.
Separation of pigments of Capsicum annuum on an octadecylsilica column.
Detection wavelength 440 nm, flow rate 1 mL/min. Gradient: A = bidistilled
water; B = acetone and C = tetrahydrofuran. Step 1: 15% of A and 85% of
B during 10 min; Step 2: 5% of A and 95% of B in 3 min; Step 3: 5% of
A and 95% of B during 10; Step 4: 2% of A and 98% of C in 5 min; Step
5: 2% of A and 98% of C during 30 min. Capxanthin = peak 6;
Zeaxanthin = peak 10; β-carotene peak 21.
Page 468
Because of their different nutritional values, many efforts were devoted to the separation of the
geometric isomers of carotene [ 609,610]. Adsorption [611] and RP-HPLC [ 612] were successfully
employed for these types of separations. A calcium hydroxide stationary phase has also been applied for
the separation of carotene isomers [ 613]. Carotene isomers were extracted from carrot baby food and
fresh tomatoes and then separated on an activated alumina column deactivated with 3% of water.
Phytofluene and phytoene were elueted firstly with n-hexane; α- and β-carotene were elueted with n-
hexane:acetone (99:1 v/v), and the other carotenes were removed from the column with n-
hexane:acetone (97:3 v/v). N-hexane or the mixtures of
Figure 3.86.
HPLC separation of a phytofluene mixture obtained
from fresh tomato using 100% hexane following
exposure to sunlight. Tentative identification of peaks:
(1) and (2) unidentified cis-, (3) 15-cis-, (4) and (5)
unidentified cis-, and (6) all-trans-phytofluene.
the publisher and the corresponding author,
Page 469
n-hexane and acetone, p-methylanisole, or benzyl ether were used as isocratic eluents. Carotene isomers
were detected with a diode array detector at 325, 400, 440, and 455 nm. The six geometric isomers of
phytofluene were well separated using n-hexane eluent (Figure 3.86). The method was suitable for the
detection of the effect of sunlight on the isomer composition, as demonstrated in Figure 3.87. The
method was proposed for the separation of acyclic and cyclic geometric isomers of carotenes containing
5, 7, or 11 aliphatic double bonds.
Not only HPLC, but also supercritical fluid chromatography, was applied for the separation of the
geometric isomers of α- and β-carotenes [ 614,615 ].
Figure 3.87.
(A) HPLC separation of a carotene mixture obtained from processed carrot baby
food using 1.4% benzyl ether in hexane; (B) HPLC separation of the same
mixture exposed to iodine and sunlight using the same chromatographic
conditions. Tentative identification of peaks: (1)-(3) unidentified cis-,
(4) 15-cis-, (5) an unidentified cis-, and (6) all-trans-carotene.
Page 470
3.3—
Separation of Food Additives
A wide variety of organics and inorganics has been used as food additives for different purposes such as
the enhancement of flavor and the improvement of physicochemical parameters and products. Becuase
of the highly different chemical and physical characteristics of food additives, many chromatographic
techniques were employed for their separation and quantitative determination.
3.3.1—
Gas-Liquid Chromatography
It was established many years ago that potassium bromate improves the baking parameters of flour by
oxidizing gluten. Dough treated with potassium bromate better retains carbon dioxide, resulting in a
taller loaf. A sensitive GC -MS method was developed for the determination of the quantity of bromate
in bread [ 616].
The scheme of the sample preparation procedure is shown in Figure 3.88. Separation of the α-
bromomethyl-benzenemethanol derivative was performed on a DB-1 (methyl silicone) capillary column
(30 m × 0.25 mm I.D., film thickness 0.26 µm). Helium was used as the carrier gas. The initial oven
temperature was 40°C for 2 min, increased to 230 min at 10°C/min, and final hold 10 min. The
relationship between detector response and bromic acid ion was linear between 0.03 µg/g and 0.50
µg/g, the coefficient of correlation being 0.998. The recovery values varied between 91.3% and 98.1%,
with a CV of 6.5-11.1%. The detection limit was 0.03 µg/g. The residues of bromate in bread are listed
in Table 3.85. The data indicate that residual bromate can be detected in bread with higher initial
bromate concentrations. It was further established that the simultaneous addition of ascorbic acid
significantly decreased the amount of residual bromate (Table 3.86). Because of the good repeatability
and sensitivity, the method was proposed for the detection of small quantities of bromate in bread.
The performance of GC and HPLC-inductive-coupled plasma-mass spectrometry (ICP -MS) for the
determination of potassium bromate in bread has also been compared [ 617]. Samples for GC -ECD
analysis were prepared by extracting potassium bromate of crumbed breads with water, adsorbed on
DEAE Sephadex A-25, and cleaned with subsequent washing with methanol ethanol, 5% v/v aqueous
ethanoic acid and water. Bromate was eluted with 30% aqueous potassium chloride solution. The eluate
was reacted with styrene monomer and used for GC-ECD. Samples for HPLC-ICP-MS analysis were
prepared similarly, and the aqueous extract of bread was further cleaned on 3 mL of SAX Bond Eluts
coupled in series, conditioned with 3.0 mL of methanol, 5.0 mL of water, 5.0 mL of 5 M ammonium
acetate, and 5.0 mL of water. An aliquot of aqueous extract (5.0) was applied to the Eluts and washed
Page 471
Figure 3.88.
Analytical procedure for bromate in bread.
Taylor & Francis and the corresponding author.)
with 5.0 mL of water and 2.5 mL of 0.2 M ammonium acetate. Bromate was eluted with 2.5 mL of 0.5
M ammonium acetate. Separation was performed on a Dionex IonPac CS5 column with a CG5 guard
column. The mobile phase was 0.5 M ammonium acetate. Typical GC and HPLC chromatograms are
shown in Figure 3.89. The peak of bromate is well separated in both
Page 472
TABLE 3.85. Residues of Bromate in Bread (sponge and dough method).
Added Bromate * Residual Bromate in the Bread **

(µg/g on flour weight) (µg/g)
0 ND
10 (13.0)*** ND
30 (38.9)*** ND
40 (51.8)*** 0.03 ± 0.01
50 (64.8)*** 0.09 ± 0.02
60 (77.7)**** 0.33 ± 0.06
* Indicate the amounts as HBrO3.

**n = 5.
*** Indicate the amounts of KBrO3 in parentheses.
chromatographic systems without any interferences from the accompanying matrices. This finding
indicates that both methods can be used for the determination of bromate residues in bread. The
quantitative results are given in Table 3.87. The quantitative results using either GC or HPLC are not
significantly different, indicating that both methods are suitable for the determination of bromates in
bread samples.
Capillary GC has also been used for the quantitative determination of propionic acid and propionates in
bread and bread products [ 618]. Extraction was carried out by homogenizing 5 ± 0.01 g of crumbed
sample with 50 mL of ethyl acetate, 1 mL of orthophosphoric acid, and 10 g of anhydrous sodium
sulfate. The mixture was filtered, and the residue was extracted again with 50 mL of ethyl acetate and
filtered. The combined extracts were used for GC analysis. Separation was performed on an FFAP
fused-silica capillary column (15 m ×
TABLE 3.86. Residues of Bromate in Bread (short process method).

Residual Bromate in the Bread ** (µg/g)
Added Bromate * Combination with Ascorbic

(µg/g on flour weight) Bromate Alone Acid (50 µg/g)
0 ND ND
50 ND ND
75 ND ND
100 0.13 ± 0.06 ND
125 1.51 ± 0.35 0.44 ± 0.35
150 6.17 ± 1.17 2.60 ± 0.23

* Potassium bromate was added to flour as KBrO3.
** n = S.
Page 473
Figure 3.89.
(I) GC-ECD chromatogram of bread baked with flour fortified to contain 50 mg
potassium bromate/kg flour. Retention time of the chloro -bromo-styrene derivative
of bromate is marked A. This corresponds to 398 µg potassium bromate/kg bread.
(II) HPLC-inductively coupled plasma (ICP)-MS chromatogram of bread baked
with flour fortified to contain 50 mg potassium bromate/kg flour. This
corresponds to 344 µg/kg bread. (A) Elution of bromate and (B) elution of bromide.
(Reprinted from Reference [ 617] with permission from Taylor
& Francis and the corresponding author.)
0.32 mm I.D., film thickness 0.25 µm). Hydrogen was the carrier gas, and 3-methylbutanoic acid was
used as internal standard. Injector and FID temperatures were 220°C and 250°C, respectively. The
column temperature program was: 50 to 115°C at 8°C/min, to 230°C at 39.9°C/min, and final hold 5
min. Some characteristic chromatograms are shown in Figure 3.90. As can be seen on the
chromatograms, the acids are well separated from each other and from the coextracted impurities. The
recovery values varied between 106% and 96%, the CV being 0.7-1.1. Because of the high sensitivity,
precisness, and separation capacity, the method was proposed for the determination of propionic acid
and propionates in bread, rolls, pita breads, and hamburger buns.
To modify the nutrition value of triacylglycerols, a low-calorie triacylglycerol product called Salatrim
was developed. Salatrim is an interesterification product of triacetin, tripropionin, tributirin, and
hydrogenated vegetable oils. HPLC with an evaporative light-scattering detector and GC were equally
successfully used for the determination of the composition of Salatrim [ 619,620,621]. High-temperature
capillary GC (HTCGC) has also been employed for the analysis of Salatirm [ 622]. Fat was extracted
from the ground samples by SFE: a 0.5-g sample was extracted with CO 2 (density 0.8 g/mL, pressure
365 bar,
Page 474
TABLE 3.87. Comparison of Results for IHR Breads by GC-ECD and HPLC -ICP-MS Methods.
Number of Mean Standard Deviaton
Method Sample Analyses (µg/kg) (µg/kg) CV (%)
GC -ECD IHR 0* 1 <12 0 0
IHR 50 ** 6 426 75 18
HPLC IHR 0* 5 <12 0 0
ICP-MS IHR 50 ** 5 396 48 12
* Bread baked with wholemeal flour (which does not contain added bromate).
** Bread baked with the same flour fortified to contain 50 mg potassium bromate/kilogram flour.
Data corrected for blank contribution and recovery.
flow rate 4 mL/min) at 80°C. GC-FID separation was carried out on a Chrompack SIM-DIST CB
fused-silica capillary column (5.0 m × 0.32 mm I.D., film thickness 0.1 µm) connected with a
deactivated fused-silica precolumn (0.5 m × 0.53 mm I.D.). The column temperature varied from 100°C
to 150°C at 15°C/min, then to 350°C at 10°C/min, and final hold 2 min. Hydrogen was used as the
carrier gas, and the detector temperature was 375°C. Slightly different conditions were used for
HTCGC, coupled with positive chemical ionization mass spectrometry: the same type of columns were
used, but the length of the analytical and precolumn were 10 m and 0.8 m, respectively. The
temperature program was: from 50 to 150°C at 18°C/min, then to 350°C at 12°C/min, and final hold 10
min. It was found that the recovery (93-106%) and the CV (1-3.2%) of the SFE method is similar to
those of AOAC standard methods, indicating that SFE can be used as a valuable alternative for the
extraction of fat from various matrices. HTCGC separated the triacylglycerols of Salatrim and other fats
well, as demonstrated in Figure 3.91. The quantitative results and the statistical parameters of the
analyses are compiled in Table 3.88. The data prove that the method is suitable for the accurate and
precise determination of the generic Salatrim content in food products.
The composition of meat-like flavorings was studied with gas chromatography combined with
olfactometry (GCO) [ 623]. The retention index determined on two different GC columns, the odor
quality, and the flavor dilution (FD) factor of the components of savory and grilled flavorings are given
in Table 3.89. The data clearly show that more than one volatile compound contributes to the aroma
composition of both savory and grilled flavorings. The retention order of volatiles was different on OV-
1701 and FFAP columns, proving the different retention characteristics of the capillary columns. The
comparative data of both flavorings are presented in Table 3.90. The data prove again the complex
aroma composition of the flavorings, and they further indi-
Page 475
Figure 3.90.
Chromatograms of hamburger bun extracts: (a) containing < 10 mg/kg, (b) containing
375 mg/kg, and (c) containing 1480 mg/kg propionic acid. Peak 1 = acetic acid;
peak 2 = propionic acid; peak 3 = 3-methylbutanoic acid internal standard.
(Reprinted from Reference [ 618] with permission from Taylor
& Francis and the corresponding author.)
cate that the aroma composition considerably depends on the dry or dissolved state of the flavorings.
The artificial sweetener aspartame was determined in tabletop sweeteners and soft drinks by using
pyrolysis-GC-MS (Py-GC-MS) [ 624]. Tabletop sweeteners were powdered, and softdrinks were
evaporated in vacuum before analysis. Approximately 0.2-1.5 mg of dry residue was used for Py-GC-
MS. Pyrolysis was generally performed at 600°C for 5 s. Separation was carried out on a Supelco SPB-
5 capillary column (30 m × 0.32 mm I.D., film thickness 0.25 µm). Oven temperature was 50°C for 10
min and then increased to 300°C at 5°C/min. The Py-GC-MS profile of some samples are shown in
Figure 3.92. The peaks of the pyrolysis products are well separated from each other and for the peaks
originated from the other components present in tabletop sweeteners and softdrinks. It was proposed
that the method can be used for the rapid determination of aspartame in various food products after
improving quantitation parameters.
Page 476
Figure 3.91.
Comparative HTCGC profiles of the crude fat extracts obtained from
(A) chocolate-coated butter cookie made with Salatrim 24/4SO
(18.56%), butter fat (8.88%), and cocoa butter (0.74-0.89);
(B) chocolate-coated butter cookie made with Salatrim 23SO (19.17%),
butter fat (8.41%), and cocoa butter (0.76-0.92), and (C) reference butter fat.
Peak at RT = 13.1 min is internal standard triundecanoin. Triacylglycerols
with acyl carbon number less than 42 (components with retention time less
than 17.00 min) are triacylglycerols originating from Slatrim 24/4SO,
Salatrim 23SO, and butter fat in chromatograms A and B. Triacylglycerols
at an acyl carbon number of 46 (retention time 18.2 min) originating from
butter fat were used as reference peak to calculate the ratio (R b46 ) of
triacylglycerols 30 -42 and that of an acyl carbon number of 46.
Numbers indicated in the chromatograms are acyl carbon numbers.
Page 477
TABLE 3.88. Salatrim Contents in Foods by the Alternative Fat Extraction—HTCGC Analysis (four
parallel determinations).
Theoretical* Accuracy ** Total
Baked Products Average SD CV (%) (%) (%) Error ***
1 cookie 3.93 0.11 0.11 4.00 –0.1 0.36
2 cookie 9.64 0.10 1.05 10.00 –0.4 0.62
3 cookie 15.22 0.53 3.50 16.00 –0.8 2.15
4 cookie 4.53 0.16 3.44 4.00 0.5 0.94
5 cookie 9.42 0.05 0.57 10.00 –0.6 0.72
6 cookie 16.48 0.17 1.00 16.00 0.5 0.91
7 cookie **** NA NA NA 0 NA NA
Confectionary products, nondairy type

8 chocolate-coated cookie 13.55 0.10 0.74 14.36 –0.8 1.07
9 chocolate-coated cookie 17.27 1.13 6.53 17.24 0.0 2.94
Confectionary products, dairy type

10 chocolate -coated
butter cookie 17.12 0.32 1.89 18.56 –1.4 2.28
11 chocolate -coated
Butter cookie
frozen dessert 18.60 0.79 4.23 19.17 –0.6 2.60
12 chocolate ice cream 10.24 0.28 2.70 10.00 –e0.2 0.95
* Theoretical value (or true value); % Slatrin by calculation from formulation.

**Accuracy expressed as the difference between the calculated average and the theoretical value divided by
theoretical value.
*** Total error (E), E =/ x-u/ + 2.58 SD.
**** Not for Salatrim content analysis.
NA = not available.
3.3.2—
Thin-Layer Chromatography
Because of their simplicity, various TLC methods have also found application in the separation of food
additives. Thus, sorbic, benzoic, and dehydroacetic acid was determined in fruit juice, iced tea, and soda
by direct spotting of samples onto the TLC plates [ 625]. TLC was combined with SPE for the
determination of the mixture of sorbic and benzoic acids in low concentrations [ 626]. Octadecylsilica
SPE columns were conditioned with one column volume of methanol followed by one column volume
of 0.1 M HCl. Samples were acidified to pH = 2.0 with concentrated HCl, and passed through the SPE
column at ca. 3 mL/min. The column was washed with 2 × 1 mL of 0.1 M HCl and dried in vacuum.
Acids were removed by 2.0 mL of dichloromethane. The organic phase was concentrated and spotted
onto precoated C18 layers. Eluent was methanol:0.5 M NaCl (1:1).
TABLE 3.89. Odorants of the Savory and Grilled Flavoring Identified by Aroma Extract Dilution Analysis (FD 5).
Retention Index
No. Compound OV-1701 FFAP Odor Quality (GCO) Savory
1 1-Hexen-3-one ** 860 1120 Musty, metallic, fatty —
2 Hexanal*** 884 1110 Green, malty
3 2-Methyl-3-furanthiol *** 931 1325 Meaty, roasty, sweet
4 Trans-2-Methyl-3-tetrahydrofuranthiol ***, **** 992 1315 Savory, onion-like
5 2-Furfurylthiol*** 1000 1450 Roasty, sulphury 20480
6 3-(Methylthio)-Propanal (methional)*** 1044 1465 Cooked potato, sweet
7 (E)-2-Heptenal *** 1065 1335 Fatty, musty —
8 1-Octen-3-one ** 1068 1315 Mushroom-like
9 Trimethylpyrazine*** 1078 1415 Roasty, earthy —
10 Unknown 1080 1310 Catty, sulphury
11 2-Acethylthiazole*** 1137 1665 Roasty
12 Unknown (M = 134, C6H 14SO) 1156 1715 Savory, onion-like
13 (E)-2-Octenal *** 1170 1440 Fatty, musty —
14 2-Methyl-2-(methyldithio)-propanal **** 1192 1570 Fatty, smoky —
15 S-(2-methyl-3-furyl)-ethanethioate***,**** 1238 1700 Meaty, roasty
16 (Z)-2-nonenal ** 1258 1515 Fatty, cucumber-like

TABLE 3.89.
Retention Index
No. Compound OV-1701 FFAP Odor Quality (GCO) Savory
17 S-(2-furfuryl)-ethanethioate *** 1271 1785 Roasty, sulphury
18 (E)-2-nonenal*** 1274 1545 Fatty, leather-like
19 2-Furfurylmethyl-disulphide*** 1320 1820 Smoky, sulphury
20 (E,E)-2,4-nonadienal*** 1350 1715 Fatty
21 (E)-2-decenal*** 1380 1655 Fatty, soapy
22 4-Acetyloxy-2,5-dimethyl-3(2H)- 1430 2005 Caramel-like, savory

furanone***,****
23 (E, E)-2,4-decadienal*** 1453 1820 Fatty, fried potato
24 (E)-2-undecenal*** 1483 1760 Soapy, fatty
25 Bis-(2-Methyl -3-furyl)-disulphide *** 1635 2170 Meaty, roasty, sweet

* Simultaneous distillation-extraction under static vacuum. FD factors were determined on the OV-1701 capillary.
*** Identification by comparing with the reference substance on the basis of: RI on two capillaries, MS (EI, CI) and odor quality.
** Identification by comparing with the reference substance on the basis of: RI on two capillaries and odor quality.
**** Identification on the basis of: MS (EI, CI, HR) and NMR ( 1H, 13C, DQF-COSY, NOESY).
Amsterdam, The Netherlands.
Page 480
TABLE 3.90. Comparison of Savory and Grilled Food Flavorings by Static Headspace GCO.
Savory Grilled
Flavoring* Flavoring**
Compound (as eluted

on the FFAP capillary) Solid Aqueous Solid Aqueous
2,3-Butanedione (buttery) ** 10 1 20 5
Hexanal** 20 1 10 0.2
1-Hexen-3-one ** — — 1 2
Unknown (RI = 1210, metallic,
vegetable-like) — — 2 5
1-Octen-3-one** 2 0.2 0.5 0.2
Trans-3-Methyl-3-tetrahyrdofuranthiol ** — <0.4 *** — 1

Unknown (RI = 1385, metallic,
vegetable-like) 20 5 5 1
1-Nonen-3-one (mushroom-like) **** — — 2 2
Trimethylpyrazine** — — 10 —
2-Furfurylthiol** 1 <0.2 *** <0.5 *** <0.2 ***
Methional** 1 <0.2 *** 1 <0.2 ***
Acetic acid (acidic) ** 5 — 10 —
(Z)-2-Nonenal** 20 — 5 5
(E)-2-Nonenal** 5 0.2 1 0.2
2-Methyl -2-(methyldithio)-propanal ** — — 2 1
Butaboic acid (rancid) ** 20 — 20 —
(E)-2-Decenal ** — — 10 —
2-Acetylthiazole ** 20 5 10 5
S-(2-Methyl -3-furyl) -ethanethioate** 5 5 — —
(E, E)-2,4-Decadienal** — — 5 —
4-Acetyloxy -2,5-dimethyl -3(2H)-furanone ** 0.5 5 10 —
4-Hyroxy-2,5-dimethyl -3(2H)-furanone** 5 — 10 —
(caramel -like, sweet)

*The lowest headspace volume (mL) required to detect the odorant by GCO is given for the powder
and its aqueous solution.
**Identification based on the data reported in Table 3.89.
*** The compound was still odor-active in this sample.
**** The compound was tentatively identified.
NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.
After development, the plates were dried and evaluated by a TLC densitometer (225 nm for benzoic and
267 nm for sorbic acid). The RF values of benzoic and sorbic acids were 0.67 and 0.57, respectively.
The coefficient of correlation between the peak area and the quantity of organic acids was 0.992, and
the reproducibility varied between 0.50%-5.0%. Recovery was 95.0 -109%. It was stated that the good
validation parameters of the method make
Page 481
Figure 3.92.
Py-GC-MS profile (total ion current) of tabletop
sweetening tablets, tabletop sweetening powder
and softdrink. Peak: 1 = toluene; 2 = ethylbenzene;
3 = ethenylbenzene; 4 = benzeneacetonitrile;
5 = benzenepropanenitrile; 6 = phenylalanine ethylester;
7 = diphenylethane;
10 = 2-(2-aminopropionylamino)-3-phenylpropionic
acid methyl ester; 11 = unknown;
12 = 2-(2,5-dioxopyrrolidin-1-yl)-4-phenylbutiric
acid methyl ester;
13 = 2-(3-amino-2,5-dioxopyrrolidin-1-yl)-4-phenylbutiric
acid methyl ester; 14 = 2-(5-benzyl -3,6-dioxopiperazin-2-yl)-acetic acid.
Page 482
it suitable for the quantitative determination of benzoic and sorbic acids in beverages, and the method
can be a valuable substitute of the HPLC methods used for this purpose.
The content of the flavor and taste enhancer MSG of some food products was also determined by TLC,
and the results were compared with those of enzymic, HPLC, and CE methods [ 627]. TLC was
performed on silica layers using a threefold ascending development. The spots were detected with a
ninhydrin reagent and evaluated by a densitometer at 520 nm. Glutamate was derivatized with 9-FMOC
before HPLC analysis, and the derivative was separated on a C18 column (250 × 4.6 mm I.D.) using
gradient elution. Solvents A and B were acetate buffer:methanol:tetrahydrofuran (80:10:10) and acetate
buffer:methanol:tetrahydrofuran (40:59:0.5), respectively. The gradient was 0 min, 34% B; 9 min, 36%
B; 20 min, 45% B; 30 min, 60% B; 41 min, 99.9% B. The flow rate was 1.5 mL/min, and the excitation
and emission wavelengths of the fluorescence detector were 265 nm and 315 nm, respectively. The
dansyl derivative of glutamate was prepared for CE analysis. The capillary was 50 m × 50 µm. Running
buffer was 20 mM borax with 102.5 mM SDS. Separation was carried out at 25 kV. Dansylated
glutamate was detected at 214 nm. Typical chromatograms of a meat sample are shown in Figure 3.93.
The chromatograms indicate that the interference from other sample constituents is the highest in TLC
and negligible in both HPLC and TLC. The MSG content of various samples determined by the
enzymatic and chromatographic methods is given in Table 3.91. The TLC method was proposed for the
industrial quality control laboratories as a rapid, inexpensive, and sufficiently accurate analytical
procedure.
Polyphosphates in seafoods have also been detected by TLC [ 628]. Cod (Gadus morhua) and sea scallop
(Placopecten magellanicus) were used without pretreatment and with dipping in 10% sodium
tripolyphosphate (STP) solution for 1 min and 10 min. The drip loss of the samples and the
homogenizate of cod with water (1:2) were used for the experiments. Phosphates were separated on
cellulose layers using a mixture of 200 mL of 2-propanol, 175 mL of 1-propanol, 25 g of trichloroacetic
acid, 1 mL of 88% ammonia, and 125 mL water as eluent. After development, the plates were dried at
room temperature, and the spots were detected with molybdenum blue reagent (light blue spots on dark
blue background). Plates were dried again for 2 h in the dark and sprayed with 288 g of sodium
pyrosulfite, 10.5 g sodium sulfite, and 1 g of methylaminophenol dissolved in 1 L of water. Plates were
dried again in the dark for 1 h (blue spots on white background). The mean Rf values of phosphates and
the standard deviation are presented in Table 3.92.
Tripolyphosphate and pyrophosphate were well separated from each other and from the spots of
monobasic and dibasic phosphates that eluted together. This rapid and inexpensive method was
proposed for the detection and mislabeling of fresh seafood in both the seafood industry and regulatory
agencies.
Page 483
Figure 3.93.
Chromatographic determination of MSG in a meat sample with (A) TLC, (B) HPLC, and (C) CE.
Page 484
TABLE 3.91. Comparison of the MSG Content (g/100 g) of Some Food Products,
Determined Using the Enzymic Method (EM), Quantitative TLC, HPLC, and CE.
Product EM TLC HPLC CE
Meat ragout 1 0.531 0.651 0.579 0.499
Meat ragout 2 0.062 0.092 0.085 0.089
Frankfurter 1 0.225 0.232 0.218 0.245
Frankfurter 2 0.009 0.003 0.005 0.003
Mortadella 1 0.299 0.333 0.378 0.312
Mortadella 2 0.321 0.345 0.312 0.299
Mortadella 3 0.289 0.305 0.298 0.300
Salami 1 0.411 0.455 0.411 0.423
Spice 1 0.543 0.567 0.523 0.545
Spice 2 0.256 0.295 0.266 0.257
Tortellini 1 0.200 0.250 0.233 0.254
Tortellini 2 0.621 0.671 0.625 0.645
Instant soup 1 4.021 4.036 4.569 4.499
Instant soup 2 14.980 15.000 14.654 3.989
Tomato paste 1 0.367 0.381 0.321 0.299
Tomato paste 2 0.896 0.920 0.934 0.912
Meat paste 1 0.167 0.195 0.189 0.178
Meat paste 2 0.040 0.080 0.067 0.054
Instant potatoes 0.210 0.290 0.245 0.267
3.3.3—
As food colorants show negligible volatility, their separation and quantitative determination can be
carried out only with liquid chromatographic methods. An interesting application of LC is the
separation of sugar colorants on a sucrose column [ 629]. Sugar colorants and synthetic colorants have
also been separated on sugar crystals [ 630]. Samples were prepared by dissolving 5 g of sugar in 5 mL
of distilled water, then 1 g of fructose and 10 mL of ethanol:water:HCl (50:49:1) and 30 g/L of NaCl
were added. A column of 370 × 15 mm I.D. was filled with 22 g of sugar crystals of 0.15-0.30 mm. The
col-
TABLE 3.92. R f, Values for Sodium Salts of Various Phosphate Solutions (n = 10).
Tripolyphoshate Pyrophosphate Monobasic Dibasic

Mean 0.12 0.29 0.48 0.48

International.
Page 485
TABLE 3.93. Gradient Elution Timetable for the Separation of Sugar Colorants on
Sucrose Crystals.
Time (min) Eluent A (%) Eluent B (%) Eluent C (%)
0–30 100
30 –40 100–0 0–100

40 –90 100
90–100 100–75 0–25
100–230 75–0 25–100
230–250 100
Eluent A: 1-propanol:ethanol (70:30 v/v) containing 0.3% of 2 M NH 3; eluent B:

methanol containing 0.3% of 2 M NH 3; eluent C: methanol: acetonitrile (70:30 v/v)
containing 0.5% of 1 M sulphuric acid.
Reprinted from Reference [ 630] with permission from Verlag Dr. ALbert Bartens.
umn was washed with methanol:acetonitrile (70:30 v/v) containing 0.1% of 1 M sulphuric acid (1
mL/min for 10 min) and then with 1-propanol:ethanol (70:30 v/v) containing 0.3% of 2 M NH3 (2
mL/min for 50 min). A new column was prepared for each run. The gradient elution is shown in Table
3.93. Peaks were detected by a diode array detector in the wavelength range of 220-500 nm. The
method separated five different colorants (g1-g5) from various sugar products, as demonstrated in
Figure 3.94. The chromatograms indicate that the content of various colorants and their ratio depend on
the type and character of the product under investigation. The peak areas of various syrups, molasses,
raw sugars, and liquors are given in Table 3.94. It was assumed that this technique allows not only the
separation of sugar colorants in different groups, but also the determination of their affinity to sugar
crystals. Moreover, the method can be successfully used for the evaluation of the efficacy of various
decolorization systems used in refining processes.
The food colorant annatto has also been determined in high-fat dairy products, margarine, and hard
candy by HPLC [631]. Annatto was extracted from hard candy by dissolving 20.0 g of uncrushed candy
in 50.0 mL of distilled water and extracting the solution with 50.0 mL of chloroform containing 0.5% of
acetic acid. Extraction was repeated twice with 25 mL of the same solution. The extraction procedure of
annatto from cheese, butter, and margarine is shown in Figure 3.95. Separation was carried out on a C18
column (250 × 4.6 mm I.D., particle size 5 µm) using an isocratic eluent methanol:2% aqueous acetic
acid (9:1). The flow rate was 1.0 mL/min, and the detection wavelength was 500 nm. The method
separated the color components of annatto well, as demonstrated in Figure 3.96. The chromatograms
indicate that the RP -HPLC method provides a quasi-baseline separation of α- and β-norbixin and α-
and β-bixin in cheese and cheese products. The concentration of norbixin and bixin in various dairy
products is shown in Table 3.95. The recovery varied
Page 486
Figure 3.94.
Chromatograms at 330 nm of (A) cane refinery molasses, (B) cane syrup, and (C) affination syrup,
(Reprinted from Reference [ 630] with permission from Verlag Dr. Albert Bartens.)
Page 487
TABLE 3.94. Peak Areas (%) of Various Syrups, Molasses, Raw Sugars, and Liquors.
Colorant Affination Cane Molasses Beet Molasses Cane
Group Syrup Syrup Cane Syrup Beet (Brazil)
g1 31.4 31.7 28.4 39.3 8.7 20.5
g2 37.4 44.0 20.8 61.7 69.7 63.8
g3 12.3 5.4 22.9 0.0 2.2 6.4
g4 14.5 10.8 24.0 0.0 10.4 4.0
g5 4.4 8.1 3.9 0.0 9.0 5.3

Cane Cane Beet Raw Carbonated Fine
(Cuba) (Columbia) (Germany) Liquor Liquor Liquor
31.3 28.4 16.8 29.6 36.6 43.5
42.4 49.3 71.2 37.4 43.0 10.6
8.5 7.6 3.1 10.5 3.5 9.6
13.0 8.1 4.8 12.2 4.5 6.5
4.8 6.6 4.1 10.3 12.4 29.8
Reprinted from Reference [ 630] with permission from Verlag Dr. Albert Bartens.
between 92.6% and 93.2%, proving the reliability of the method. However, it was not stated that the
RP-HPLC method seems to be suitable for the separation and quantitative determination of the
components of annatto in high-fat dairy products.
Various HPLC methods have been frequently applied for the determination of aspartame in food
products using precolumn derivatization [ 632] or CLND [633]. Both traditional and microbore C18
columns (150 × 4.6 mm I.D. and 150 × 2 mm I.D.) were used for the separation of nitrogen-containing
compounds in soda drinks. The mobile phase consisted of aqueous 0.2% H3PO4, pH adjusted to 2.2 with
0.05 M KH 2 PO4 and methanol (82:18 v/v); the flow rate was 0.050 mL/min. Solutes were
simultaneously detected by UV (214 nm) and CLND by splitting the effluent in the ratio of 0.45-0.55
(UV). Nitrogen-containing and UV absorbent compounds were well separated and detected by the
method in diet citrus soda and diet cola beverages. Some characteristic chromatograms are shown in
Figure 3.97. It was established that HPLC-CLND easily detects compounds with a nitrogen atom in the
molecule, and other compounds did not interfere with the detection; the method is especially suitable
for the detection of these types of compounds in commercial softdrinks.
Another RP-HPLC method was developed and successfully applied for the determination of aspartame
in beverages, milk products, and sweets [ 634]. Beverages were degassed and filtered, sweets were
dissolved in bidistilled water and filtered, and dairy products were mixed with water and then filtered
before analysis. Separation was carried out on a C18 column (250 × 4 mm I.D.,
Page 488
Figure 3.95.
Procedure for the extraction of annatto from cheese, butter, and margarine.
Taylor & Francis and the corresponding author.)
Page 489
Figure 3.96.
HPLC chromatogram of (A) a Canadian
wine cheese extract and (B) a process
cheese spread dissolved in methanol.
Mobile phase, methanol—2.0% acetic
acid (9:1). Detector wavelength, 500 nm.
from Taylor & Francis and the corresponding author.)
Page 490
TABLE 3.95. Annatto in Commercial Samples of Bulk Cheese, Cheese Spread, Process
Cheese, and Butter.
Total Norbixin * Total Bixin*
Sample (µg/g) (µg/g)
Process cheese spread 1.1 ± 0.1 5.9 ± .2
Process cheese slices 2.5 ± 0.2 ND
Skim milk cheese 15.6 ± 0.4 ND
Canadian white cheese 16.8 ± 0.5 5.1 ± 0.2
Cheddar, medium 18.2 ± 0.5 ND
Canadian Colby 21.2 ± 0.6 ND
Cheddar, sharp 68.8 ± 1.8 ND
Sample (ng/g) (ng/g)
Butter Brand A 199 ± 12 914 ± 25
Brand B 12 ± 5 ND
Brand C1 14 ± 5 19 ± 5
Brand C2 19 ± 5 27 ± 5
Brand D 17 ± 5 33 ± 5
* Average of two or three determinations; three extractions/determinations.
particle size 10 µm) guarded by a 25 × 4 mm I.D. precolumn filled with the same support. Isocratic
eluent consisted of 12.5 mM KH2PO4 and methanol (70:30 v/v) with the pH adjusted to 4.5 by
orthophosphoric acid. The flow rate was 1.2 mL/min, and the detection wavelength was set to 217 nm.
The results of HPLC and enzymatic determination of aspartame are compared in Table 3.96. The data
prove that the aspartame contents determined by HPLC and enzymatic methods are commensurable.
The recovery of both methods was 93-102%, and the sensitivity was also similar. It was found that
these methods can be used for the quantitative determination of aspartame in various food products.
An interlaboratory study was performed for the evaluation of the statistical parameters of the
determination of benzoic acid in orange juice [ 635]. Samples of orange juice were centrifuged, and 1 mL
of supernatant was passed through a C18 cartridge preconditioned with 2 mL methanol, followed by 5
mL of water. The cartridge was washed with 3 mL of 2% acetonitrile in n-hexane; the cartridge was
dried, and benzoic acid was eluted with 3 mL of methanol. Benzoic acid was separated on a C18 column
(250 × 4.1 mm I.D., particle size 10 µm, fitted with a similar guard column). Isocratic eluent consisted
of acetonitrile: 0.05 M KH 2PO 4 with the pH adjusted to 2.3 by 83% of orthophosphoric acid (40:60 v/v).
The flow rate was 0.7 L/min, and the detection wavelength was 230 nm. Benzoic acid eluted at 7.26 ±
0.05 min. The CVs of the retention time and response factor were 0.7% and 1.1%, respectively. The
other valida-
Page 491
Figure 3.97.
Split HPLC-CLND and UV detection
of diet cola #3 with 1 mL/min flow rate:
(A) saccharin, (B) caffeine (C) aspartame
and (D) sodium benzoate.
tion parameters of the interlaboratory study are given in Table 3.97. The results indicated that the
method cannot be used for the detection of benzoic acid at concentrations lower than 0.5 ppm because
the variability is high, probably because of the natural occurrence of benzoic acid in citrus fruits.
Sucrose polyester in salad dressing has also been determined by HPLC using an evaporative light-
scattering mass detector [ 636]. Extraction was
Page 492
TABLE 3.96. Results of the Determination of Aspartame in Food Products.

Sample HPLC (mg/L) Enzymatic Method (mg/L)
Beverage 1 31 34
2 426 429
3 471 487
4 19 23
5 18 21
6 92 92
Yogurt 1 43 39
2 86 89
3 59 61
4 34 34
5 74 84
Sweetener 1 163 161
2 178 179
Sweets 1 267 268
2 96 95
Reprinted from Reference [ 634], copyright © 1995. Wissenschaftliche

performed by homogenizing 3 g of dressing, 20 mL of 2-propanol, 4.5 g of anhydrous MgSO4 , and 20

mL of methylene chloride. The homogenate was filtered and the residue was washed with 2 × 15 mL of
methylene chloride. The residue was homogenized again with 5 mL of 2-propanol and 20 mL of
methylene chloride and treated as described above. The filtrate was collected, evaporated to dryness,
and redissolved in methylene chloride. Acylglycerols and sucrose polyster were separated by gel
permeation chromatography using four columns connected in series (Ultrasphere 1000 Å, 300 × 7.7 mm
I.D., Ultrasphere 500 Å, 300 × 7.7 mm I.D., µStryragel 500 Å, 300 × 7.8 mm I.D.;
TABLE 3.97. Precision Measures of Results for Determination of Benzoic Acid

Added to Orange Juice. *
Mean Recovery
Added, ppm ppm % sr SR RSDr (%) RSDR(%)
10 9.61 96.1 0.461 0.665 4.79 6.92
4 3.78 94.5 0.184 0.255 4.87 6.74
3 3.01 100.3 0.159 0.276 5.28 9.16
1 1.01 101.0 0.084 0.161 8.27 15.97

0.5 0.57 114.0 0.113 0.159 19.91 27.90
* S r and S R standard deviations for repeatability and reproducibility, respectively.
RSDr and RSDR are relative standard deviations for repeatability and
reproducibility, respectively.
International.
Page 493
Figure 3.98.
Reversed-phase liquid chromatogram of an extract of
French salad dressing: (A) internal standard peak (sucrose
octaacetate); (B) sucrose polyester peak. An injection volume
of 10 µL with a gradient program and an evaporative
light -scattering mass detector were used.
(Reprinted from Reference [ 636 ] with permission,
copyright © 1995, by AOAC International.)
and µStryragel 100 Å, 300 × 7.8 mm I.D.). Eluent was methylene chloride, and the flow rate was set to
1.0 mL/min. A fraction collected between 27 min and 29.2 min was collected, evaporated to dryness,
and used for further HPLC analysis using a C 18 column (250 × 4.6 mm I.D., particle size 5 µm). Sucrose
polyester was separated with gradient elution composed of methylene chloride, acetonitrile, and 2-
propanol at 40°C. Initial eluent composition was 45.5% of methylene chloride, 52.5% of acetonitrile,
and 2% of 2-propanol. After 5 min, the composition was changed to 98% of methylene chloride and 2%
of 2-propanol and remained unchanged for 6 min. Sucrose octaacetate was used as internal standard.
Sucrose polyester eluted as a well-defined peak from the C18 column (Figure 3.98). Recoveries varied
between 90.5% and 103%, indicating that the method can be safely used for the determination of
sucrose polyester in various salad dressings.
Page 494
Oxalic acid can be used for the whitening of cork stoppers. Since oxalic acid shows marked toxicity, its
determination is of considerable importance. Because of the highly polar character of oxalic acid, an
ion-exchange HPLC method was employed for its determination with UV [ 637] or electrochemical
detection [ 638]. Cork stoppers were granulated (4.0 mm) before the extraction procedure. Oxalate was
extracted by 12% of ethanol for 24 h at 40 ± 2°C, and the filtrate was used for HPLC analysis.
Separation was performed on an anion-exchange column (300 × 7.8 mm I.D.) using 0.1% aqueous
phosphoric acid as eluent at a flow rate of 0.5 mL/min. The detector voltage was +1.25 V (glassy
carbon working electrode). The peaks of oxalic acid (9.33 min), ethanol (24.23 min), and other
components present in the extract were separated well during the chromatographic process, as
demonstrated in Figure 3.99. Recoveries and RSDs varied from 92.85-103.6% and 5.90-8.20%,
respectively. The oxalic acid content of natural cork, treaded cork, and finished cork stoppers are listed
in Table 3.98. The data prove that the oxalic acid content of the finished cork stoppers is very near to
that of natural cork; that is, the whitening process does not cause a health hazard for the users. It was
stated that the high sensitivity, accuracy, and precision of the method makes it suitable for the
determination of oxalates in cork in both industry and quality control organizations. The
monocarboxylic polyether monensin is used as a feed additive for cattle. An HPLC method was
developed for its determination in bovine tissues and milk [ 639]. Monensin was extracted from tissues
by sonificating or homogenizing 10 g of minced tissue with 75 mL of methanol:water (85:15 v/v). The
mixture was centrifuged, and a 10% NaCl solution was added to the supernatant (50 mL for muscle and
liver tissue, 40 mL for kidney, and 60 mL for fat tissue). The tissue was extracted with 2 × 35 mL of
dichloromethane, and the organic phase was separated, evaporated to dryness, and redissolved in 7 mL
of dichloromethane. An aliquot of 5 mL maximum of the solution was passed through a silica SPE
cartridge preconditioned with 3 mL of dichloromethane. The cartridge was washed with 10 mL of
dichloromethane and eluted with 5 mL of dichloromethane:methanol (95:5 v/v). The eluent was
evaporated in nitrogen stream and redissolved in the HPLC eluent. Milk samples (40 mL) were
sonicated with 160 mL of methanol and centrifuged after 10-15 min. The supernatant was mixed with
60 mL of NaCl solution and extracted with 2 × 70 mL of dichloromethane. Further purification steps
were the same as for bovine tissues. Separation was performed on a C 18 column (250 × 4.6 mm I.D.,
particle size 5 µm). The mobile phase was methanol:water:acetic acid (940:60:1). Postcolumn
derivatization of monensin was carried out with vanillin reagent (20 mL of concentrated sulfuric acid,
950 mL of methanol, 30.0 g of vanillin) at 98°C. Monensin derivatives were detected at 520 nm.
Typical chromatograms of bovine liver and milk samples are shown in Figure 3.100. The sensitivity of
the method was higher for milk (5 ppb) than for bovine muscle, liver, kidney, and fat (25 ppb).
Page 495
Figure 3.99.
(A) Chromatogram of oxalic acid standard (10 µg/mL).
Peaks: O = oxalic acid; E = ethanol (attenuation 8).
(B) Chromatogram of an ethanolic extract of untreated
cork stopper. Peaks: O = oxalic acid; E = ethanol
(attenuation 4). (C) Chromatogram of an ethanolic
extract of cork stopper treated with oxalic
acid. Peaks: O = oxalic acid; E = ethanol (attenuation 8).
Page 496
TABLE 3.98. Oxalic Acid Content of Natural Cork, Treated Cork, and Finished Cork
Stoppers.
Oxalic Acid Content (µg/g)
Product Mean Standard Deviation
Natural cork 120.6 18.0
Treated cork 633.2 80.2
Finished cork stoppers 173.8 15.4
The recovery was between 80% and 88%. The good validation parameters, the specificity, accuracy,
precision, and short analysis time make a method suitable for the analysis of monensin in bovine tissues
and milk.
3.3.4—
Capillary Zone Electrophoresis and Micellar Electrokinetic Capillary Chromatography
Due to its high separation capacity and unique retention characteristics, CZE and MECC have also
found extensive application in the analysis of food additives.
Various red food dyes (wanthene and azo dyes) were successfully separated by CZE using sodium
dodecyl sulfate or β-cyclodextrin as buffer additives [640]. Separation was performed on a fused-silica
capillary (50 cm × 75 µm I.D., effective length 37.5 cm, analytical voltage 10 kV, capillary temperature
25°C, hydrostatic injection, diodearray detection between 190 nm and 600 nm). Buffer was a mixture of
25 mM sodium phosphate (pH 8.0) and 25 mM sodium borate (pH 8.0) (1:1 v/v). Separation was
improved by adding 10 mM sodium dodecyl sulfate or 10 mM β-cyclodextrin to the buffer. The method
separated the synthetic dyes well, and it was proposed as an additional assay method for these colorants.
Six synthetic food dyes with different colors were also separated by CZE [ 641]. Separation was
performed in a fused-silica capillary (60 cm × 75 µmI.D., total and effective length 40 cm; background
electrolyte 20 mM borate buffer, pH = 7-9; electromigration injection 4 kV for 14 s; analytical voltage
25 kV; detection wavelength 220 nm; capillary temperature 18°C). Samples have to be dissolved in the
background buffer for successful separation. Typical electropherogram is shown in Figure 3.101. The
baseline separation of the synthetic color pigments can be achieved in 10 min, showing the high
separation power of this relatively simple CZE method. The parameters of the linear regression
equation are compiled in Table 3.99. It was stated that the method is simple and suitable for the
separation of these colorants, shows acceptable accuracy and reproducibility, and can be used as an
additional
Page 497
Figure 3.100.
Typical chromatograms of (A) bovine liver and (B) bovine milk samples.
(Reprinted from reference [ 639] with permission, copyright
method for the analysis of these food additives. MECC has also been used for the analysis of synthetic
colors [642].
A CZE method was developed for the determination of artificial sweeteners (cyclamate, saccharin,
dulcin, and aspartame) in low-joule foods such as diet cola and low-joule cordials and jam [ 643].
Beverages were diluted, and the jam was mixed with water and filtered before CZE analysis. α-
hydroxyisobutyric acid was used as the internal standard. Separation was performed
Page 498
Figure 3.101.
Typical electropherogram of a mixture of six synthetic pigments;
pH = 9.0 Peaks and concentrations: 1 = phenol (20.0 ppm),
2 = brilliant blue (21.4 ppm), 3 = indigo carmine (20.0 ppm),
4 = sunset yellow (24.7 ppm), 5 = amaranth (15.2 ppm),
6 = Ponceau 4R (18.3 ppm), 7 = tratrazine
(21.7 ppm) 9 = unknown.
kind permission from Elsevier Science—NL, Sara Burgerhartstraat
with an uncoated fused-silica capillary column (75 cm × 75 µm I.D., effective length 50 cm, electrolyte
1 mM hexadecyltrimethylammonium hydroxide and 10 mM sodium benzoate pH 6.6, voltage -20 kV,
capillary temperature 28°C, loading under vacuum, indirect detection at 254 nm). An electropherogram
of the separation of articial sweeteners is shown in Figure 3.102. Except for saccharin, artificial
sweeteners formed well-defined symmetric peaks on the electropherogram, proving the good separation
power of the method. The data showed good agreement with the results of the standard AOAC
gravimetric method, indicating that the method is suitable for the analysis of this class of food additives.
MECC has also been employed for the determination of artificial sweeteners in low-joule softdrinks and
other food products, and the results were compared with those of HPLC [ 644]. Samples for MECC
analysis were diluted with water, the internal standard dehydroacetic acid was added, and the mixture
was filtered. An uncoated fused-silica capillary column (75 cm × 75 µm I.D., effective length 50 cm)
was used for the separation. The voltage was
Page 499
TABLE 3.99. Regression Equations for Six Synthetic Food Colorants in the
Concentration Range 2-50 ppm.
Colorant a b r
Brilliant blue 3530.04 –10679.1 0.99778
Indigo carmine 1669.30 –4873.26 0.99898
Sunset yellow 7099.71 –33844.6 0.99752
Amaranth 6901.51 2572.56 0.99622
Ponceau 4R 8472.33 6020.40 0.99599
Tratrazine 6769.94 –24133.8 0.99786

y = ax + b, where y is the amount of colorant in ppm, x is the corresponding peak area
integrated (µVs) and r is the correlation coefficient.
Netherlands.
20 kV, and the capillary was thermostatted at 27°C. Buffer consisted of 0.05 M sodium deoxycholate,
0.01 M potassium dihydrogenorthophosphate, 0.01 M sodium borate (pH 8.6). Artificial sweeteners
were detected at 220 nm. HPLC separation was carried out on a C 18 column (10 cm × 8 mm I.D.)
equipped with a C18 precolumn. The mobile phase was acetonitrile:0.01 M potassium phosphate with
the pH adjusted to 4.0 with orthophosphoric acid. Sweeteners were detected at 220 nm (254 nm for
acesulfame-K). Characteristic chromatograms are shown in Figure 3.103. The MECC method separated
each compound well from each other and from the possible impurities present in the samples. The
concentrations of solutes determined by both MECC and HPLC methods are listed in Table 3.100. The
data indicate that the results obtained with MECC are comparable with those obtained with HPLC. The
repeatability of the methods is similar; however, MECC is faster and less expensive than the HPLC
method.
Steviol glucosides such as stevioside, rebaudioside A, rebaudioside C, and dulcoside A in stevia

sweeteners were also determined by CZE, and the results were compared with those of HPLC [ 645].
Sweeteners were dissolved in deionized water, diluted with the HPLC mobile phase, and purified on a
C8 cartridge. HPLC separation was carried out on an amino column (250 × 4 mm I.D.) using
acetonitrile:water (80:20 v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection wavelength
was 210 nm. The optimal separation conditions on the fused-silica capillary (50 cm effective length ×
50 µm I.D.) were: 50 mM sodium tetraborate (pH 9.3):acetonitrile (55:45 v/v) buffer, detection 210 nm,
voltage 16.5 kV, ambient temperature. The CZE method separated the stevia sweeteners well, as
demonstrated in Figure 3.104. The results of CZE were in good agreement with those of HPLC. Since
the CZE method is simple, rapid, and accurate, it can be used as an alternative method for the separation
and quantitation of steviol glycosides in stevia sweeteners.
Page 500
Figure 3.102.
Electropherogram showing the separation of cyclamate, acesulfame-K,
aspartame, alitame, and saccharin using a 10 mM sodium benzoate, 1 mM
hexadecyltrimethylammonium hydroxide electrolyte. α-Hydroxyisobutyric
acid (a -HIBA) was used as internal standard. The x-axis gives
the migration time in minutes.
from Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)
Sorbic acid, frequently used as a preservative, has also been detected in various food products by
capillary isotacophoresis [ 646,647]. Samples of softdrinks and wine were diluted with an aqueous
Na 2SO4 solution to an end concentration of 1 mM. Juice and fruit concentrates and marmalade were
diluted in a similar manner, sonificated, and filtered. Margarine (0.35-0.40 g) was dissolved in 15 mL
of n-hexane and was extracted with 2 × 10 mL of 1 mM aqueous solution of 2,2-bis(hydroxymethyl)-
2,2',2''-nitriloethanol. Separation was performed on an isotachophoretic analyzer using a fluorinated
ethylenepropylene copolymer capillary tube (250 mm × 0.3 mm I.D., effective length 200 mm). Buffer
consisted of 100 mM of 2-(N-morpholino)-ethanesulfonoic acid, 10 mM of 2,2-bis(hydroxymethyl)-
2,2',2" -nitriloethanol and 0.2 % w/v polyethylene glycol 5,000,000. Detection wavelength was 254 nm,
and the voltage was ca. 400 V/cm. The concentrations of sorbic acid in various food products
determined by this method are given in Table 3.101. The method showed good validation parameters,
the recovery was between 98% and 102%, the RSD was less than 5%, and the correlation coefficient of
the linear relationship between the detector signal and concentration of sorbic acid was over 0.996.
Benzoic and sorbic acids were simultaneously determined in foods by
Page 501
Figure 3.103.
Electropherograms of (A) low-joule cola 1, (B) low-joule cola 2,
(C) low-joule cola 3 and (D) standard solution with a buffer consisting
of 0.05 M sodium deoxycholate, 0.01 M potassium dihydrogenorthophosphate,
0.01 M sodium borate (pH 8.6). The x-axis gives the migration time in minutes.
Peaks: 1 = caffeine; 2 = dulcin; 3 = alitame; 4 = aspartame;
5 = dehydroacetic acid; 6 = sorbic acid; 7 = benzoic acid; 8 = saccharin; 9 = acesulfame-K.
Page 502
TABLE 3.100. Comparison of MECC and HPLC Quantitation of Asparatame, Benzoic Acid,
and Caffeine in Low-Joule Softdrinks Showing RSDs between Instruments with a Sodium
Deoxycholate-Modified Buffer.
Amount (mg/L)
Aspartame Benzoic Acid Caffeine

Sample MECC HPLC MECC HPLC MECC HPLC
Cola 1 510 530 170 165 140 130
RSD (%) 0.5 3.0 0.9 3.0 0.7 1.0
Recovery (%) 104 87 112 88 112 91
Cola 2 440 105 170 160 100 100
Cola 3 450 430 175 175 90 95
Cola 4 470 450 170 165 85 85
Cola 5 * 335 335 150 150 60 60
Cola 6 ** — — 320 320 80 80

Lemonade 1 405 420 265 260
RSD (%) 2.1 3.4 0.7 1.1
Recovery (%) 105 106 103 100
Lemonade 2 480 470 155 155
Lemonade 3 395 415 180 175
Orange 1 450 440 230 235
RSD (%) 0.5 1.2 1.2 1.3
Recovery (%) 100 94 104 97
Orange 2 335 335 170 165
Lemon 1 395 395 230 230
RSD (%) 0.5 0.0 1.0 0.5
Lemon 2*** 295 295 190 190

* This sample also contained acesulfame-K (MECC 45 mg/L, HPLC 40 mg/L).
** This sample contained saccharin (MECC 75 mg/L, HPLC 80 mg/L) and cyclamate.
*** This sample also contained sorbic acid (MECC 50 mg/L, HPLC 50 mg/L).
MECC and HPLC [ 648]. Samples of juice and fruit drinks were diluted with the solution of the internal
standard (dehydroacetic acid), the pH was adjusted to be 9 with 1 M NaOH, and the mixture was
filtered. Jams and preserves were treated similarly.
Sorbic acid and benzoic acid were extracted from cheese slices and dips by steam distillation, and the
distillate was treated as described above. MECC separation was performed in a fused-silica capillary
(68 cm × 75 µm I.D., effective length 43 cm) using +25 kV and UV detection at 230 nm. The capillary
was thermostatted at 27°C. Buffer consisted of 0.05 M SDS and 0.02 M disodium hydrogen
orthophosphate (pH 9.2). HPLC separation was carried out in a C 18 column (300 mm × 3.9 mm I.D.)
using an aqueous solution of KH 2PO 4 and K2HPO4, each at 2.5 g/L. The flow rate was 1 mL/min, and
the
Page 503
Figure 3.104.
Capillary electropherograms of stevia sweeteners:
(A) sample 1 and (B) sample 2. Buffer, 50 mM sodium tetraborate
(pH 9.3): acetonitrile (55:45 v/v) buffer, detection 210 nm,
voltage 16.5 kV, ambient temperature. Peaks: 1 = dulcoside A (DA);
2 = stevioside (SS); 3 = rebaudioside C (RC); 4 = rebaudioside A (RA).
Page 504
TABLE 3.101. Examples of the Determination of Sorbic Acid in Various Food Products.
Determined
(mg/kg)
Maximum Permitted
Food Products A B (mg/kg)
Fanta (soft drink) 203.5 206.2 200
Sprite (soft drink) 195.6 194.8 200
Diplomat (wine) 146.2 146.1 200
Athena orange (juice concentrate) 137.0 136.9 300
Athena lemon (juice concentrate) 2.4 2.5 —
Apricot (marmalade) 118.5 119.4 400
Halvarine (margarine) 180.6 178.9 200
A, B = peak height measurements from the integrator and the recorder, respectively.
detection wavelength was the same as for MECC. The internal standard, sorbate, and benzoate were
separated well by the MECC method, and no interference from the accompanying matrix was observed
(Figure 3.105). The concentrations of sorbic and benzoic acid in various foods determined by MECC
and HPLC are presented in Table 3.102. The results obtained by MECC and HPLC are comparable, for
the majority of products the repeatability of both methods being similar. Because the MECC method is
faster, more efficient, and less expensive than the HPLC, it was proposed for the determination of
sorbic and benzoic acid in various food products.
Page 505
Figure 3.105.
Separation of dehydroacetate (internal standard), (2) sorbate, and (3) benzoate in a fruit drink using a buffer
consisting of 0.05 M sodium dodecylsulphate and 0.02 M disodium hydrogen phosphate,
pH 9.2. The x-axis gives the migration times in minutes.
Page 506
TABLE 3.102. Comparison of the Amounts of Sorbic Acid and Benzoic Acid Present in a
Variety of Beverages and Foods Determined by MECC and HPLC.*
Sorbic Acid Benzoic Acid % CV
Sample (mg/L) MECC HPLC MECC HPLC MECC HPLC

Orange juice Brand 1 370 380 — — 1.40 0.12
Brand 2 150 150 — —
Brand 3 320 340 — —
Brand 4 360 420 — —
Apple juice Brand 1 350 370 — — 1.74 0.61
Brand 2 170 180 160 190
Brand 3 210 200 230 240
Brand 4 280 270 — —
Cordials Brand 1 — — 390 350 0.73 0.40
Brand 2 — — 440 400
Brand 3 — — 350 350

Brand 4 — — 390 400
Diet cordial Brand 1 — — 440 450 0.92 1.47
Brand 2 70 60 — —
Brand 3 — — 100 90
Brand 4 — — 370 340
Fruit drink Brand 1 270 280 170 160 1.25 * 44 *
(25% juice) Brand 2 410 410 — —
Brand 3 240 240 120 120
Brand 4 250 250 120 120
Soft drinks Brand 1 — — 220 260 1.28 1.29
Brand 2 — — 170 170
Brand 3 — — 130 140

Brand 4 — — 150 140
Low-alcohol Brand 1 170 220 — — 1.06 0.50
wine Brand 2 — — 240 280
Cheese Brand 1 960 830 — — 1.8 1.52
slices Brand 2 1680 1830 — —
(mg/kg) Brand 3 790 810 — —

Brand 4 1140 1240 — —
Dips (mg/kg) Brand 1 1270 1250 — — 1.05 1.91
Brand 2 460 450 — —
Brand 3 810 800 — —
Low-joule Brand 1 960 930 — — 1.68 1.17
jam (mg/kg) Brand 2 — — 400 400

*Repeatability data, expressed as % CV, from seven replicate determinations of one
sample of each type of product is also shown.
** Percent CV for sorbic acid.
Page 507
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products, J. Planar Chromatogr., 8, 1995, 117-121.
628. Krzynowek, J. and Panunzio, L.J., Practical application of thin-layer chromatography for detection
of polyphosphates in seafood, JAOAC Int., 78, 1995, 1328-1332.
629. Bento, L.S.M., Separation of sugar colorants through sucrose chromatography, Int. Sugar J., 95,
1993, 391-396.
630. Bento, L.S.M., Separation of sugar colorants using chromatographic columns containing sucrose
crystals, Zuckerind., 120, 1995, 123-130.
631. Lancaster, F.E. and Lawrence, J.F., Determination of annatto in high-fat dairy products, margarine
and hard candy by solvent extraction followed by high-performance liquid chromatography, Food Add.
Contam., 12, 1995, 9-19.
632. Hayakawa, K., Schilp, T., Imai, K., Higuchi, T. and Wong, O.S., Determination of aspartic acid,
phenylalanine, and aspartylphenylalanine in aspartamecontaining samples using a precolumn
derivatization HPLC method, J. Agric. Food Chem., 38, 1990, 1256-1260.
633. Fujinari, E.M., HPLC -CLND application to quantitative aspartame and other nitrogen containing
compounds in beverages. In: Shelf Life Studies of Foods and Beverages, Chemical, Biological and
Nutritional Aspects (Ed.: Charalambous, G.), Elsevier Science Publishers B.V., The Netherlands, 1993,
pp. 1033-1048.
634. Gromes, R., Schnellbacher, B., Siegl, Th. and Vatter, Th., Determination of aspartame in foods
with a new enzymatic test and with HPLC, Deutsch. Lebensmitt.-Rundsch., 91, 1995, 171-174.
635. Lee, H.S., Liquid chromatographic determination of benzoic acid in orange juice: interlaboratory
study, JAOAC Int., 78, 1995, 80-82.
636. Chase, G.W., Jr., Akoh, C.C. and Eitenmiller, R.R. Liquid chromatographic analysis of sucrose
polyester in salad dressing by evaporative light-scattering mass detection, JAOAC Int., 78, 1995, 1324-
1327.
637. Huang, A. and Tanudjaja, L., Application of anion-exchange high performance liquid
chromatography in determining oxalates in taro (Colocasia esculenta) corms, J. Agric. Food Chem., 40,
1992, 2123-2126.
638. Carvalho, F., Bastos, M.L., Remiao, F. and Ferreira, M.A., Determination of oxalates in cork
stoppers by HPLC with electrochemical detection, Am. J. Enol. Viti., 46, 1995, 63-66.
639. Moran, J.W., Turner, J.M. and Coleman, M.R., Determination of monensin in edible bovine
tissues and milk by liquid chromatography, JAOAC Int., 78, 1995, 668-673.
Page 546
640. Suzuki, S., Shirao, M., Aizawa, M., Nakazawa, H., Sasa, K. and Sasagawa, H., Determination of
synthetic food dyes by capillary electrophoresis, J. Chromatogr. A, 680, 1994, 541-547.
641. Liu, H., Zhu, T., Zhang, Y., Qi, S., Huang, A. and Sun, Y., Determination of synthetic colourant
food additives by capillary zone electrophoresis, J. Chromatogr. A, 718, 1995, 448-453.
642. Thompson, C.O., Trenerry, V.C. and Kemmery, B., Determination of synthetic colours in
confectionery and cordials by micellar electrokinetic capillary chromatography, J. Chromatogr. A, 704,
1995, 195-201.
643. Thompson, C.O., Trenerry, V.C. and Kemmery, B., Determination of cyclamate in low joule foods
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644. Thompson, C.O., Trennery, V.C. and Kemmery, B., Micellar electrokinetic capillary
chromatographic determination of artificial sweeteners in low-joule soft drinks and other foods, J.
Chromatogr. A, 694, 1995, 507-514.
645. Liu, J. and Li, S.F.Y., Separation and determination of stevia sweeteners by capillary
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Page 547
Index
acetic acid, 23, 26, 355, 390, 401
actin, 274, 275
actinin, 274, 275
adhumulone, 411
adlupulone, 411
Adzuki bean, 199
almond, 199, 227
almond oil, 103
amaranth, 498
amperometric detector (PAD), 12, 28
amylase, 46, 377
amylopectin, 44
amylose, 44
anchovy, 212, 214
androstanone, 434
androsterone, 432, 434, 435
animal fats, 53
annatto, 485, 488, 490
anthocyanins, 452, 454
apple, 210
arabinose, 39, 40, 45
aroma components of wine, 389, 401
aroma profil of boiled chicken breast meat, 431, 432

artifical sweeteners, 497, 499, 503
ascorbic acid, 299, 300, 301, 303, 304, 305, 306, 309
asparagus, 307
Aspartame, 475, 487, 490, 497
Association of Official Analytical Chemists (AOAC) method, 60
avenasterol, 123, 125, 126
avocado oil, 103
azo dyes, 496
baby milk, 15, 20, 155, 156, 302, 316
bacery products, 149
bacon, 142
banana, 227
barley, 46, 354
bean, 199, 200, 201, 227, 352
beef, 218, 219, 220, 227, 272, 438
beer, 30, 307, 309, 334, 390, 403, 404, 405, 407
beet, 28, 29
beetroot, 307
benzoic acid, 492, 506
benzyl benzoat, 366
biscuit, 24, 27, 152
bixin, 485
blueberry extract, 305
borage oil, 108
borneol, 371
bovine kidney, 494
bovine liver, 494

bovine muscle, 494
brassicasterol, 136
brassinolide, 123
bread, 24, 27, 227, 470, 472
brilliant blue, 498
broccoli, 307
Brussel sprouts, 307
buckthorn, 84, 86
butter, 111, 115, 425, 438, 485, 490
butter cake, 111
cabbage, 209
cacao, 199
cadinene, 366
Page 548
caffeic acid, 337, 385, 388
caffeine, 420, 421, 422
calcium, 13
Candida cylindacea, 100
cane syrup, 487
canola oil, 97
canonical discriminant analysis, 32
campestadiene, 131
campestenol, 136
campesterol, 67, 68, 69, 118, 123, 124, 125, 126, 131, 136
capillary gel electrophoresis (CGE), 9, 196
capillary electrophoresis (CE), 8, 196
capillary isoelectric focusing (CIEF), 9
capillary isotachophoresis (CITP), 9
capillary zone electrophoresis (CZE), 9, 196
capric acid, 67, 68, 69, 402
caprylic acid, 402
capsaicin, 378, 379, 382-383
capsici seed, 199
Capsicum annuum, 375, 377, 378, 380, 455, 457, 458, 459, 460, 461, 462, 464, 466, 467
caramel, 450, 454
caraway fruit, 378
carbohydrate polimer, 37, 44
carboxylic acid, 20, 23, 356, 390, 391
carotenoids, 101, 312, 314, 455, 457, 458, 459, 460, 461, 462, 463, 468
carrot, 44, 210, 307, 455, 457, 465, 469
carveol, 378
carvone, 378
caryophyllene, 328
casein, 183, 235, 236, 239, 241, 242, 244, 252, 262, 264, 265, 294
cassia, 365, 366, 367, 368
catechin, 337, 388
cauliflower, 307
cellobiose, 45
cheese, 142, 145, 147, 150, 151, 223, 238, 245, 357, 425, 426, 427, 438, 453, 485, 488, 489, 490, 506
chestnut, 199
chlorogenic acid, 416, 419, 420
chicken, 102, 142, 227, 430, 433
chocolate, 223, 438, 451, 477
cholecalciferol, 79
cholesterol, 51, 67, 68, 69, 85, 91, 93, 113, 115, 118, 120, 122, 123, 124, 127, 128, 129, 130, 131, 136
cholestane, 67, 68, 69
cholesterol ester, 85, 86, 91
ciceritol, 38
cider, 400, 401, 402, 403
cineol, 371
cinnamaldehyde, 366
cinnamon, 364, 365, 366, 367, 368
cinnamyl acetate, 366
cinnamyl alcohol, 366
cironellol, 397
citric acid, 13, 20, 359, 387, 390
citronellal, 371
clementine, 210
clerosterol, 136
cloudberry (Rubus chamaemorus), 80, 86
cocoa beans, 106, 476
cocoa butter, 107, 108, 476
coconut, 427, 428
coconut oil, 97, 118
Coffea arabica, 37, 38, 199, 346, 414, 415, 416, 417, 418
Coffea canephora var. Robusta, 37, 38, 199, 346, 414, 415, 416, 417, 418
coffee, 37, 38, 199, 346, 413, 414, 415, 416, 417, 418, 419
cohumulone, 412
cola drink, 15, 20, 501
color pigment, 331
colupulone, 411, 412
content of ascorbic acid, 304, 307, 309
convicine, 349, 352
cooking oil, 439, 440
coriander seed oil, 392
corn, 174, 453, 465
corn oil, 94, 95, 99, 100, 438
cottonseed oil, 96, 103, 438
coumaric acid, 337, 388
coumarin, 366
coumestrol, 339
creatinin, 275
cucumber, 307
cyclamate, 497
D
dansyl derivatization, 173, 178, 183, 184
decanal, 371, 378

Page 549
dehydroascorbic acid, 301, 302, 304
dehydroisoascorbic acid, 301, 302, 304
demosterol, 67
densitometer, 88
detector, 3, 8, 27, 51, 408
diacylglycerol, 93
dietary fibers, 44, 45
diglycerides, 92
dihydrobenzoic acid, 337
dihydrocarvone, 378
dimethyl sulphide, 408, 410
distribution constant, 3
distribution of fatty acids in several food products, 65
dulcin, 497
durum-wheat semolina, 283, 285, 291
egg, 85, 91, 122, 169, 286, 288, 290, 294, 295, 296, 438
electroosmotic flow (EOF), 9
elemene, 378
ergocalciferol, 79
ergosterol, 67
erythorbic acid, 305, 306, 307
essential oils, 446, 448
ethanol, 20, 22, 24, 25, 26
ethyl caprylate, 67
ethyl deconoate, 67
ethyl vanillin, 369

eugenol, 327, 329, 366
European Economic Community (EEC) Commission Regulation, 23
Fanta, 504
fatty acid, 53, 56, 57, 59, 61, 66, 85, 91
ferulic acid, 384, 385
filamin, 274
fish, 166, 168, 212, 213, 215, 249, 252, 253, 255, 275, 277, 430
fish oils 78, 94, 103
flatfish, 277
flavonoids, 341, 342
flaxseed phenols, 325
folates, 312
Fourier transform infrared spectroscopy (FT-IR), 53
Frankfurter, 484
free amino acids, 165, 175, 179, 180, 182, 191, 202, 204, 206
fructose, 16, 20, 23, 24, 31, 38, 39, 40
fruit juice, 12, 14, 15, 16, 17, 299, 300, 306, 307, 309, 335, 341, 342, 352, 358, 359, 390, 442, 477, 492,
502
fruit of Cucurbitaceae, 119, 123, 124
fumalic acid, 390
furaldehyde, 403, 405
Gadus morhua, 425, 429, 482
galactilipids, 92
galactose, 17, 38, 40, 45
galactosidase, 13
galactosyl, 92
galacturonic acid, 45
gallic acid, 337, 388
garlic, 209, 227, 385, 386, 387, 453
gas-chromatography (GC), 1
gas-chromatography combined with isotope ratio mass spectrometry, (GC/IRMS), 54, 55
gas chromatography-Infrared Spectrometry (GC-IR), 52
gas chromatography-Mass Spectrometry (GC-MS), 46
gel-permeation chromatography (GPC), 7
gentisic acid, 384, 388
genuine beef, 102
geranial, 371, 378
geraniol, 371
geranyl acetate, 371
ginger, 369, 370, 371, 372, 373
gliadin, 289, 291, 292
glucose, 16, 17, 23, 24, 26, 38, 39, 40, 44
glucovanillin, 367, 369, 379
glutamic acid, 191
gluten, 281
glutenin, 281
glycerol, 20, 21, 22, 23, 24, 26
glycolic acid, 390
grape, 210
grapefruit, 210
green bean (Phaseolus vulgaris), 96, 100, 198
green capsicum (pepper), 307, 385
grilled snack chips, 63,

H
hake, 212, 214
headspace sampling, 2
heptadecanoic acid, 67
Page 550
hesperidin, 341, 342
high-performance liquid chromatography (HPLC), 6
high-performance thin-layer chromatography (HPTLC), 6
hydrogenated fatty acids, 70
hydroxybenzoic acid, 385, 388
hydroxycinnamaldehyde, 365, 367
honey, 27, 29, 32, 33
hop, 403, 409, 411, 412
humulene, 328, 411
Humulus lupulus, 403, 409
indigo carmine, 498
indole, 432, 434, 435
inositol phosphates, 345, 346, 348, 349, 351
invert sugar, 27
isoamyl acetate, 397
isoascorbic acid, 301, 303, 304
isocitric acid, 13, 359
isoeugenol, 371
isoflavones, 335, 338
isorhamnetin, 337
isotachophoresis 9
lactose, 14
lactic acid, 25, 26, 355, 390
lactones, 427, 428

lanosterol, 69
lard, 102, 438
lemon, 210, 446
lentil (Lens culinaris), 34
lettuce, 307
light scattering detection, 30, 103
lignan, 339
limonone, 397
linalool, 397, 401
linoleic acid, 67, 105, 106
linolenic acid, 52, 62, 67, 105, 106, 108
linseed oil, 94
liver, 317
lutein, 314
magnesium, 13
maize, 48, 118
maize germ oil, 62
malic acid, 13, 17, 20, 23, 25, 355, 360, 387, 390
malt, 39, 346, 403, 407, 408
maltoheptaose, 30, 31
maltohexaose, 30, 31
maltopentaose, 30, 31
maltose, 30, 31
maltotetraose, 30, 31
maltotriose, 30, 31
mandarin, 449
mannose, 31, 38
margarine, 54, 116, 316, 320, 427, 428, 438, 488, 504
marine oils, 79, 82, 83
martini, 332
mayonnaise, 438
meat ragout, 484
meat sauce, 62
mercaptoethanol, 221, 222, 286
methylglyoxal, 51
methylmethionine, 407, 408
micellar electrokinetic capillary chromatography (MECC), 9
milk, 14, 56, 60, 74, 75, 85, 87, 89, 90, 115, 153, 154, 155, 156, 230, 243, 258, 259, 260, 261, 263, 264,
267, 291, 294, 297, 310, 423, 424, 494
mineral water, 357
miristic acid, 139, 402
molasses, 361, 362, 486
monoacylglycerol, 93
monoglycerides, 92
monosodium glutamate, 187
monounsaturated triacylglycerol, 108
muscle, 127
musts, 17, 66, 160
mutton, 102
myosin, 274
myrcene, 378
myricetin, 337
naringin, 341, 342
narirutin, 341, 342

nerol, 371
nicotinic acid, 307
nonadecanoic acid, 67, 68, 69
nonanoic acid, 67, 68, 69
nonpolar fatty acid, 70
nonsaponifiable lipid, 66
norbixin, 485
nucleosides, 405, 406, 407
oat, 282, 284
odorants of the Savory and Grilled Food Flavoring, 478

Page 551
olfactometry, 474
oligosaccharides, 28, 31, 32
olive oils, 62, 66, 94, 99, 100, 118, 130, 131, 132, 133, 134, 135, 136, 137, 140, 438, 439
onion, 307
optothermal window (OW), 53
orange, 446
orange syrup, 20
organic acid, 20
Origanum vulgare, 385
oxalic acid, 494
palm oil, 137, 141
palmitic acid, 67, 104, 137
peanut, 134, 414, 415
pear, 210
pepper, 227
perillaldehyde, 378
petitgrain oil, 446, 447
phenolic compounds in wine, 336, 337, 347, 387
phenylisothiocyanate, 159
pholyphosphates, 482
phosphatidylcholine, 144
phosphatidylethanolamine, 145
phospholipid, 92, 138, 143, 144, 146, 147
phthalaldehyde, 176, 181, 182, 220, 222, 223
pig, 270, 432, 435
Pimpinella anisum, 385

pinene, 378
plant oil, 62
pork, 142, 227, 273
pork fat, 98
portocatechuic acid, 385, 388
potassium bromate, 470, 471, 472
potato, 24, 307, 438, 484
potato (peeled), 307
pregnenolone, 129
propionic acid, 20
pumpkin seed oil, 62, 98
pyrogallic acid, 388
quercetin, 337
quinic acid, 356
quinoa (Chenopodiun quinoa Willd) seed, 88, 91, 92
radish, 307
raffinose, 38, 39, 40
rainbow trout oil, 87
rapeseed oil, 62, 76, 138, 438
raspberry, 442, 443, 444
ravioli, 62
red raspberry (Rubus idaeus), 12
resolution, 4
resveratrol, 342, 343, 344
resveratrol glucoside, 343, 344

retention index, 4
retention time, 8
retinol, 79, 315
reversed-phase HPLC, 7
reversed-phase TLC, 6
rhamnose, 40, 45
rhoifolin, 341, 342
rice, 228
root of Cruciferae, 119, 123, 124
sabinene, 378
saccharin, 497
salad dressing, 491, 493
salami, 484
Salatrim, 473, 476, 477
Salmo fario, 425
salycylic acid, 384, 385
sardine oil, 438
scampi, 276
sea buckthorn (Hippophae rhamnoides), 69
sea scallop, 482
seed, 24
sesame oil, 438
shark (Cetrophorus equamosus), 69, 81
shark liver oil, 73
shikimic acid, 356
shrimp, 142, 176, 276

silylated rapeseed oil, 77
sitosterol, 67, 68, 69, 114, 118, 123, 124, 125, 126
size-exclusion chromatography (SEC), 7
skatole, 432, 434, 435
sodium, 13
solid-phase extraction (SPE), 89
solid-phase microextraction (SPME), 46
sorbic acid, 500, 504
sorbitol, 13
soup powder, 153, 193
soya-based infant formula, 302
soybean, 246, 268, 286

Page 552
soybean oil, 62, 76, 78, 79, 94, 95, 99, 100, 102, 104, 105, 125, 137, 138, 139, 140, 146, 147, 169, 170,
438
spectroscopic method, 8
sphingomyelin, 143
spinasterol, 123, 124
Sprite, 504
squalene, 79
stachyose, 39, 40
starch, 45, 47, 48, 49, 50
stearic acid, 104, 105, 106, 139
sterol, 66, 67
steviol glucosides, 499, 503
stigmastadiene, 129, 130, 131, 132, 133
stigmasterol, 67, 68, 69, 114, 118, 124, 125, 126, 128, 130
strawberry, 453
succinic acid, 20, 23, 26
sucrose, 13, 16, 28, 38, 40
sucrose polyester, 491
sulphur compound of garlic, 385
sunflower speed (Helianthus annus L.) oil, 64, 95, 99, 100, 134, 115, 125
sunset yellow, 498
sweetening powder, 481
synapic acid, 384, 385
synthetic pigments, 498
syringic acid, 337, 384, 385
syrup, 20, 32, 34, 35, 36, 37
T
tannic acid, 355
tartaric acid, 20, 23, 26, 355, 390
tea, 340, 357, 363, 413, 415, 416, 420
terpineol, 371, 401
tetradecanoic acid, 67
theobromine, 421
theophylline, 421
theoretical plate number, 5
thiazolidine, 437
thin-layer chromatography (TLC), 5
thiols, 353
thistle oil, 62
Thymus vulgaris, 375
titin, 274
tocopherol, 80, 113, 115
tocopherol acetate, 113, 115
tomato, 444, 445, 455, 456, 457, 465, 468, 484
tortellini, 484
total carbohydrate profil of coffee, 41, 42, 43
total fat of various foods, 64
transesterification system, 112
triacylglycerols, 91, 94
tridecanoic acid, 67
triglycerides, 92
trigonelline, 420
triolein, 157, 158
tropomyosin, 274
troponin, 274, 275
turmeric, 371, 374, 375, 376
undecanoic acid, 67
urethane derivaves, 109, 110, 111
uridine, 352
vanilla, 366, 370
vanillic acid, 369, 384, 388
vanillin, 370, 388
vegetable oils, 52, 55, 56, 57, 76, 94, 97, 98, 99, 114, 117, 136, 138
veratric acid, 389
verbascose, 38
vermouth, 332, 334
vicine, 349, 352
vinegar, 357, 386, 388, 389
Vitamin A, 313, 315, 319, 320, 322, 323
Vitamin B1, 310
Vitamin B12, 310, 311
Vitamin D, 322, 323
Vitamin E, 322, 323
volatile compounds in wine, 393
wanthene, 496
water-insoluble vitamin, 312
waxes, 100
wheat, 62, 281, 283, 285, 291, 292

wheat germ oil, 62, 115, 118
wine, 15, 16, 17, 21, 23, 25, 26, 160, 185, 186, 199, 201, 205, 207, 220, 224, 225, 307, 309, 328, 330,
334, 342, 352
xylitol, 31
xylolose, 31
xylose, 31, 40, 44
yoghurt, 13, 20, 23, 227
Zingiber officinale Roscoe, 369
zingiberenol, 371
zucchini, 307

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Chromatography in Food Science and Technology

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Chromatography in Food Science and Technology

Chromatography in Food Science and Technology

Technomic Publishing Company, Inc.

Copyright © 1999 by Technomic Publishing Company, Inc.

No part of this publication may be reproduced, stored in a

Printed in the United States of America

Main entry under title:

A Technomic Publishing Company book

Library of Congress Catalog Card No. 99-62434

Chapter 1. Theory and Practice of Chromatography 1

1.1 Gas Chromatography 1

1.2 Liquid Chromatography 5

1.3 Capillary Electrophoresis 8

Chapter 2. Macrocomponents in Foods 11

2.1 Separation of Carbohydrates 11

2.2 Separation of Lipids 51

2.3 Separation of Amino Acids, Amines, Peptides and Proteins 158

Chapter 3. Microcomponents in Foods 299

3.1 Vitamins and Provitamins 299

3.2 Miscellaneous Bioactive Microcomponents 324

3.3 Separation of Food Additives 470

N-acetyl neuraminic acid

simultaneous distillation–extraction without concentration

Thin-layer chromatography (TLC) is a planar chromatographic technique extensively used as a rapid

The relationship between capacity factor and Rf is

Separation by electrophoresis is obtained by differential migration of solutes in an electric field. In CE,

where ∈ = dielectric constant, ζ = zeta potential, and η = bulk viscosity.

Various capillary electrophoretic methods are discussed more profoundly in references

1. Carbohydrates (in mono-, oligo-, and polymeric forms)

2. Triglycerids and other compounds of lipoidic character

3. Nitrogen-containing compounds such as amino acids, peptides, and proteins

An interesting application of the HPLC determination of carbohydrates is the measurement of the

TABLE 2.3. Sucrose, Glucose, and Fructose Contents in Fruit Juices.

Internal 73.5 ± 1.01 1.4

Glucose — — IS — 60.2 3.8 31.5 4.4

Sucrose IS — 80.0 3.4 7.8 7.6 63 2.3

Maltose — — — — 7.0 7.0 IS —

Lactos 20.9 3.5 42.6 4.7 IS — — —

Fructose (g/L) 20.28 0.0368 0.18 0.5

Glycerin (g/L) 5.29 0.0244 0.46 1.3

Ethanol (g/L) 66.76 0.2398 0.36 1.1

Reprinted from Reference [ 39] with permission.

Sr 0.11796 0.14506 0.12157 0.43660

0.029 xi 0.024 xi 0.16 %vol

0.036 xi 0.027 xi 0.18 %vol

Ethanol * RI 9.16 0.95 9.16 0.73

Baking Powder Biscuit

Crumb ND ND 2.55a ND 0.67a

End of proof 1.32a 0.92b ND 1.48b ND

Crumb 1.31a 0.62b ND 2.01c ND

End of fermentation 1.15a 0.67b ND 1.72b 0.59a

End of proof 0.52b 0.10b, c ND 1.88b 0.49a

Crumb 0.44b 0.06c ND 2.04b 0.52a

fermentation 0.00a 0.00a ND 0.00a ND