Eur. J. Lipid Sci. Technol. 106 (2004) 621–648 DOI 10.1002/ejlt.

200400986 621

Manuela Buchgraber Triacylglycerol profiling by using chromatographic

Franz Ulberth
Hendrik Emons techniques
Elke Anklam
Triacylglycerols (TGs) make up the major part of naturally occurring fats and oils. The
European Commission, composition and fine structure of TGs determine to a large extent the functionality of
DG Joint Research Centre, fats and oils as food ingredients and the physiological effects of fats and oils as com-
Institute for Reference Materials ponent of the human diet. Analysis of intact TGs is usually performed by chromato-
and Measurements,
graphic methods. In this article the application of gas-liquid chromatography, high-
Geel, Belgium
performance chromatography in normal and reversed phase mode, thin-layer chro-
matography and supercritical fluid chromatography for the qualitative and quantitative
determination of TGs is reviewed. Emphasis is put on those factors that are decisive for
obtaining reliable quantitative data. Furthermore, techniques for the stereospecific
analysis of the fatty acid distribution along the glycerol backbone of TGs are presented
briefly.

Keywords: Triacylglycerols, analysis, GLC, HPLC, TLC, SFC, silver-ion chromatogra-

phy, stereospecific analysis.

1 Introduction The stereochemistry of TGs can be described by the

Edible fats and oils are complex mixtures containing a
wide range of compounds. They are mainly composed of
triacylglycerols (TGs), diacylglycerols (DGs), free fatty
acids (FFAs), phospholipids, and other minor compo-
nents. Any of the components contributes to the texture
and flavour of natural fats and oils. However, the most
important group of compounds is represented by TGs,
which are in chemical terms trihydric alcohols esterified
with fatty acids (FAs). The molecular structure of each in-
dividual TG species can be described by a few basic
attributes:
– the total carbon number (CN), which is the sum of the
alkyl chain lengths of each of the 3 FAs,
– the degree of unsaturation in each FA, and
“stereospecific numbering” (sn) system as recommended
by a IUPAC-IUB commission on the nomenclature of
glycerolipids [1]. The carbon atoms of the glycerol part are
numbered 1–3. In a Fischer projection of a natural L-glyc-
erol derivative, the secondary hydroxyl group is shown to
the left of C-2. The carbon atom above this then becomes
C-1, that below becomes C-3 and the prefix sn is placed
before the stem name of the compound. A single mole-
cular species is identified by listing the sn-1, sn-2 and
sn-3 positions in order.
The analysis of the TG composition of an oil or fat is a very
challenging task because an enormous number of indi-
vidual TG species is possible due to the large number of
possible FA combinations on the glycerol backbone
(Tab. 1). For example, a fat containing only 2 different FAs

– the position and configuration of the double bonds in
each FA.
Moreover, each TG species may be differentiated in
regiospecific/stereospecific isomers by determining the
exact position of the 3 FAs on the glycerol backbone. The
trihydric alcohol glycerol itself has a plane of symmetry.
However, a TG molecule shows optical activity when the 2
primary hydroxyl groups are esterified with different FAs.
Correspondence: Franz Ulberth, European Commission, DG
Joint Research Centre, Institute for Reference Materials and
Measurements, Retieseweg 111, 2440 Geel, Belgium. Phone:
+32-14-571600, Fax: +32-14-590406; e-mail: franz.ulberth@cec.
eu.int
Review Article
results in the theoretical number of 8 possible isomers.
However, most seed fats from plants contain 5–10 differ-
ent FAs. Simple seed oils composed of e.g. 5 different FAs
may give 125 individual TG molecules. Vegetable oils
consist of fewer FAs than animal carcass fats, milk fats or
fish oils. They seldom contain odd-numbered, branched-
chain, or unsaturated FAs with fewer than 16 carbon
atoms. Animal fats may be much more complex consist-
ing of 10–40 different FAs [2].
More than 400 different FAs have been identified as
components of milk fat. Most of those FAs arise from
ruminal microbial metabolism and thus are unique to
ruminant fats. Theoretically, they could be incorporated
into the TG structure to form 64 million different mole-
cules. However, because the enzymes responsible for

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622 M. Buchgraber et al. Eur. J. Lipid Sci. Technol. 106 (2004) 621–648

Tab. 1. Number of triacylglycerol species in fat contain-
ing6different fatty acids.
Fatty acids Number of triacylglycerols
All isomers No optical No isomers
isomers
x x3 (x31x2)/2 (x313x212x)/6
2 8 6 4
3 27 18 10
4 64 48 20
5 125 75 35
10 1000 550 220
20 8000 4200 1540
40 64000 32800 11480
milk fat synthesis place FAs on specific positions of the
glycerol backbone, the total is much less than 64 millions
[3].
The biosynthetic pathways of TGs from various origins lead
to species specific arrangements of the FAs within a glyc-
eride. The major biosynthetic pathway for TGs in both plant
and animal tissues is the alpha-glycerophosphate or sn-
glycerol-3-phosphate pathway in which sn-glycerol-3-
phosphate is acylated in turn at positions sn-1 and sn-2 by
specific transferases to form phosphatidic acid. The en-
zyme phosphatidate phosphatase removes the phosphate
group and the resulting 1,2-diacyl-sn-glycerol is acylated
by a further acyltransferase to form a triacyl-sn-glycerol [4].
The glyceride structure of vegetable oils is quite specific;
saturated FAs are concentrated almost entirely in the pri-
mary positions (sn-1 and sn-3), while the sn-2 position of
TGs of seed oils is greatly enriched in polyunsaturated FAs,
and monoenoic acids are relatively evenly distributed.
Since the sn-3 position is the last to be esterified, the less-
common FAs tend to be concentrated in this position.
The distribution of FAs in fats from animal tissues differs
appreciably according to the animal species and the
location of the adipose tissue in the body. However, in
most species, saturated FAs tend to occupy mainly posi-
tion sn-1, though substantial amounts of oleic acid can
also be found at that position. Unsaturated acids and
certain medium-chain saturated acids (myristic acid) are
mainly concentrated in the sn-2 position. There was some
preference for the longer-chain FAs to be located in posi-
tion sn-3, as reviewed by Ruiz-Gutierrez and Barron [5].
Two-thirds of the FAs in milk fat are medium- and long-
chain saturated FAs. The sn-1 position is almost entirely
occupied by long-chain FAs, whereas sn-3 is dominated
by butyric acid, caproic acid and oleic acid. In fact, nearly
all of the butyric and caproic acids are exclusively found in
the sn-3 position [3].
The qualitative and quantitative determination of TG mix-
tures is important in a variety of fields. Natural fats and oils
possess a characteristic and more or less unique pattern
of TGs. These patterns are used increasingly in the food
industry to confirm authenticity, detect adulteration, and
to unravel the composition of mixtures [5–7]. Adulteration
of expensive oils and fats such as olive oil, milk fat or
cocoa butter has always been a serious problem because
of the economic advantages of partly replacing high-
priced fats and oils with low-priced oils without labelling
the product accordingly. From the points of view of both
commercial value and health, the authenticity of the var-
ious categories of olive oil is of great importance. Olive
oils command a higher price than other vegetable oils,
due to their popularity and high costs of production. Olive
oil must conform to certain legal limits, analytical param-
eters and restrictions, and must satisfy precise sensorical
panel test scores [8]. The nutritional and commercial
importance of milk fat has prompted extensive studies of
its TG composition [9–12]. The official EC method for the
determination of the purity of milk fat is based on TG
analysis by packed column gas liquid chromatography
(GLC) [13]. Likewise, for identity control purposes of
cocoa butter the knowledge of the TG profile of fats and
oils has become an indispensable tool. TG analysis is the
method of choice for detection and quantification of
cocoa butter equivalents in cocoa butter [14–16].
Moreover, the knowledge of the TG structure and of par-
ticular FAs on the glycerol backbone is becoming
increasingly important in lipid chemistry for the under-
standing of lipid metabolism and for formulating nutritious
food products. For example structured lipids have
become an important topic in lipid chemistry since enzy-
matic synthesis makes it possible to incorporate selected
acyl moieties at specific positions of the glycerol back-
bone unlike its chemical synthesis counterpart, which
does not exhibit such selectivity [17]. Structured lipids
can be considered as TGs that have a predetermined
composition and distribution of FA groups on the glycerol
backbone. The use of structured lipids as food ingre-
dients or nutritional supplements requires that methods
for their analysis are available.
Experimental evidence that dietary fat can influence car-
cinogenesis, coronary heart disease and inflammatory
alterations has been accumulated for more than 30 years.
The observed effects of reduced mortality from coronary
heart disease in people with a habitual consumption of
fish oil focused attention on the structure of these impor-
tant oils in the last years [18].
In partially hydrogenated fats, the number of FA isomers is
dramatically increased by the appearance of geometrical,
i.e., cis and trans configuration, and various positional

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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
isomers. The analysis of trans FAs in hydrogenated oils
has become more and more important, since their con-
sumption has raised questions regarding their safety.
There is a common trend towards a lower trans content in
hardened fat products. Analytical methods capable of
determining both the TG composition and the trans con-
tent simultaneously are required; however, with currently
available techniques this is not possible at the moment.
From a technological point of view, the TG molecular
species profile represents a key to the understanding of
several physical properties of an oil or fat. In food prod-
ucts that contain significant levels of oil, the behaviour of
the product can depend on the TG composition of the oil.
For example cocoa butter is the only continuous phase in
chocolate, thus responsible for the dispersion of all other
constituents and for the physical behaviour of chocolate
[19]. TGs are the main components of cocoa butter.
Unique to cocoa butter is its brittleness at room temper-
ature and its quick and complete melting at body tem-
perature. The knowledge of the TG composition of cocoa
butter has resulted in a much better understanding of the
physical behaviour of chocolate.
The possibility of altering the physical properties of fats
has aroused considerable interest in recent years. World-
wide liquid oils are predominantly produced, and there is
a substantial demand for hard fats. There are only two
ways to satisfy this demand, i.e., hydrogenation of liquid
oils and the use of naturally occurring hard fats to produce
spreadable fats by blending or interesterification [20].
Information on the TG profile is needed to control these
processes.
A variety of chromatographic techniques have been suc-
cessfully used for TG analyses including GLC, high-per-
formance liquid chromatography (HPLC), thin-layer chro-
matography (TLC), and super critical fluid chromatogra-
phy (SFC) [21]. In the present paper a general overview on
the principles of TG analysis using such methods is given.
Additionally, methods for stereospecific analysis of TGs,
which are important tools with which the nutritional quality
of TGs can be established, are reviewed. A few illustra-
tions presented in this review have been the result of our
own work.
2 Methods
2.1 Gas liquid chromatography
The analysis of oil samples by GLC has progressed
immensely from its conception by James and Martin [22]
in 1952 to the present state-of-the-art. In the past 50
years, GLC has played an important role in the analysis of
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Triacylglycerol profiling 623
oils and fats. Kuksis and McCarthy [23] published the first
GLC analysis of natural TGs in 1962. They analysed milk
fat TGs and found that it gave the most complex elution
pattern of all natural fats. At that time glass or stainless
steel packed columns were used for GLC studies of TGs.
A new stage in the GLC of TGs was represented by the
introduction of capillary columns in the 1970s. Applica-
tions of glass capillary columns in the analysis of TGs
were for the first time reviewed in 1976 [24]. The authors
presented the current situation in glass capillary column
chromatography with commercial instruments. Addition-
ally, the progress in production, connection and
advanced applications of glass capillary columns was
surveyed. Tvrzicka and Mareš [25] summarised a number
of advantages of capillary columns over packed columns,
i.e., better separation efficiencies, quantitative recovery,
reproducibility of retention data, shorter analysis times,
and longer lifetimes of chemically bonded stationary
phases. Nowadays, fused silica capillary columns pro-
tected by a coating of temperature resistant polyimide are
generally used.
Another distinct advantage of GLC over other competing
techniques for TG analysis is the availability of a simple
but universal detector, i.e. the flame ionisation detector
(FID), responding to organic carbon in a more or less non-
discriminatory fashion. Moreover, the detector response
is linear over several decades.
Selectivity in GLC is governed by the chemical nature of
the stationary phase. As already mentioned, much of the
early work in the analysis of TGs employed packed col-
umns with thermostable apolar stationary phases of the
dimethyl polysiloxane type (OV-1, OV-101, OV-3, SE-30,
SE-54, SP-2100, Dexsil 300). In general, columns packed
with silicone stationary phases did not permit the
separation of saturated from unsaturated components of
the same chain length. Separations were based solely on
differences in vapour pressure, which are correlated to
the molecular masses of the compounds. Some separa-
tion of TGs according to the degree of unsaturation in
addition to chain-length has been achieved on packed
GLC columns containing polar stationary phases [26].
Packed column GLC will not be covered in this review.
This has been done authoritatively by Precht [27].
Due to the rapid progress of capillary column technology
for GLC packed columns are rarely used any longer.
Nonetheless, the official European Community (EC)
method for the determination of milk fat is based using the
TG composition as determined on a packed column [13].
This method, which has been developed by Precht [28], is
based on an evaluation algorithm (“TG formulae”) using
TG data obtained by chromatographic determination of
the TGs present in milk fat as input variables. For the
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624 M. Buchgraber et al.
Fig. 1. GLC separation of the triacylglycerol profile of a
milk fat sample obtained by using the following experi-
mental conditions: Ultimetal HT Simdist CB
5 m60.53 mm60.15 mm; N2 15 kPa; temperature gra-
dient (80 7C, 1 min, 50 7C/min, 190 7C; 1 min, 6 7C/min,
350 7C; 6 min).
established evaluation procedure only quantitative data of
TG groups with the same CN are needed, a result that can
easily be delivered with packed column GLC. Equivalent
separations can be obtained by using short capillary col-
umns (3–5 m) coated with thermostable polysiloxane pha-
ses [29, 30]. Fig. 1 shows a typical chromatogram of milk
fat TGs obtained with a 5 m60.5 mm ID metal column
resulting in the same elution pattern as obtained by a
packed column. For the evaluation of milk fat purity by the
described “TG formulae” approach the odd-numbered
TGs appearing between the major even-numbered TG
peaks have to be added to the respective preceding peak.
TG profiling of edible fats and oils by using short capillary
columns is nowadays a well-established practice and is
routinely used by industry for product specification pur-
poses and process control.
The highly efficient separation of individual TGs became
possible due to the application of thermostable stationary
phases based on polysiloxane with methyl, phenyl, and
cyanopropyl groups attached to the surface of capillary
columns. The availability of cross-linked and chemical-
bonded phases improved the properties of columns con-
siderably by extending the column lifetime and reducing
baseline drift at elevated temperatures [31]. Initially, non-
polar stationary phases of the polysiloxane type were
mostly used. Those phases, which are marketed under a
variety of different brand names (OV-1, OV-101, SE-30,
DB-1, HP-1, Ultra-1, SPB-1, CP-Sil 5 CB, Rtx-1, BP-1,
AT-1, etc) for 100% dimethyl polysiloxane and OV-3,
SE-54, DB-5, HP-5, Ultra-2, SPB-5, CP-Sil 8 CB, Rtx-5,
BP-5, AT-5, etc for the slightly more polar 5% phenyl/95%
dimethyl polysiloxane stationary phase, allow only sepa-
ration according to the CN of TGs, even in capillary
columns.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
Fig. 2. GLC separation of the triacylglycerol profile of a
cocoa butter sample obtained by using the following
experimental conditions: DB-5HT 15 m 6 0.32 mm 6
0.1 mm; H2 3 mL/min; temperature gradient (50 7C/min–
300 7C; 30 7C/min–350 7C).
Column lengths usually vary from a few meters (3–5 m) to
25–30 m and the film thickness should be between 0.1
and 0.2 mm. Normally, no useful resolution according to
the total number of double bonds within the constituent
FAs of TGs can be obtained on such columns, although
some partial separations were reported [32, 33]. For
example, Geeraert et al. [34] separated coffee oil on a
15-m glass column coated with OV-101, where the CN54
was separated into 3 fractions according to the number of
unsaturated FAs in the molecule, but not by the number of
double bonds within each acid. Collomb et al. [35] also
separated TG groups differing with respect to the number
of unsaturated FAs of different oils and fats, i.e., palm,
rapeseed, soybean, etc. on a DB-5 column. On apolar
columns unsaturated TGs eluted before saturated ones.
The reason for this consists in the remarkable difference
in vapour pressure between saturated and unsaturated
FAs. Among unsaturated FAs differences are too small to
effect further separations, thus fractionation according to
the degree of unsaturation is not possible. On apolar col-
umns the elution sequence for TGs with a given CN is:
UUU, SUU, SSU, SSS (S – saturated FA, U – unsaturated
FA).
Fig. 2 gives as an example a chromatogram of the
separation of cocoa butter TGs on a relatively short cap-
illary column coated with a DB-5 non-polar liquid sta-
tionary phase. The chromatogram shows three well
separated components, namely, the TG groups with
CN50, CN52, and CN54. For example the CN52 peak
consists primarily of 1-palmitoyl-2-oleoyl-3-stear-
oylglycerol (POS) but includes also 1-palmitoyl-2,3-di-
oleoylglycerol (POO), 1-palmitoyl-2-linoleoyl-3-stear-
oylglycerol (PLS) and 1-palmitoyl-2-linoleoyl-3-oleoyl-
glycerol (PLO).
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
Progress in separation efficiency of TGs has been
achieved by using capillary columns coated with more
polar polysiloxane phases containing a higher proportion
of phenyl groups (50–65%). The use of temperature
resistant medium polarity capillary columns enhances the
resolution power largely, and allows determining individ-
ual TG species. High temperature GLC on medium polar-
ity stationary phases has been extensively applied for the
comprehensive separation of TGs in a wide range of fats
and oils [36–43].
Geeraert et al. [44, 45] first used columns coated with a
polarisable 50% phenyl methyl polysiloxane stationary
phase (RSL-300) for the analysis of TGs in 1984. On phen-
ylmethylsiloxane stationary phases (DB-17ht, HP-17,
CP-Sil 24 CB, Rtx-50, SPB-50, BPX-50, RTx-65TG) the
CN number sequence is retained but now unsaturation
contributes to the fine structure of each CN fraction [46–
49]. Additionally, there are some dedicated capillary col-
umns available which are coated with proprietary phases
such as the CB-TAP column; its selectivity indicates a
65%-phenyl, 35%-methylpolysiloxane coating. The per-
formance of most of the columns commercially offered is
equivalent [50].
The polarity and selectivity for double bonds of capillary
columns coated with chemically bonded polarisable
phenyl methyl polysiloxanes increase with increasing
temperatures. Commercially available columns withstand
temperatures up to 365–390 7C. At such high tempera-
tures the protective polyimide coating of fused silica
capillaries starts to become brittle, thereby not only
increasing the chances that the column breaks but also
increasing the permeability of oxygen into the column. If
the column is exposed to traces of oxygen at the high
temperatures necessary to elute TG, its lifetime is drasti-
cally reduced, in severe cases to only a few hours.
Aluminium clad columns were proposed as an alternative
and meanwhile surface deactivation technology has made
such advances that even stainless steel capillary columns
(Silcosteel, Ultimetal) are offered for TG analysis.
Mayer et al. [48] described the preparation of fused silica
capillary columns coated with 75% diphenyl/25% di-
methyl polysiloxane, a stationary phases with an even
higher polarity than currently available medium polarity
columns. The increased polarity enabled a slightly
improved resolution of TGs that differ only by one double
bond, e.g. SOO and OOO. Nevertheless, separation of all
single complex mixtures could not be achieved within a
single run.
Capillary columns coated with high polarity stationary
phases of the cyanopropyl polysiloxane type (OV-275,
Silar 10C, CP Sil88, SP-2340, Rtx-2330), which are used
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Triacylglycerol profiling 625
with great success for the separation of saturated/unsat-
urated FAs, do not tolerate the high temperatures neces-
sary for TG work. Nevertheless, such columns (SP-2330,
Rtx-2330) have been used for the separation of tri-
methylsilyl ether (TMS) derivatives of diacylglcerols
obtained from phospholipids by enzymatic dephos-
phorylation [51, 52]. This technique can yield valuable
information for the study of the structure of phospholip-
ids, although HPLC seems to be the preferred technique
for such analyses.
Fig. 3 shows the analysis of the TG profile of cocoa butter
on a 25 m60.25 mm CB-TAP column coated with 0.1 mm
of the proprietary CB-TAP stationary phase [46]. In com-
parison to the profile obtained on a non-polar column
(Fig. 2) a much more detailed picture of the TG composi-
tion is obtained. Besides the CN separation, the TGs are
also separated according to their degree of unsaturation.
Thus, for CN52 the fully saturated PPS fraction is eluted
first, followed by POP and PLP. However, positional iso-
mers such as POP and PPO remain unresolved. The
chromatogram shows that the cocoa butter TGs are largely
made up of POP, POS and SOS species. In contrast, a
polyunsaturated soybean oil TG profile shows a much
Fig. 3. GLC separation of the triacylglycerol profile of a
cocoa butter sample obtained by using the following
experimental conditions: CB-TAP 25 m 6 0.25 mm 6
0.1 mm; H2 0.8 mL/min; temperature gradient (100 7C;
50 7C/min–300 7C; 30 7C/min–340 7C) Abbreviations:
PPP: tripalmitin, MOP: 1-margaroyl-2-oleoyl-3-palmi-
toylglycerol, PPS: 1,2-dipalmitoyl-3-stearoylglycerol,
POP: 1,3-dipalmitoyl-2-oleoylglycerol, PLP: 1,3-dipalmi-
toyl-2-linoleoylglycerol, PSS: 1-palmitoyl-2,3-distearoyl-
glycerol, POS: 1-palmitoyl-2-oleoyl-3-stearoylglycerol,
POO: 1-palmitoyl-2,3-dioleoylglycerol, PLS: 1-palmitoyl-
2-linoleoyl-3-stearoylglycerol, PLO: 1-palmitoyl-2-lino-
leoyl-3-oleoylglycerol, SSS: tristaerin, SOS: 1,3-dis-
tearoyl-2-oleoylglycerol, SOO: 1-stearoyl-2,3-dioleoyl-
glycerol, SLS: 1,3-distearoyl-2-linoleoyl glycerol, OOO:
triolein, SLO: 1-stearoyl-2-linoleoyl-3-oleoylglycerol,
SOA: 1-stearoyl-2-oleoyl-arachidoylglycerol, AOO: 1-
arachidoyl-2,3-dioleoylglycerol.
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626 M. Buchgraber et al.
Fig. 4. GLC separation of the triacylglycerol profile of a
soybean sample obtained by using the following experi-
mental conditions: CB-TAP 25 m60.25 mm60.1 mm; H2
100 kPa; temperature gradient (270 7C; 2.5 7C/min–350 7C).
Fig. 5. GLC separation of the triacylglycerol profile of a
milk fat sample obtained by using the following experi-
mental conditions: CB-TAP 25 m60.25 mm60.1 mm; H2
100 kPa; temperature gradient (270 7C; 2.5 7C/min–
350 7C).
more complex structure, especially in the CN 54 group
(Fig. 4). The chromatogram shows also three groups,
separated according to their chain length (CN separation)
and moreover each CN group is split up due to the polarity
differences in the TG. Polarity increases with the degree
of unsaturation in the FA (Ln...LO.S) and with the
total number of double bonds in the TGs (LLL.OLL.
OOL.OOO) [44, 45]. The major TG species are PLO, PLL,
OOL, SLL, OLL, and LLL. By using a polar stationary
phase a very complex TG pattern is obtained for milk fat.
Milk fat contains short-chain FAs such as butyric acid and
long-chain FAs. Triacylglycerol species containing both
short- and long-chain FAs are resolved in the order of
decreasing chain length of the short-chain FAs. As polar-
ity increases with decreasing chain-length of the constit-
uent FAs, TGs containing butyric acid are eluted later than
those containing caproic acid, which in turn are eluted
later than caprylic acid containing TGs. This chain-length
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
specific effect diminishes with FA chain length . caprinic
acid. Over 200 peaks are distinguishable in the TG profile
of milk fat (Fig. 5).
The inherent separation power of polarisable capillary
columns for TGs may lead to identification problems.
Preliminary identification of individual fractions can be
based on analysis of oils and fats with known composi-
tion, literature data or data obtained by other methods.
One of the major difficulties in any chromatographic
method is the prediction of the retention time or retention
volume for a compound where no primary standard is
available for identification purposes. A linear relationship
between the logarithms of retention times (tR) and carbon
numbers (CN) of the members of a homologous series,
which can be used to identify components in a chroma-
togram, was described as early as in 1954 [53]. The clas-
sical retention indices have been replaced by equivalent
chain length (ECL) number [54], or the equivalent carbon
number (ECN) [55]. The ECL concept was originally intro-
duced for the GLC analysis of FA methyl esters and was
later adopted for HPLC. Values of ECL were found to be
useful for the identification of TGs of fats and oils by cap-
illary GLC [39]. Hyphenated techniques, where the chro-
matographic instrument is coupled to a spectrometer,
usually a mass spectrometer, are nowadays available in
many laboratories and used for identification purposes. A
number of authors have covered this aspect for TG anal-
ysis [56–58].
High separation efficiency is the most striking advantage
of capillary GLC for TG work. The technique has therefore
found wide application in various fields. Examples of
capillary GLC employing medium polarity stationary pha-
ses along with the applied chromatographic conditions
are listed in Tab. 2. However, critics of the technique may
advocate the view that discrimination during sample
introduction may occur and highly unsaturated compo-
nents are lost to a varying degree due to the harsh tem-
perature conditions which are necessary to elute TGs
from the column [69, 70]. The injection technique has
been considered as being most critical for obtaining high
accuracy and precision, since at this stage discrimination
against less volatile high molecular weight compounds
can occur while the sample is being transferred from the
syringe to the column (syringe discrimination) [71, 72].
Many authors [32, 40, 42, 72, 73] published detailed
studies concerning the effect of the injection technique on
the recovery of TGs.
Basically, capillary injection techniques can be divided
into 2 groups, i.e., direct on-column injection (OCI) and
injection into an externally heated vaporiser (split/splitless
injector) [25]. Both techniques can be used in several
modifications, differing in the construction of the injector
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648 Triacylglycerol profiling 627

Tab. 2. GLC conditions used for the determination of the triacylglycerol profile of various matrices.

Sample Column Stationary Oven temperature program Injection Carrier Pressure Ref.
dimension phase mode gas

Butterfat 15 m60.25 mm; 0.1 mm PhMeSi 65% 200 7C; 4 7C/min; 360 7C PTV split H2 124 kPa [40]
Synthetic TG mixture 25 m60.32 mm; 0.25 mm DiMeSi 80 7C; 20 7C/min; 200 7C; PTV He 365 kPa [59]
8 7C/min; 370 7C
Palm oil 25 m60.25 mm; 0.1 mm PhMeSi 340 7C; 1 7C/min; 355 7C hot OCI H2 69 kPa [42]
Chicken fat, pork fat, horse 25 m60.25 mm; 0.12 mm PhMeSi 50% moveable OCI [60]
fat, mutton fat, beef
tallow, butter oil, human
breast milk
Palm oil, cottonseed oil, 25 m60.25 mm; 0.12 mm PhMeSi 50% moveable OCI H2 100 kPa [45]
soybean oil, coffee oil
Olive oil 25 m60.25 mm; 0.1 mm CB-TAP, 340 7C; 1 7C/min; 360 7C split He 130 kPa [36]
PhMeSi
Plasma 25 m60.25 mm; 0.12 mm PhMeSi 50% 40–290 7C; 10 7C/min; OCI H2 50–100 kPa [61]
RSL-300 330 7C;1 7C/min; 360 7C
Butter fat 25 m60.25 mm; 0.1 mm PhMeSi 280 7C; 3 7C/min; 355 7C split He 100 kPa [47]
CB-TAP
Cocoa butter, sal fat, palm 25 m60.32 mm; 0.1 mm PhMeSi 340 7C; 1 7C/min; 360 7C split He 100 kPa [47]
oil, palm mid fraction CB-TAP
Butter fat 25 m60.25 mm; 0.1 mm PhMeSi 65% 330 7C; 2 7C/min; 360 7C moveable OCI H2 140 kPa [62]
PU 4550/11
Palm oil 15 m60.53 mm; 0.1 mm PhMeSi 65% 345 7C, 1 7C/min; 365 7C aut. cold OCI He 20 kPa [38]
Olive oil, soybean oil 25 m60.25 mm; 0.1 mm PhMeSi 50% 330 7C; 1 min; 1 7C/min; automatic H2 100 kPa [37]
OV-17 355 C; 4 min split
Cheese samples 25 m60.25 mm; 0.1 mm PhMeSi 280 7C; 1 min; 3 7C/ PTV He 110 kPa [41]
CB-TAP min;350 7C
Olive oil 25 m60.25 mm; 0.1 mm PhMeSi 33 7C; 1 min; 1 7C/min; H2 [63]
CB-TAP 344 7C
Blood samples 25 m60.25 mm; 0.1 mm PhMeSi 65% 350 7C; 1 min; 0.5 7C/min; Split He 130 kPa [64]
Quadrex 360 7C; 6 min
400
Ham fat; interesterified 25 m60.2 mm; 0.1 mm DiPhDiMeSi PTV H2 [65]
cocoa butter hot split
Reference TGs, olive, corn, 25 m60.25 mm; 0.1 mm PhMeSi 50% 335 7C; 1 7C/min; 360 7C Split He 160 kPa [66]
hazelnut, sunflower oils
Peanut, olive, patua pulp, 25 m60.25 mm; 0.1 mm PhMeSi 50% 330 7C; 1 min; 1 7C/min; Split H2 100 kPa [67]
bacaba OV-17-OH 340 7C; 9 min
Goat milk fat 2.5 m60.25 mm; 0.1 mm PhMeSi 65% 210 7C; 8 7C/min/355 7C; aut. split/ He 70 kPa [68]
RTx-65 10 min splitl.
Synthetic TG mixture 15 m60.32 mm; 0.2 mm PhMeSi 75% 60 7C; 25 7C/min; 280 7C; H2 [48]
SOP-75 4 7C/min; 390 7C

and in the temperature at the time of sample introduction.
In capillary GLC most of the injection techniques used,
have the drawback that the sample has to be transferred
from the injection zone to the capillary column inlet at the
oven entrance. This drawback can be suppressed by
injecting the sample directly into the capillary column.
Grob [32] reported that OCI was the technique of choice,
reducing or eliminating thermal decomposition, mass
discrimination, and other effects associated with classical
split/splitless injection. They found that TGs were recov-
ered to ca. 80% in comparison to an n-alkane (n-penta-
decane), while recovery of TGs was reduced for splitless
or split injection (Fig. 6). Considering that the FID re-
sponse for tricaprin and tristearin is about 83% and 89%,
respectively, of that of an n-alkane TGs were almost
quantitatively recovered by using on-column injection for
sample introduction. For the vaporising injector TG losses
of 14–20% (splitless injection) and 25–50% (split injec-

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628 M. Buchgraber et al.
Fig. 6. Peak areas and their standard deviation for tri-
acylglycerols normalised on the n-pentadecan as
obtained with different injection techniques.
tion) relative to the peak area of n-pentadecane were
observed, most likely due to insufficient elution out of the
syringe needle of the high boiling compounds. Standard
deviations of data obtained with splitless injections were
9–13%, and 15–30% for split sampling. However, when
another TG was used as an internal standard instead of
n-pentadecane, the mean values obtained for the
remaining TGs were fairly accurate and precise for split-
less injection, but still unacceptable for split injection.
Consequently, classical split injection for TG analysis was
mostly mentioned in connection with qualitative analysis,
while the majority of the authors advised not to use this
injection mode for quantitative purposes.
The originally employed cold on column injector was
found to be less useful for the analysis of TGs, because it
required a low initial column temperature, depending on
the type of the solvent used. As an alternative a moveable
OCI was described, where the first few centimetres of the
column can be temporarily moved out of the oven during
injection, the sample is injected at ambient temperature
and then pushed back into the hot oven to start the
separation process [34]. Cold OCI at high oven tempera-
ture was made possible by cooling the first few centi-
metres of the column inside the oven with compressed air
during injection. By switching off the air supply the sam-
ple is heated to the oven temperature within a few sec-
onds [42].
Hinshaw and Seferovic [40, 49] and Garcia Regueiro et al.
[74] demonstrated that a programmed temperature
vaporisation (PTV) injector is an equivalent alternative to
an OCI. Temperature programming of the external
vaporiser, starting at a temperature below the solvent
boiling-point to a temperature high enough to transfer the
analyte(s) to the column, can be regarded as an attempt
to combine the advantages of the traditional on-column
and split/splitless injection techniques. The results were
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
compared to those obtained with OCI. PTV injection in
both cold split and solvent venting modes were excellent
sample introduction systems for high temperature work.
A main advantage over cool OCI, which still gives the
most accurate results, is that the column can be main-
tained at a relatively high temperature during injection.
Quantitative analysis in chromatography is generally
based on the relationship between the mass of the ana-
lysed substance and the detector response. The differ-
ence between the theoretical and experimental findings
can be quantitatively expressed as mass correction factor.
Quantitative aspects of the separation of TG on fused silica
capillaries were studied in detail by Mareš and Hušek [69]
and Mareš [75]. They reported a dependence of the
recovery of TGs, and thus their correction factors, on sev-
eral parameters such as injection technique, column
quality, carrier gas flow-rate and the amount and molecular
mass of the substance. The main conclusion of this study,
which is also in agreement with the findings of Carelli and
Cert [76], was that the extent of TG loss depends to a large
extent on its elution time. Chromatographic parameters
leading to shortening the elution time of a component
(shorter column, thinner stationary phase film, higher car-
rier gas velocity, using hydrogen instead of other carrier
gases, steeper temperature gradient) improved con-
siderably its recovery rate. On the other hand, optimisation
of operational parameters has to take into consideration
separation efficiency, which might be ruined by forcing the
solutes through the chromatographic process. Optimising
a capillary GLC system for TG separation is therefore not a
simple compromise between resolution and analysis time,
but a delicate balance between separation efficiency and
recovery of late eluting TG.
Flame ionisation detector response factors depend to a
certain extent on the ratio of the fuel gases [77]. When
hydrogen is used as a carrier gas the varying flow-rate
during temperature programmed operation may therefore
also influence FID response factors [78], in particular
when short capillary columns and older GC equipment,
which does not have the possibility to control the carrier
gas flow, is used.
Mechanistic aspects leading to losses of TGs in the cap-
illary column were studied by Grob [79]. He demonstrated
that losses are primarily results of thermal degradation
and not due to polymerisation, which leads to involatile
products. In this process free FAs and diglyceride-like
compounds, probably enol esters, are formed. The gen-
eral recommendation to minimise losses was to keep
thermal stress to a minimum by eluting the compounds as
rapidly as possible and at the lowest elution temperature
feasible. A careful optimisation of operating parameters
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
Fig. 7. Flame ionisation detector response factors of
synthetic triacylglycerols.
allows a nearly non-discriminatory sample introduction,
which can reduce losses of individual TGs. However, they
can not be eliminated entirely. Thus, a lot of care is nec-
essary to check for losses that may occur during the
separation process.
Geeraert and Sandra [45] stated that the response factors
for TGs, except for highly unsaturated substances, are
almost equal to one by using OCI and a 25-m polarisable
capillary column, and that no special attention need to be
paid to this subject, whereas Termonia and co-workers
[42] reported that a higher bleeding level of phenyl-
methylsilicone phases resulted in lower response factors.
This is quite in contrast with another report, where recov-
ery of triolein (OOO), trilinolein (LLL) and trilinolein
(LnLnLn) from a 25-m medium polarity capillary column
dropped to 94%, (OOO), 70% (LLL) and 35% (LnLnLn),
assuming that tristearin was recovered to 100% [80]. Al-
though losses, in particular for the polyunsaturated TGs,
were quite high, they were very reproducible and could be
controlled by proper calibration. Nevertheless, this high-
lights the necessity to check response factors for every
newly purchased capillary column carefully and to re-
calibrate the instrument frequently during routine use;
otherwise variable and large errors can not be excluded.
Recently, results of two intercomparison studies for TG
analysis by GLC, where the performance of different
sample introduction techniques, i.e. OCI, split injection
and PTV injection, was compared, were made available.
Experimentally determined FID response factors of syn-
thetic TG mixtures formed the basis of the assessment
[81]. None of the laboratories in those studies obtained
unity response factors for TGs normally present in cocoa
butter. FID response factors deviated to a considerable
degree and were in the range of 0.8–1.3 (Fig. 7). However,
the results of the two method intercomparison studies
suggest that all injection techniques considered were
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Triacylglycerol profiling 629
equivalent in terms of repeatability and accuracy, as
judged by the magnitude of the obtained FID response
factors. OCI, which is generally thought of as a nondis-
criminatory sample introduction technique and recom-
mended as best practice for TG analysis, did not give
results superior to those of PTV or the split injection
techniques. The outcome of the collaborative trial under-
pins that GLC conditions have to be selected carefully if
an acceptably accurate quantitative result has to be
achieved.
2.2 High-performance liquid chromatography
HPLC is a separation process based on the selective
interaction of solutes between a solid, stationary phase
and a liquid, mobile phase. In principle, two different
separation modes, i.e., normal-phase (NP) and reversed-
phase (RP) are applied in lipid analysis. Normal-phase
chromatography is characterised by the reversible
adsorption of solutes to polar groups of the solid adsor-
bent (silica gel, alumina, Florisil), while RP chromatogra-
phy is based on the partition of solutes between the
mobile phase and the stationary phase which has a suit-
able “liquid” phase deposited as a thin film, or chemically
bonded to the surface of an inert solid support. Octadec-
ylsilyl (C18) modified silica gel is the most commonly used
material for the preparation of RP columns. In NP-HPLC a
solvent system, less polar than the stationary phase, is
used, in contrary to RP-HPLC, which uses a solvent sys-
tem more polar than the stationary phase.
2.3 Normal phase HPLC
Attempts have been undertaken to use NP-HPLC for the
separation of TGs, but only with limited success [82–84].
The elution order of TGs in NP-HPLC is determined by the
degree of saturation and by the CN. Saturated TG species
elute before the more unsaturated homologues. Normal-
phase separation systems do not have the resolving
power of their RP counterpart and are not commonly
used for TG separations. However, NP chromatography,
either in column chromatography, thin-layer chromatog-
raphy or HPLC format, is an indispensable tool for lipid
class separation [85]. TGs can be readily resolved from
diacylglycerols, monoacylglycerols, sterylesters, sterols,
free fatty acids and phospholipids [86].
2.4 Reversed phase HPLC
The general usefulness of RP-HPLC for the resolution of
molecular species of neutral TGs has now been well
established. Separations are obtained by partition be-
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630 M. Buchgraber et al. Eur. J. Lipid Sci. Technol. 106 (2004) 621–648

tween relatively non-polar stationary phases of the C18 Tab. 3. Classification of triacylglycerols of the 5 most
type and polar mobile phases, mostly mixtures of aceto- abundant fatty acids (P – palmitic acid; S – Stearic acid; O
nitrile with acetone or chlorinated solvents. Thus, the elu- – oleic acid; L – linoleic acid; Ln – linolenic acid) of vege-
tion order of the solutes is reversed compared to NP- table oils according to their ECN and number of double
bonds [88].
HPLC. Numerous practical applications of RP-HPLC for
TG separations have been described [87–106]. ECN 36 38 40 42 44 46 48 50 52 54
NDB
The separation pattern of TGs on RP columns differ pro-
foundly from those obtained by GLC, where TGs are 0 PPP PPS PSS SSS
principally separated according to differences in molecu- 1 PPO PSO SSO
lar mass (CN separation). In GLC CN groups do not over- 2 PPL PSL SSL
POO SOO
lap, even on medium-polarity columns, while this is the
3 PPLn PSLn SSLn
case with RP-HPLC, where components are separated
POL SOL
according to the combined effect of the chain-lengths of OOO
the FA moieties contained in a given TG species plus their 4 POLn SOLn
degree of unsaturation; each double bond reduces the PLL SLL
retention by the equivalent of about 2 carbon atoms. OOL
Mechanisms of retention of TGs in RP-HPLC have been 5 PLLn SLLN
thoroughly reviewed by Nikolova-Damyanova [107]. OOLn
Retention indices have found widespread use in lipid OLL
6 PLnLn SLnLn
analysis, especially for the identification of molecular
OLLn
species of TGs. A number of parameters such as partition LLL
number (PN), ECN, and theoretical carbon number (TCN) 7 OLnLn
have been suggested for describing chromatographic LLLn
properties of TG molecules and determining their elution 8 LLnLn
order. 9 LnLnLn

The concept of the ECN has been introduced by Plattner

et al. [108]. They extended the concept of ECL [54], which
has found widespread use for characterising the retention Tab. 3 gives a classification of the TGs of the 5 most
behaviour of fatty acid methyl esters in GLC, to HPLC. abundant FAs of vegetable oils according to their ECN
This information can be utilised to identify an unknown and number of double bonds.
peak eluting with a given retention volume from an HPLC
As already mentioned the effect of a double bond does
column. The ECN system is based on the observation that
not exactly equate the contribution made by 2 methylene
the CN is linearly related to the logarithm of the retention
groups. El-Hamdy and Perkins [110] introduced a more
volume of a TG in isocratic HPLC. Each double bond
specific term, i.e., the TCN. The TCN value can be deter-
present in a TG decreased the retention volume to ap-
mined either from a plot of the logarithm of the capacity
proximately that of a saturated TG with 2 carbon atoms
factor (k’) versus CN of saturated TGs or calculated from
less. Thus, the ECN was defined by the equation:
the following equation:

ECN = CN 2 2n TCN = CN 2 a1n1 2 a2n2 2 a3n3

where the coefficients a1, a2 and a3 can be calculated by

where CN is the sum of the carbon atoms in the aliphatic
residues and n is the sum of the double bonds per TG. In
essence, this concept corresponds to the older term par-
tition number (PN) as defined by Litchfield [109]. Mole-
cules with identical ECN values are called critical pairs,
which are difficult to separate. For example, TGs con-
taining FAs in the combinations C16:0-C16:0-C16:0,
C16:0-C16:0-C18:1, C16:0-C18:1-C18:1 and C18:1-
C18:1-C18:1 tend to elute together. Using contemporary
HPLC hardware those “critical pairs” are nowadays
separable, and the formula is only used as a rough guide
to what may elute in the same region of a chromatogram.
multiple regression analysis using reference TGs, and n1,
n2 and n3 are the number of double bonds attributable to
oleic, linoleic and linolenic acid residues, respectively.
Podlaha and Töregård [111] developed a graphical sys-
tem for tentative identification and prediction of retention
times for TGs separated by RP-HPLC. They demon-
strated a linear relationship between the ECN and the CN
of TGs, which showed the same unsaturation character-
istics. They found straight, parallel lines for different ho-
mologous series of TGs when plotting CN against ECN.
Several groups [112–114] reported a similar approach for

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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
gradient elution conditions, i.e., a non-linear elution pro-
gramme giving a linear relationship between ECN and
retention times.
A scheme for identifying TG species that is based on the
additivity of solution free energies of saturated and un-
saturated acyl residues was described by Goiffon et al.
[115, 116] and later on confirmed by Podlaha and Tö-
regård [111] and Sempore and Bezard [117]. This
approach is based on the calculation of partial ECN
values for each individual FA component of a TG from the
ECN values of simple model TGs. Combining those partial
ECN values can approximate the elution behaviour of
complex TG occurring in natural fats and oils.
Highest separation efficiencies have been achieved on
HPLC columns packed with chemically bonded C18 pha-
ses. However, C5 and C8 alkyl RP-columns have also
been occasionally employed. Most silica packings con-
sist of spherical particles; the particle diameter ranges
from 3–10 mm. A comparison of various columns and of
the effect of the difference in particle size on TG separa-
tion was investigated by El-Hamdy and Perkins [110].
They observed that a 5 mm LC-18 column was the most
efficient of the columns evaluated. Furthermore, they
found out that separation is improved by increasing the
carbon loading of the silica particles (usually between 10–
30%) smaller particle size of the silica particles and an
increase in mobile phase polarity. The most widely used
columns are 250 mm long, with an internal diameter of
4–4.6 mm (Tab. 4). Enhanced separations have been
obtained by connecting 2 or 3 columns in series. A sam-
ple chromatogram of the TG profile of cocoa butter
obtained by RP-HPLC on two 25064.6 mm Hypersil ODS
columns in series is represented in Fig. 8.
Fig. 8. RP-HPLC profile of cocoa butter: two
25064.6 mm; 5 mm Hypersil ODS; flow 1 mL/min; tem-
perature 30 7C; gradient from 80 ACN:20 DCM-46 ACN:54
DCM over 60 min; 10 mL 5% CB in chloroform.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Triacylglycerol profiling 631
Octadecyl-modified silica materials and pre-packed col-
umns of different dimensions can be purchased from a
large number of manufacturers and suppliers. Although
most of them claim to offer C18 modified silica, variation
with regard to retention characteristics exists due to dif-
ferences in pore size, surface area, carbon load, mono-
meric or polymeric C18, degree of end-capping, etc.,
which may complicate the comparability of published
retention data.
In RP-HPLC the mobile phase has a main effect on the
separation of TGs through competition between the
mobile phase and the TGs for the stationary phase and
the increase or decrease in TG solubility in the mobile
phase, which can enhance or retard their separation. The
research groups of Plattner [84, 108, 138] and Perkins
[110, 139] studied the effect of column packing, mobile
phase, and their interactions in order to optimise the
potential of RP-HPLC for separation of lipids. In principle,
mobile phases are composed of a “weak” organic sol-
vent, mostly acetonitrile and a “strong” organic modifier,
mostly acetone or alternatively chlorinated solvents
(dichloromethane, chloroform, 1,2-dichloroethane), ben-
zene, or tetrahydrofurane, which is added to improve the
solubility of TGs in the mobile phase. The most widely
employed solvent system is a mixture of acetonitrile with
acetone [93, 112, 117, 126, 136, 140]. Acetone improves
the separation of critical pairs of TGs and acetonitrile is
able to interact with the p electrons of double bonds,
thereby effecting separation of unsaturated species.
Dotson et al. [129] studied the effects of changes in
mobile phase polarity on critical pair separation using a
C18 column and a refractive index (RI) detector. They
confirmed that critical pairs, resulting from the approx-
imate equivalence of one double bond to a chain short-
ening by 2 methylene units, were effectively separated
with acetone/acetonitrile as mobile phase in isocratic
mode. However, a drawback for acetone/acetonitrile is
that saturated long-chain TGs are not sufficiently dis-
solved. TGs up to tripalmitin can be analysed properly
with acetone/acetonitrile (70:30); the solubility for trisatu-
rated higher molecular mass compounds drops drasti-
cally in this mobile phase [119]. A further increase of the
acetone proportion concomitantly increases the solvent
strength of the mobile phase to a degree that separation
of early eluting substances is severely compromised. As
the solubility of long-chain TGs improves considerably at
elevated temperatures, programming of the column tem-
perature was proposed as a means to improve separation
of long-chain TG [94, 127, 141]. Chloroform, which is an
exceptionally good solvent for lipids, could replace ace-
tone [119]. n-Propionitrile is also a good alternative to
acetone/acetonitrile [88, 90, 111, 123, 127].
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632 M. Buchgraber et al. Eur. J. Lipid Sci. Technol. 106 (2004) 621–648

Tab. 4. RP-HPLC conditions used for the determination of the triacylglycerol profile of various matrices.

Sample Sample Column dimension Stationary phase Mobile Phase flow rate Detector Ref.
solvent (components) [mL/min]

Coconut oil THF; AC 25064.6 mm; 10 mm Partisil ODS-1; MeOH-AC-iProp-ACN RI [110]

octadecyl-silica
Coconut oil THF; AC 25064.6 mm; 10 mm Partisil ODS-2; MeOH-AC-iProp-ACN RI [110]
octadecyl-silica
Coconut oil THF; AC 25064.6 mm; 6–7 mm Zorbax-ODS; MeOH-AC-iProp-ACN RI [110]
octadecyl-silica
Coconut oil THF; AC 15064.6 mm; 5 mm LC-8; octyl-silica MeOH-AC-iProp-ACN RI [110]
Coconut oil THF; AC 15064.6 mm; 5 mm LC-1; methyl-silica MeOH-AC-iProp-ACN RI [110]
Coconut oil THF; AC 15064.6 mm; 5 mm LC-18; octadecyl-silica MeOH-AC-iProp-ACN RI [110]
ACN-Hex-iProp 15064.6 mm; 5 mm Nucleosil C18 0.8 UV 215 nm [122]
Cocoa butter Tol 25064 mm; 5 mm Lichrosorb RP18 PPN RI [123]
Palm oil, shea butter, sal PPN; Tol 26250 mm; 5 mm Lichrosorb RP18 PPN 0.5 RI [111]
fat, Dhupa oil, mango oil,
Illipe butter, cocoa but-
ter, coconut oil
cocoa butter, corn, cotton 25064.6 mm; 5 mm RSil C18 HL PPN RI [88]
seed, rapeseed, ha-
zelnut, safflower, soy-
bean, sunflower oils
Cocoa butter, palm, CHCl3 25064.6 mm; 5 mm Lichrosorb RP18 AC-ACN 1.5 [118]
linseed, olive, rapeseed
oils
Olive oil 25064.6 mm; 5 mm ODS Zorbax FID [124]
25064.6 mm; 5 mm ODS Zorbax FID [124]
6066.2 mm; 3 mm ODS Zorbax ACN-DCM FID [124]
Cocoa butter; soybean oil, CHCl3 25064.6 mm; 5 mm ODS Zorbax ACN-DCM FID [125]
olive oil
Butter AC 5064.6 mm 1 Spherisorb-5-ODS2 EtOH-ACN 1.5 ELSD [126]
25064.6 mm
Butter AC 5064.6 mm 1 Spherisorb-5-ODS2 AC-ACN 1.5 RI [126]
25064.6 mm
Butter AC 5064.6 mm 1 Spherisorb-5-ODS2 EtOH-ACN-Hex 1.5 UV 225 nm [126]
25064.6 mm
Soybean, coconut oils, Hex-iProp 25064.6 mm; 5 mm Lichrospher 100 RP18 EtOH-ACN 1 ELSD [113]
butter , standard TGs
Sunflower oil, linseed oil, CHCl3 25064 mm; 5 mm HIBAR Lichrospher 100 CH- AC-ACN 0.7 ELSD [119]
olive oil, horse fat 18/2
Olive, soybean, sunflower, 50064 mm; 5 mm Lichrosorb RP18 PPN 1 RI [90]
wheat germ oils
Soybean, sunflower, olive, PPN-ether 25064 mm; 5 mm Hibar RP18 PPN 0.6 [127]
palm, palm kernel, co-
conut oils, cocoa butter,
butter fat
Peanut oil AC 25064 mm; 3 mm HIBAR Lichrospher 100 AC-ACN 1.2 RI [117]
CH-18
Butter oils 2625064 mm; 5 mm LiChrospher 100 CH-18/2 ACN-AC FID [112]
Soybean oil Hex, PE, ChCl3, 15064.6 mm; 5 mm Spherisorb-5-ODS2 ACN-MTBE UV 215 nm [92]
MBTE
Soybean oil, olive oil, DCM-ACN 15064.6 mm; 3 mm Spherisorb ODS II DCM-ACN 0.6 ELSD [91]
cocoa butter, cocoa
butter equivalents
Soybean oil, corn oil DCM-ACN various columns DCM-ACN 0.6 ELSD [128]
Mix of interesterified palm- 25064–6 mm; 5 mm Spherisorb ODS II ACN-AC 0.6–1.6 RI [93]
palm kernel oil, coconut
oil, linseed oil

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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648 Triacylglycerol profiling 633

Tab. 4. Continued.

Sample Sample Column dimension Stationary phase Mobile Phase flow rate Detector Ref.
solvent (components) [mL/min]

Corn oil, rapeseed oil, EtOH-iOct 25068 mm; 5 mm LiChrospher 100 RP18 ACN-EtOH-iOct ELSD [7]
soybean oil
Summer butter fat AC 25064 mm; 4 mm Lichrospher 100 RP18 AC-ACN 1 RI [94]
Olive, soybean, sunflower, 2650064.9 mm; 5 mm Zorbax C-18 ACN-DCM 0.8 FID [95]
corn, cottonseed, pump-
kinseed, peanut, saf-
flower, rapeseed, palm
oils
Human milk AC-ACN 25064.6 mm; 5 mm Supelcosil LC-18 AC-ACN RI [129]
Avocado CHCl3 2625064.6 mm; 3 mm Spherisorb ODS II AC-ACN 0.8 ELSD [120]
Soybean oil, milk fat CHCl3 25064.6 mm; 3 mm Spherisorb ODS II ACN-DCM, ACN-AC, 1.2 ELSD [96]
ACN-AC-MTBE
Olive oil, rapeseed oil AC 25064.6 mm; 5 mm Supelcosil LC-18 ACN-AC 1 RI [97]
Soybean, rapeseed, palm, EtOH-iOct 25064.6 mm; 5 mm Supersphere RP-100 ACN-iOct to ACN-EtOH-iOct 1.5 ELSD [98]
linseed, fish oils
Hydrogenated palm kernel 25064 mm; 4 mm Supersphere RP-18 ACN; iProp; ACN-iProp-Hex 1 ELSD [99]
and palm oil
Hydrogenated palm kernel 25064 mm; 4 mm Supersphere RP-18 ACN-iProp-Hex; EtOH-Hex 1 ELSD [99]
and palm oil
Seed oils Hex 2650064.9 mm; 5 mm Zorbax C-18 ACN-DCM 0.5 FID [130]
Olive oil 2506150 mm; 5 mm Supelcosil LC-18 AC-ACN 1.5 RI [131]
12564 mm; 3 mm Spherisorb ODS2 ACN-PPN 0.6 RI [132]
Evening primrose oil, CHCl3 25064.6 mm; 5 mm Supelcosil LC-18 ACN-iProp 1 UV 210 nm [100]
borage oil
Ewe, cow, goat milk fat Hex 250 1 15064.6 mm; Spherisorb ODS-2 AC-ACN 0.9 ELSD [133]
3 mm
30064 mm; 5 m 1 Wakosil 5C18N 1 AC-ACN 0.7 ELSD [134]
25064 mm; 4 mm Supersphere RP-18
Palm olein AC 25064 mm; 5 mm RP-18 AC-ACN 1 RI [101]
Synthetic TGs iProp//DCM 25064.6 mm; 5 mm Ultrasphere C18 MeOH-iProp 1 PDA 205 nm [102]
Sardine fish oil Hex 25064.6 mm; 3 mm Spherisorb ODS2 AC-ACN 1 ELSD [103]
Soybean oil 2625064.6 mm; 5 mm Inertsil ODS-80A ACN-DCM 0.6 ELSD; FID [104]
19 almond cultivars 25064.6 mm; 5 mm Simmetry C18 AC-ACN 1.5 RI [135]
Olive oil, homogeneous CHCl3-iProp 24464 mm; 5 mm Supersphere 100 RP-18 ACN-iProp-Hex; EtOH-Hex 0.8 DAD-ELSD [121]
reference TGs
AC 15063.9 mm; 10 mm Bondapak C18 ACN-AC 0.5 RI [106]
Coffee bean oil AC 25064 mm; 4 mm Supersphere 100 RP-18 ACN-AC 1 RI [136]
2610063 mm; 3 mm Inertsil ODS ACN-AC 0.43 ELSD [137]

Abbreviations: THF – Tetrahydrofurane; MeOH – Methanol; AC – Acetone; iProp – iso-Propanol; ACN – Acetonitrile; Hex –
Hexane; PPN – Propionitrile; Tol – Toluol; ChCl3 – Chloroform; DCM – Dichloromethane; EtOH – Ethanol; PE – Petroleum-
ether; MBTE – Methylbutylether; iOct – iso-Octane.

A mobile phase gradient, where the proportion of the
organic modifier is increased to vary the solvent strength
in a predetermined way (linear, concave, etc), is nowa-
days the preferred option for attaining optimal resolution
of TGs by RP-HPLC. However, acetone and chlorinated
solvents can only be used in isocratic mode even when a
UV detector is employed. Both show strong absorbance
at wavelengths where TGs are detected. In such circum-
stances other modifiers, i.e. ethanol [113, 126], methyl-
butylether [92], n-propionitrile [132], or iso-propanol [100]
have been proposed. The choice of the mobile phase
depends on its compatibility with the available detection
mode.
Many published methods employ RI detectors and iso-
cratic solvent systems [88, 90, 94, 97, 111, 117, 126, 129,
132, 135, 140]. RP-HPLC using acetone/acetonitrile as
mobile phase and RI detection was found to be an effec-

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634 M. Buchgraber et al.
tive system for the resolution of the TG mixture repre-
sented by different vegetable and animal fats. However,
total time of analysis was about 90 min [142]. Although the
RI detector is nearly universal, it does have the drawback
of low sensitivity and of different response towards satu-
rated and highly unsaturated TGs. The most significant
disadvantage of the RI detector is the fact that solvent
gradients cannot be used and thus optimum separation is
not always achieved [143, 144]. For simple oils composed
of only a few TG species, separation is easily achieved by
using isocratic elution; for more complex mixtures, such
as milk fat, the need for gradient elution limits the choice
of the detector. The use of a UV detector permits gradient
elution [100], although some baseline drift can occur in
the absorption region used for lipids [145]. Herslöf [122]
reported that in many cases an enhanced sensitivity
compared to the RI detector could be expected. Meas-
urements should be carried out in the region where the
ester chromophore absorbs, i.e. 200–230 nm. However,
as the contribution of double bonds to the optical density
is predominant at low wavelengths the column effluent
has to be monitored at 220 nm to avoid interferences from
(poly) unsaturated TGs [146]. Another shortcoming of the
UV detector is that many suitable solvents such as
chloroform, acetone, ethylacetate or toluene cannot be
used since they absorb strongly between 200 and 220 nm,
which overlaps with the detection region of TGs. Some of
the disadvantages of using an UV-detector can be avoi-
ded by brominating the TGs prior to their separation. In
particular, bromination leads to a better separation of the
monounsaturated TGs from the di- and poly-unsaturated
TGs [88].
Another type of detector that can be used with mobile
phase gradients is the transport FID. The eluent from an
HPLC column reaches a moving wire or belt that is
passed into the flame ionisation chamber after evapora-
tion of the solvent. Phillips et al. [124, 125] used the FID for
the quantification of TGs in cocoa butter, soybean oil, and
olive oil. Differences in the response of individual species
were small and did not require the use of response fac-
tors. Those findings were not confirmed by Nurmela and
Satama [112], who found a variable response for different
TGs, although the variation was less than with UV detec-
tion. The detection limit for the FID was in the ng range.
Even though good sensitivity and baseline stability was
reported for this type of detector [95], it is no longer
commercially available.
A detection method that is not affected by changes in the
mobile phase composition, thus permitting a wider range
of solvent programming, is the evaporative light scatter-
ing detector (ELSD) [147]. The ELSD detects solutes on
the basis of light scattering, after nebulisation of the elu-
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
ate and removal of the solvent by evaporation. It is com-
patible with gradient elution and gives no baseline drift for
non-aqueous volatile solvents [126]. Carelli and Cert [76]
showed that HPLC using solvent gradient and ELSD is an
appropriate technique for the separation of oils containing
a complex TG mixture. The main limitations are that (i) the
sample must be less volatile than the solvent and (ii) the
lack of linearity [113, 147]. The latter seems to be related
to the droplet size inhomogeneity, which depends on
surface tension, viscosity, density and flow-rate of the
mobile phase; it also depends on the nebuliser gas flow-
rate and on the geometry of the nebuliser. Quantification
may represent a problem, since the response function is
not rectilinear at very low or high concentrations. In newer
and improved versions of this type of detector this short-
coming has been alleviated. In a study dealing with avo-
cado TGs the ELSD response was linearly related to the
amount of sample injected over a range of 10–70 mg [120].
Buchgraber et al. [50] reported that by optimising the
instrumental parameters carefully and setting up proper
calibration conditions reliable quantitative results can be
obtained for cocoa butter TGs. In order to proof authen-
ticity of cocoa butter Anklam et al. [148] employed HPLC
equipped with an ELSD. HPLC with an acetonitrile/
dichloromethane gradient and ELSD gave excellent
separations of TGs of animal and vegetable fats [149].
Neff et al. [104] used a newer version of the ELSD and
investigated it as a quantitative HPLC detector for TG
analysis of oils from genetically modified soybean vari-
eties. The ELSD results were compared to those obtained
using a moving belt FID. They demonstrated that the
ELSD produced quantitative results without the need for
response factors of individual TGs. They observed that
the ELSD gave a linear response for sample masses of
soybean oil TGs over the range of 10–50 mg per HPLC
injection. The ELSD proved also its usefulness for the
study of structured lipids (mixed medium long-chain TGs)
[137].
A RP-HPLC method using RI detection has been collab-
oratively tested using various vegetable oils as test
samples and the procedure was standardised as IUPAC
method 2.324 [150]. Quantitative results had to be
reported for ECN 40–50 for sunflower oil and ECN 42–50
for olive oil. The relative standard deviation under re-
producibility conditions varied to a large extent
with maximum values of 92.6% for ECN 50 (mean value
1.3% m/m) in sunflower oil and 21.1% for ECN 42 (mean
0.6% m/m) in olive oil down to 2.4% for ECN 46 (mean
22.5% m/m) in sunflower oil and 1.6% for ECN 46 (mean
21.2% m/m) and ECN 48 (mean 64.7% m/m). It was
concluded that those results were acceptable and one
possibility of application of the method is the detection
of adulteration.
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
2.5 Thin-layer chromatography
In TLC the stationary phase is coated onto glass or a
plastic plate. The sample, either liquid or dissolved in a
volatile solvent, is deposited as a spot on the stationary
phase. Mostly, silica gel is used as an adsorbent. Since
TLC is a very simple and cheap technique it has been the
basic separation method used by lipid chemists for the
analysis of TG for many years. In principle, there are three
main modes in usage, i.e., NP-TLC, RP-TLC and silver-
ion TLC. In principle, the same separation mechanisms,
elution solvents, and elution orders are followed as for the
respective HPLC modes.
2.6 Normal-phase TLC
Normal-phase TLC separates lipid classes according to
their overall polarity and is still in use to isolate and purify
TG fractions of natural lipid mixtures [151–153]. In order to
obtain a more accurate estimate of the TG composition of
cow milk Breckenridge and Kuksis [154] used NP-TLC for
the pre-separation of milk fat TGs, which yielded 3 dis-
tinct fractions. Total TGs of milk fat were separated into
high, medium and low molecular mass TGs on the basis
of the average molecular mass of the constituent FA. This
observation was confirmed by Arumughan and Naraya-
nan [155], who separated buffalo milk fat by TLC [156].
The working group of Steele showed that separation of
hydrogenated milk fat by TLC also yielded 3 fractions
[157, 158]. Many solvent systems and qualitative visuali-
sation reagents are available for NP-TLC, with the actual
selection depending largely on personal preference.
2.7 Reversed-phase TLC
The working group of Kaufmann [159, 160] has introduced
RP-TLC into TG analysis in which TGs are distributed be-
tween a non-polar stationary phase and a relatively polar
mobile phase. Reversed-phase TLC separates TG classes
into groups according to their overall polarity, resulting in
separations according to CN, in the same way as RP-
HPLC. Therefore, RP-TLC is only occasionally used
nowadays and has largely been supplanted by RP-HPLC.
The lipophilic stationary phase is held on a layer of an inert
support, i.e., either silica gel or Kieselguhr. Earlier TLC
plates were impregnated with paraffin oil [161], whereas
improved RP-TLC plates used RP-18 or RP-8 plates. RP-
TLC is less efficient for the separation of critical pairs
compared to RP- HPLC.
As TGs lack chromogenic groups, visualisation and
quantification is a crucial point. Several staining reagents
such as rhodamine B [159], iodine vapors [159], iodine-a-
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Triacylglycerol profiling 635
cyclodextrine [162, 163] or phosphomolybdic acid [164]
were not suitable for densitometric quantification. Since
charring is most often used for direct densitometric
determination of TGs it is impossible to use long-chain
hydrocarbons and liquid paraffin as stationary phase. By
treating a silica gel or Kieselguhr layer with dimethyldi-
chlorsilane a lipophilic C2 layer is obtained, which allows
the use of charring reagents. Reference TGs were suc-
cessfully separated and could be visualised by treating
with charring reagents and carbonisation via heating.
Recently, a detailed overview regarding the application of
quantitative RP-TLC to TG analysis was given by Niko-
lova-Damyanova [165–167].
2.8 Silver-ion chromatography
Silver-ion chromatography, also dubbed argentation
chromatography, utilises the property that silver ions form
reversible complexes with p-electrons of unsaturated
centres in organic molecules, enabling separation
according to the number, the geometrical configuration
and the position of double bonds in a molecule. The
complexes are of the charge-transfer type in which the
unsaturated compound acts as an electron donor and the
silver ion as the electron acceptor [168, 169]. They are
formed transiently and are in kinetic equilibrium with the
native olefin.
TGs are separated according to the overall degree of
unsaturation of the constituent FAs [170, 171]. A TG mix-
ture made up of FAs with 0–3 cis double bonds can be
separated into 20 unsaturation classes in the following
order of chromatographic mobilities:
SSS.SSM.SMM.SSD.MMM.SMD.MMD.SDD.
SST.MDD.SMT.MMT.DDD.SDT.MDT.DDT.STT
.MTT.DTT.TTT
where S, M, D, and T denote saturated-, monoenoic-,
dienoic-, and trienoic-acyl residues [165, 170–172]. The
position of the residue in the TG molecule is only indica-
tive and does not follow the stereospecific numbering
convention of TGs. In the case of TG classes with an
equal number of double bonds in the molecule, e.g. SMM
and SSD, the latter, where the double bonds are within a
single acyl moiety, will have a lower mobility [170].
In most applications silica gel or cation-exchange mate-
rial has been used to immobilise the silver ions to serve as
stationary phase either in TLC or in HPLC.
Addition of AgNO3 to silica gel slurry and spreading this
on a TLC glass plate is probably the simplest variant of
silver-ion chromatography. Moreover, this is the only
technique where the concentration of silver ions in the
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636 M. Buchgraber et al.
stationary phase can be controlled exactly. However,
above a certain minimum, the Ag1 concentration does not
seem to influence the separation characteristics pro-
foundly. In earlier reports, a silver content of 10–30% has
been considered essential for good resolution [173].
However, such plates are very sensitive to light and this
greatly hampers detection; in situ quantification is practi-
cally impossible with such plates. Today, the impregna-
tion with high percentages of silver salts is rarely used. It
has been shown that contents higher than 5% in the layer
have no effect on retention and resolution [165]. The res-
olution is much more influenced by the choice of the
mobile phase.
Pre-coated silica gel plates may be impregnated either by
dipping in a AgNO3 solution or by spraying such a solution
evenly onto the plates [174, 175]. Very efficient plates
have been obtained by a dynamic impregnation tech-
nique, where the pre-coated plate is developed in aceto-
nitrile containing AgNO3 and also a fluorescence dye
(phloxin) [176]. This indicator is immobile when the plate is
developed in n-hexan/toluene and acts like a fluorescent
ion exchanger in the Ag1-form, thereby not only facilitat-
ing separation, but also detection of separated com-
pounds when viewed under UV light.
Several developing systems have been used for separat-
ing TGs. Components with up to 6 double bonds were
well separated with 1% methanol in chloroform [173].
Resolution of TG mixtures containing more highly unsa-
turated species was improved when adding 5% methanol
to chloroform.
Silver-ion TLC has been used either on its own or mainly
for preparative isolation of fractions for subsequent eluci-
dation of the structure of oils and fats. For in situ quantifi-
cation the separated TG classes have to be visualised,
mostly by charring, followed by densitometric evaluation
[177].
The combination of silver-ion TLC and RP-TLC has been
successfully applied to unravel the TG composition of
many vegetable oils such as sunflower [178], olive, pea-
nut [167], corn, cotton seed [172], soybean or linseed oils
[179]. Many other examples where this strategy has been
followed were reviewed by Dobson et al. [180].
A main problem in applying HPLC for separations in the
silver-ion mode has been the loading of the column in
order to prepare a stable system with minimal or no
bleeding of silver ions. Columns either containing silica
gel impregnated with silver nitrate or those based on sil-
ver ions attached to cation exchangers have been used.
Silica gel columns impregnated with silver nitrate were
the first to be developed. These columns are not com-
mercially available, and much practice and skill are
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
required for their preparation [181–183]. A drawback with
this type of column is the release of silver ions by the sta-
tionary phase, limiting the lifetime of the column. More-
over, the frequent formation of corrosive silver nitrate
precipitates may damage the tubing and detection sys-
tem. Nevertheless, several working groups reported good
separations of cocoa butter, palm oil or soybean TGs
using AgNO3 impregnated silica HPLC columns [184,
185]. In principle, TGs were separated according to their
degree of unsaturation (vide supra).
One of the most useful and efficient methods available for
silver-ion HPLC has been developed by Christie [168]. He
loaded a silica-based ion-exchange HPLC column
(chemically-bonded alkylphenylsulphonic acid groups) in
situ with silver ions via the injector while pumping water
through it, before replacing the aqueous phase with
organic solvents. The advantage of silver ions attached to
cation exchanger columns is that leaching of silver ions
does not occur because the silver is held by ionic bonds.
These columns are characterised by long life-times and
reproducible separations. Meanwhile ready-made col-
umns are commercially available (ChromSpher Lipids).
In cation-exchange silver-ion HPLC TGs are separated
and eluted in a similar order as in Ag-TLC. Analogous to
silver-ion TLC elution is in ascending order according of
degree of unsaturation, with stronger retention of one
dienoic residues than 2 monoenoic residues within the
same TG molecule and equal retention of a trienoic resi-
due as of 2 dienoic residues. Retention factors were
found to increase stepwise, with a saturated di-mono-
enoic TG species (SMM) being retained 106 and trilino-
lenin 10,0006 more strongly than a di-saturated mono-
enoic species (SSM). Formation of chelate-type p com-
plexes between silver ions and double bonds, with
participation of the carbonyl oxygen in the complexation,
have been assumed to explain the separation mechanism
on silver-ion loaded cation-exchange HPLC columns
[186].
The composition of the mobile phase can have a decisive
influence on TG separation. A binary gradient of acetone
into dichloromethane/1,2-dichloroethane was sufficient
to separate TGs of fats containing small proportions of
dienoic FAs such as sheep adipose tissue [169] and palm
oil [168]. A TG separation of fats and oils containing a
higher proportion of linoleic or linolenic acid such as rat
adipose tissue, sunflower seed oil, linseed oil [169], mea-
dowfoam oil [187], evening primrose oil [188], or olive oil
[189, 190] was accomplished using a ternary gradient
system consisting of dichloromethane-1,2-dichloro-
ethane/acetone/acetone-acetonitrile. In addition, fish oils
could be well separated up to MMD using the same
method. However, TG species with up to 14 double
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648 Triacylglycerol profiling 637

Tab. 5. Silver ion HPLC conditions used for the determination of the triacylglycerol profile of various matrices.

Sample Stationary phase Column dimension Mobile phase flow rate Detection Ref.
[mL/min]

Cocoa butter, palm oil, lard ODS 3 mm 2615064.6 mm Bz (isocratic) 1.5 IR [199]
Sunflower, safflower, linseed, evening primrose, Nucleosil 5SA 25064.6 mm DCE-DCM; AC; AC-ACN 1 MD [169]
coconut oils, sheep fat, fish oil (various gradients)
Evening primrose oil Nucleosil 5SA 25064.6 mm DCE-DCM; AC; AC-ACN 1 MD [188]
(ternary gradient)
Anhydrous butterfat Nucleosil 5SA 25064.6 mm DCM-DCE; AC (binary 1 MD [114]
gradient)
Partially hydrogenated soybean and palm oils Lichrosorb RP18 25064 mm; 5 mm MeOH-iProp (gradient) 1 RI [194]
Vegetable oils Chromosphere 25064.6 mm; 5 mm Hex-ACN (isocratic) 1 FID [193]
Olive, sunflower, linseed oils Nucleosil 5SA 25064.6 mm DCE-DCM-ACN/Ac ELSD [186]
(isocratic)
TG positional isomers Chromosphere 2625064.6 mm; 5 mm Hex-ACN (isocratic) 1 UV [196]
TG positional isomers Chrompack 25064.6 mm Hex-iProp-EA; Hex-iProp- 0.8 ELSD [200]
ACN (gradient)
Partially hydrogenated rapeseed and palm oils, ChromSphere 25062 mm; 5 mm Hep-ACN 1000:1; Hep-ACN 1 ELSD [195]
TG standards 100:1 (gradient)
CLA enriched TG formulation ChromSphere 2625064.6 mm; 5 mm Hex-ACN (isocratic) 1–2 UV [198]
Symmetrical and non-symmetrical TGs ChromSphere 2625064.6 mm; 5 mm Hex-ACN 1 UV [197]

Abbreviations: Bz – Benzene; DCE – Dichloroethane; DCM – Dichloromethane; Ac – Acetone; ACN – Acetonitrile; MeOH –
Methanol; iProp – iso-Propanol; EA – Ethylacetate; Hex – Hexane; Hep – Heptane; MD – mass detector.

bonds, containing eicosapentaenoic and docosahex-
aenoic acids, co-eluted because of the presence of too
many positional isomers [191, 192].
Silver-ion HPLC is not restricted to gradient operation to
achieve useful separation of TGs. TGs from a number of
commodities including saturated fats such as coconut as
well as highly unsaturated oils such as linseed were suffi-
ciently separated when operated in isocratic mode (0.5%
acetonitrile in n-hexane) and quantitated by an FID [193];
retention times of the highly unsaturated TG fractions
were of course rather long (60–80 min).
Detailed chromatographic conditions of silver-ion HPLC
methods used in practice are listed in Tab. 5.
Silver-ion HPLC has the potential to separate geometrical
isomers of TGs. This type of separation is possible due to
the formation of weaker p-complexes of silver ions with
trans-olefins than with cis-olefins. Good separation of
hydrogenated/randomised palm fractions having a cis-
trans difference was demonstrated by Smith et al. [201]
and for partially hydrogenated rapeseed oil by Macher
and Holqvist [195]. Petersson et al. [194] described a
method for the separation and quantification of TGs in
partially hydrogenated fats rich in oleic, elaidic, palmitic
and stearic acid. Samples were first fractionated by RP-
HPLC according to partition number, collected and then
reinjected for a separation according to double bond ge-
ometry by RP-HPLC with silver ions in the mobile phase.
The method has been applied to a number of different
model TGs, and also to partially hydrogenated soybean
and palm oil. This approach was chosen to fine tune the
separation power of the system, as separation of posi-
tional isomers and symmetric/asymmetric TGs was
undesirable because this led to overlapped and broad-
ened peaks as well as to identification problems. Silver-
ion TLC is as efficient as its HPLC counterpart for the
analysis of TGs differing in the geometry of double bonds
[176, 177].
Over the past 25 years, a number of economic, health,
and consumer-driven factors have stimulated research
aimed at reducing the level of trans FAs formed during the
hydrogenation of vegetable oils. Alternatives to hydro-
genation include interesterification, blending of tropical
and liquid vegetable oils, fractionation, and, more
recently, development of structurally modified oils by
transgenic or conventional plant breeding methods.
Symmetrical disaturated TGs of the structure SUS (S –
saturated, U – unsaturated), are important components
providing functionality to vegetable butters (cocoa butter,
cocoa butter equivalents), interesterified fat blends and
structurally modified oils. Non-symmetrical TGs of the
structure SSU can significantly change the melting point
and solid fat content profiles. An Ag1-TLC procedure for
separation and quantification of TGs differing in the posi-
tion of unsaturated FAs on the glycerol backbone, such as
SMS-SSM, SMM-MSM, SDS-SSD, DSD-DDS and DMD-

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638 M. Buchgraber et al.
DDM, has been developed for a rapid determination of
confectionery fat quality [202]. Resolution was obtained
by continuous development with chloroform-methanol in
open tanks. Silver-ion HPLC was used with success for
the same purpose [168, 169, 196, 197, 200].
Silver-ion HPLC was found to be as accurate as, and
more rapid than, lipolysis/GLC for the determination of
the isomeric purities of synthesised TGs [197].
Recently, special attention has been attracted by the
regioselective separation of TGs with special emphasis
on structured species. A better understanding of the
regio-distribution of conjugated linoleic acid (CLA) iso-
mers containing TG formulations has potential applica-
tions for the correct labelling of CLA containing products
[203]. Adlof et al. [198] developed a method using silver-
ion HPLC and an isocratic solvent system to rapidly ana-
lyse CLA-enriched TG formulations.
Silver-ion chromatography is an indispensable tool for
getting a comprehensive picture about the TG composi-
tion of complex natural fats, such as milk fat, fish oil, etc.
No single chromatographic technique is powerful enough
to separate TGs down to the species level. The help-
fulness of silver-ion chromatography as a pre-fractiona-
tion technique of TGs prior to RP-HPLC, GLC and GLC-
MS studies has been appreciated by many working
groups [204–208].
2.9 Supercritical fluid chromatography
In supercritical fluid chromatography (SFC) a gas com-
pressed above its critical temperature and critical pres-
sure is used as mobile phase to elute analytes from a
chromatographic column. Supercritical fluids are char-
acterised by low viscosities and high diffusivities, thereby
shorting retention times of solutes. A further advantage of
this technique is that the universality of the FID can be
exploited for quantification, provided pure carbon dioxide
is used as the mobile phase. Laakso [209] has reviewed
the analysis of lipids by SFC. Many authors described the
use of capillary columns for the analysis of TGs by SFC
[210–215]. All those studies came to the conclusion that
even long-chain high-molecular mass TGs can be eluted
at moderate temperatures (,150 7C), thus eliminating the
problem of thermal degradation usually associated with
high temperature GLC of TG. Chester [216] published the
first report on capillary SFC in combination with FID for
the analysis of glycerides. Baseline separation of TGs
differing by 2 carbons was achieved by using a
12 m6100 mm fused silica column having a 0.10 mm film
of BP-10 (7%-cyanopropyl/7%-phenylmethyl-siloxane).
Carbon dioxide at 90 7C and linear pressure programming
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
from 19.3–23.7 MPa was used as mobile phase. Sta-
tionary phases widely differing in polarity, i.e. crosslinked
DB-5 and DB-225, were compared for their applicability
to capillary SFC of TGs [217]. On DB-5 TGs with the same
CN, i.e. SSS, OOO, LLL, LnLnLn, were not separated; on
DB-225, however, these 4 TGs did not elute in the order of
their molecular mass, but in order of increasing number of
double bonds and were well separated according to
degree of unsaturation. In principle, the elution order of
TG species is governed by the polarity of the stationary
phase, as in capillary GLC. On non-polar stationary pha-
ses TGs are separated into groups having the same
CN. Polar phases separate TG species according to their
CN and within each CN group according to the degree
of unsaturation. Use of a polar stationary phase (25%-
cyanopropyl/75%-methylpolysiloxane) even allowed
separation according to the position of double bonds in
the FA chain [218]. TGs containing either one a- or g-lino-
lenic acid residue were baseline resolved; the component
containing the g-isomer was eluted before the a-isomer.
The method was applied to the analysis of alpine currant
and black currant seed oils, giving more information on
the composition of TGs in the seed oils containing g-lino-
lenic acid.
One of the first reported applications of SFC for analysis
of glycerides was by Rawdon and Norris [219], who
separated soybean TGs by packed column SFC employ-
ing either carbon dioxide or carbon dioxide/methanol and
variable wavelength UV detection. Reports followed
describing the application of columns packed with C18 or
Ag1-cation exchange material for the analysis of standard
TG mixtures and vegetable oils [220–226]. Isocratic
modes, as well as pressure and temperature pro-
grammed modes were applied and UV, ELSD and MS
were used for detection. As is the case with RP-HPLC
methods, temperature influences to a large extent the
retention behaviour of TG in packed column SFC. It was
shown that retention of TGs increased with decreasing
temperature and that temperature was a more effective
parameter to change selectivity than the mobile phase
density [227]. Optimum separation results were achieved
at temperatures below the critical temperature of carbon
dioxide (31.3 7C). The retention behaviour of TGs was
studied in subcritical fluid chromatography at 15 MPa and
in the temperature range of 0–25 7C using a C18 column
and carbon dioxide as the mobile phase without modifier
[227]. The contributions of the CN and the number of
double bonds to the retention were similar to those in RP-
HPLC, which means that the concept of ECN can be
applied to subcritical fluid chromatography. This method
can be used to optimise the separation of various plant
oils [228]. A further study of the retention behaviour of
vegetable oil TGs was carried out in subcritical fluid
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
chromatography with acetonitrile/methanol modified car-
bon dioxide [229]. This study has highlighted the simi-
larities between RP-HPLC and subcritical FC using C18
stationary phases for TG analysis: (i) the retention order is
mainly determined by the partition number and (ii) for
compounds belonging to a series having the same total
chain length or the same partition number a linear rela-
tionship between retention behaviour and the unsatura-
tion number exists.
SFC may have some advantages over GLC and HPLC in
the analysis of TGs. In comparison to HPLC, SFC allows a
wide range of detectors to be used, including the uni-
versal FID. The fact that SFC separation of TGs is con-
ducted under mild conditions should be of advantage for
the analysis of highly unsaturated oils, which may
decompose at the high temperatures necessary to elute
them from GLC columns. However, the resolution of the
overall pattern is not as good as obtained by capillary
GLC [230] or HPLC [231]. Furthermore, the claimed
superiority of using SFC in combination with an FID for
obtaining quantitative TG data has not been underpinned
by experimental results. Although much acclaimed a
decade ago, capillary and packed column SFC is not
widely in use in lipid laboratories.
2.10 Stereospecific analysis
For many years a lot of research has been dedicated to
the development of analytical methods for determining
the distribution of FAs on the glycerol backbone of TGs.
Chromatographic methods per se fail to resolve TGs
according to the distribution of their FA moieties on the
glycerol backbone, with the exception of silver-ion chro-
matography, where limited resolution (SSU/SUS) is pos-
sible.
The simplest approach to regiospecific analysis is hydro-
lysis of TGs with pancreatic lipase, followed by isolation
of the 2-monoacyl glycerols and analysis by GLC of the
FA attached to the b-position [232–234]. This method
does not allow discriminating between positions sn-1 and
sn-3. Therefore, the proper name for this approach should
be regiospecific analysis of TG structure and not stereo-
specific analysis. The FA composition for positions sn-1
and sn-3 can be calculated by (results expressed as
mol-%):
[FA in sn-1 and sn-3] = (36[FA in intact TG] 2 [FA in sn-2])/2
Pancreatic lipase hydrolyses the primary ester bonds of
glycerides with little FA specificity. Some specificity is
seen only with fats and oils containing either poly-
unsaturated, long-chain FAs (e.g. docosahexenoic acid in
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Triacylglycerol profiling 639
fish oil TGs) or short-chain FAs (e.g. butyric acid in rumi-
nant milk fat TGs). With this exception, pancreatic lipo-
lysis can be used to determine the FA composition of the
sn-2 position of nearly all naturally occurring TGs.
For the stereospecific analysis of TGs the analyst has the
possibility choosing between three options:
(i) hydrolysis of phosphorylated diacylglycerols with
phospholipases,
(ii) separation of enantiomeric derivatives of partial glyc-
erides formed with an achiral reagent by a chiral sta-
tionary phases, and
(iii) separation of diastereomeric derivatives of partial
glycerides formed with a chiral reagent by an achiral sta-
tionary phases.
Nearly all methods are based on the formation, derivati-
sation and separation of diacyl-sn-glycerols. The first
stereospecific analysis of TG was reported in 1965 by
Brockerhoff [232]. It is an enzymatic procedure, which is
still in use although modifications and improvements have
been proposed [233–236]. In the original procedure an
equimolar mixture of 1,2- and 2,3-sn-diacylglycerols was
prepared via partial hydrolysis by chemical (Grignard
reagent) or enzymatic (pancreatic lipase) hydrolysis. Then
phosphatidylphenols produced by phosphorylation of the
purified 1,2- and 2,3-sn-diacylglycerols were hydrolysed
by the stereospecific phospholipase A2. The products
were a lysophosphatidylphenol that contained the FAs
originally present in position sn-1, unesterified FAs
released from sn-2 and the unchanged 2,3-diacyl-sn-
phosphatide, which is not recognised by the enzyme. The
FA composition of each fraction was determined after
transmethylation by GLC. Using the method introduced
by Brockerhoff [232] the distribution of FAs among the
sn-1, sn-2 and sn-3 positions of TGs from a number of
plant oils have been determined. Saturated FAs showed a
preference for external positions. Linoleic acid occurred
predominantly in the sn-2 position, as generally found in
vegetable oils [237]. Human colostrum and mature milk
TG were examined with particular attention to the location
of the very long-chain polyunsaturated FAs on the glyc-
erol backbone [238].
Phospholipase C may be used for the hydrolysis of the
synthesised phospholipids in the same way as phospho-
lipase A2 [235]. Via Grignard cleavage obtained rac-1,2-
diacylglycerols were converted to rac-phosphatidylcho-
lines by chemical means, and the 1,2-diacyl-sn-glycero-
3-phosphocholine was selectively hydrolysed by phos-
pholipase C. This allowed the separation of enantiomeric
1,2-diacyl-sn-glycerol from the remaining 2,3-diacyl-sn-
glycero-1-phosphocholine.
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640 M. Buchgraber et al.
Alternatively the 1,2- and 2,3-diacyl-sn-glycerol mixture
can be subjected to phosphorylation by diacylglycerol
kinase from E. coli to form sn-1,2-phosphatidic acids,
which can be separated from the unreacted 2,3-diacyl-
sn-glycerols by TLC on silica gel [239, 240].
The resolution of 1,2- and 2,3-diacyl-sn-glycerols, which
is the most critical step in stereospecific analysis of TGs,
may be accomplished by chromatography, either as 3,5-
dinitrophenylurethane (DNPU) derivatives on a chiral col-
umn or as diastereoisomers on a silica column.
In the first approach the stationary phase presents chiral
molecules that have been chemically bonded to a silica
gel support medium. A good separation of DNPU deriva-
tives of mono- and diacyl-sn-glycerols was achieved on a
chiral (S)-2-(4-chlorophenyl)isovaleroyl-D-phenylglycine
stationary phase (Sumipax OA-2100) chemically bonded
to aminopropyl silica support [241, 242]. Takagi and Ando
[243–245] described an alternative procedure for the
separation of enantiomeric 1- and 3-monoacyl-sn-glyc-
erols derivatives. In principle, TGs were converted to
monoacylglycerols using ethyl magnesium bromide. After
isolating the resulting monoacylglycerols by TLC on boric
acid impregnated silica gel plates the 1- and 3-monoacyl-
sn-glycerol fractions were separated using a Sumipax
OA-4100 column. The FA composition of the individual
fractions and of the original TGs was determined by GLC
and used to calculate the positional distribution on the
glycerol backbone. Thirty soybean germplasm lines were
analysed stereospecifically [246] by using the chiral col-
umn method of Takagi and Ando [243]. On a chiral Sumi-
pax OA-4100 stationary phase an improved resolution of
enantiomeric 1,2(2,3)-diacyl-sn-glycerols was obtained
[247, 248]. Saturated diacyl-sn-glycerol homologues with
total chain lengths differing by more than 6 carbon atoms
were completely separated. A further improvement in the
resolution of DNPU-diacyl-sn-glycerol enantiomers was
obtained on chiral columns using (R)-(1)-1-(1-naphtyl)
ethylamine (YMC-Pack A-KO3) chemically bonded to a
silica-gel support [249]. To avoid any overlap due to
chain-length differences, 3,5-DNPU derivatives of
1,2(2,3)-diacyl-sn-glycerols may be further separated on
a C18 column according to chain length and degree of
unsaturation prior to enantiomeric separation by chiral
phase HPLC [250].
In 1990 Laasko and Christie [251] demonstrated that a
base-line resolution of diastereomeric naphtylethyl ure-
thane derivatives of diacyl-sn-glycerols was obtained by
HPLC on a column of silica gel. Diacyl-sn-glycerols were
found to react completely with the (R)- or (S)-forms of
1-(1-naphtyl)ethyl isocyanate under the conditions
described without any acyl migration or racemisation of
the components. The TGs were subjected to partial
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
hydrolysis with a Grignard reagent (ethyl magnesium
bromide) and the products were converted to the (S)-(1)-
1-(1-naphtyl)ethyl urethane derivatives, which were
resolved into the diastereomeric forms by HPLC on 2
columns of silica gel (3 mm) in series and isocratic elution
with 0.5% 2-propanol in n-hexane. The compounds were
detected by UV spectroscopy at 280 nm. Under those
conditions, the diacyl-sn-glycerols derivatised with (S)-
(1)-1-(1-naphtyl)ethyl isocyanate eluted in ascending
order of the 1,3-, 1,2- and 2,3-enantiomers. The elution
order of 1,2- and 2,3-diastereomers was reversed when
the (R)-form of 1-(1-naphtyl)ethyl isocyanate was used for
derivatisation. The potential of the method was demon-
strated by analysing maize oil, evening primrose oil and
egg yolk TGs. The composition of FAs in the TGs were
calculated from the GLC analysis of the FAs of the intact
TGs and of the fractions of the isolated 1,2- and 2,3-dia-
cyl-sn-glycerol derivatives. The obtained results were in
close agreement with results published earlier [252].
Toschi et al. [190] carefully examined the TG composition
of olive oil samples by stereospecific analysis after partial
hydrolysis with ethyl magnesium bromide, derivatisation,
preparative chiral HPLC, transesterification, and GLC
quantification of FA methyl esters. The stereospecific
analysis, which has been successfully applied to different
kinds of edible oil [189, 253, 254], enabled the determi-
nation of the substituents at all positions of the glycerol
backbone.
Enzymatic as well as non-enzymatic methods for the
stereospecific analysis of olive oil were equivalent in
qualitative and quantitative terms as demonstrated by
Damiani et al. [239]. One method was based on the for-
mation of phosphatidic acids via diacylglycerol kinase
and the other on formation of diastereomeric naphtylethyl
urethane derivatives for the separation of sn-1,2(2,3)-
diacylglycerols obtained after Grignard hydrolysis.
3 Conclusion
The progress in chromatographic techniques has enor-
mously facilitated the analysis of TGs. TGs of edible oils
and fats have been successfully analysed by capillary
GLC, HPLC, and SFC using various detection systems. In
the present review emphasis was put on detection sys-
tems widely used in laboratories, i.e., FID, UV, RI, and
ELSD. In this paper no attention was given to MS tech-
niques which are probably less suitable for everyday rou-
tine use.
Certain chromatographic techniques which are in use for
the analysis of TGs have distinct advantages but also
disadvantages depending on the objective of the analy-
sis. The major advantage of HPLC is that it is possible to
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Eur. J. Lipid Sci. Technol. 106 (2004) 621–648
separate TGs at ambient or slightly elevated tempera-
tures, thereby obviating thermal stress on thermolabile
long-chain polyunsaturated TGs. However, high molecu-
lar mass TGs are not easy to elute from HPLC columns
due to their insolubility in a number of popular mobile
phases. A further disadvantage is that commonly avail-
able HPLC detectors are only compatible with isocratic
elution (RI detector) or the detector response is influenced
by the unsaturation of the separated substances (UV
detector), which renders quantification unreliable. Today,
GLC using capillary columns coated with high-tempera-
ture polarisable phenylmethylsilicone stationary phases
has been shown to be as effective as RP-HPLC for the
separation and quantification of saturated and mono-
unsaturated TGs. In comparison to HPLC, capillary GLC
yields higher resolution, but application of carefully
determined detector response factors is necessary to
obtain reliable quantification; this is especially true for the
analysis of oils containing polyunsaturated TGs. Both
techniques have been demonstrated as being suitable for
TG analysis of very complex TG mixtures as represented
by animal fats, in particular bovine milk fat and fish oil. To
date, complete resolution of any molecular species is not
attainable, neither by capillary GLC nor RP-HPLC. For
this reason, the complementary use of those 2 main
techniques seems to be advisable. A combination of
chromatographic methods is necessary for a complete
separation of TG species. Silver-ion chromatography
provides information on the degree and the distribution of
the unsaturation and information on symmetry. It is an
especially useful technique when used in combination
with other methods of separation.
Since dietary lipids are absorbed mainly as free FAs or as
sn-2-monoacylglycerols resulting from the action of li-
pases, the FA position in the glycerol backbone is impor-
tant for physiological and nutritional reasons. Conse-
quently, for complete structural analysis of triacyl-sn-gly-
cerol complementary enzymatic and chemical methods
have to be applied. The obtained data can be used to
detect adulteration or alteration of oils, or to tentatively
elucidate the mechanism of sn-TG biosynthesis.
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[Received: April 21, 2004; accepted: June 29, 2004]
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