FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2008; 23: 297–314

Published online 19 August 2008 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/ffj.1887

Linear retention indices in gas chromatographic

John Wiley & Sons, Ltd.

analysis: a review
Barbara d’Acampora Zellner,1 Carlo Bicchi,3 Paola Dugo,4 Patrizia Rubiolo,3
Linear retention indices in gas chromatographic analysis

Giovanni Dugo1 and Luigi Mondello1,2*

1
Dipartimento Farmaco-chimico, Facoltà di Farmacia, Università di Messina, Viale Annunziata, 98168 Messina, Italy
2
Campus-Biomedico, Via Alvaro del Portillo 21, 00128 Roma, Italy
3
Dipartimento di Scienza e Tecnologia del Farmaco, Facoltà di Farmacia, Università di Torino, Via Pietro Giuria 9, 10125
Torino, Italy
4
Dipartimento di Scienze degli Alimenti e dell’Ambiente, Facoltà di Scienze MM.FF.NN., Università di Messina, Salita
Sperone, 98166 Messina, Italy
Received 9 January 2008; Revised 10 May 2008; Accepted 22 May 2008

ABSTRACT: The main purpose of any chromatographic analysis is to resolve mixtures of compounds into less complex
mixtures or ultimately into pure components. In addition to this function, the chromatographic system can provide retention
data which serve as complementary information for the positive identification of resolved components. The need to express
gas chromatographic retention data in a standardized system has long been recognized and retention index values
presented to be a valuable parameter. Those values are mainly calculated by applying the equations proposed by Kováts,
for isothermal analysis, and van den Dool and Kratz, for programmed gas chromatographic runs. In general, these indices
denote the retention behaviour of the compounds of interest according to a uniform scale determined by a series of
closely related standard substances. The use of retention indices in the flavour and fragrance field is well-documented,
and they are widely applied for the comparison of results between laboratories, as well as to characterize stationary
phases. Copyright © 2008 John Wiley & Sons, Ltd.

KEY WORDS: gas chromatography; gas chromatography/mass spectrometry; retention index; linear retention index; flavour
and fragrance materials

Introduction must be added that as gas chromatographic separations

Since the early days of gas chromatography (GC) an
enormous effort has been devoted to the standardization
of the methods used to determine retention data, with the
main target being a wider and reliable use of collated
published results for identification purposes. Already in
1952, in their first publication on gas–liquid chromato-
graphy, A. T. James and A. J. P. Martin1 mentioned that
the retention volume of a pure substance on a certain gas
chromatographic column is a characteristic value which
could be used for the identification of sample components.
Further, in 1955, Littlewood et al.2 introduced the specific
retention volume as an attempt to make retention data
independent of temperature, stationary phase film thickness,
carrier gas flow rate and pressure drop across the column;
however, this parameter was not practically applied. From
the many proposals a general consensus has emerged
favouring the use of relative retention data expressed
with respect to the retention of a standard substance. It
* Correspondence to: L. Mondello, Dipartimento Farmaco-chimico, Facoltà
di Farmacia, Università di Messina, Viale Annunziata, 98168 Messina, Italy.
E-mail: lmondello@unime.it
are performed over a wide range of temperatures and on
stationary phases of different polarities, no single sub-
stance would fulfil the role of a universal standard.
Moreover, when the retention of the components of a
sample and the standard are grossly different, accuracy
would be impaired.
In general, the most thoroughly studied, diffused and
accepted retention index calculation methods are based on
the equation developed by Kováts in 19583 for isothermal
conditions, and on the equation propounded by van den
Dool and Kratz in 1963,4 used in the case of temperature-
programming conditions. Values calculated using the
latter approach are commonly denominated in literature
as retention index (I), linear retention index (LRI) or
programmed-temperature retention index (PTRI or IT), while
values derived from the former equation are usually
called retention index (I ) or Kováts index (KI). Retention
index calculations are applied to volatile compounds
present in numerous extracts, such as flavour and fragrance
materials, foods, drugs, petrochemicals, environmental
samples, etc.
Nowadays the use of temperature-programmed GC is
adopted in a wide range of applications. Considering that

Copyright © 2008 John Wiley & Sons, Ltd.

298 B. D’ACAMPORA ZELLNER ET AL.
on a given capillary column stationary phase, the operational
variables, such as carrier gas flow-rate, stationary phase
film thickness and linear temperature programming, can
alter an analyte’s retention time, the problems of retention
index reproducibility between laboratories may still
represent a drawback. In spite of the considerable instru-
mental advances made, the acquisition of reliable retention
index values may still be arguable. When temperature
programmes are applied with a constant heating rate
during the entire analysis, the calculated retention indices
are reliable. However, when isothermal steps are included
in the temperature programme or the rate is changed
during the analysis, the calculation of the indices is more
troublesome.5–7 It is worthy of note that discrepancies
may arise from the fact that the retention index values are
reported by many different laboratories and the operating
conditions are not always reliable. Moreover, it should be
pointed out that differences may also arise from the use
of columns coated with identical stationary phase but
manufactured by distinct companies.
During the 1980s in particular, various aspects of the
retention index system were the subject of a great number
of studies and several criteria of retention index systems
were, and are still, being developed to improve their
reliability. With time, numerous linear relationships between
the retention index and other fundamental properties,
such as boiling point,8 carbon number9 and molecular
weight10,11 were derived. The prediction of programmed
temperature retention indices is also an in-depth discussed
subject,12 involving the calculation and theoretical deter-
mination of the elution order of many compounds.
Retention index systems have been exhaustively reviewed
by Budahegyi et al.6 and Tarján et al.7
The present contribution provides an overview of the
theoretical aspects and applications of retention indices
to gas chromatographic analysis. It is certain that the
references cited in this review represent a very small part
of the studies performed on retention index theories and
their applications. Gas chromatographic retention para-
meters are briefly described; followed by retention index
calculations and systems. The application of indices to the
Rohrschneider and McReynolds phase constant determi-
nations and compound identification in flavour and
fragrance analysis are reported. Furthermore, a critical
viewpoint is discussed with respect to the indiscriminate
use of retention indices.
Principles of Gas Chromatographic
Retention Parameters
As is well known, the retention of analytes in gas chro-
matographic capillary columns results from the differential
distribution (partition) of the solutes between the stationary
liquid and the mobile gas phases.13 Solute retention in the
column, and also their resolution, are a consequence of the
Copyright © 2008 John Wiley & Sons, Ltd.
solution and dissolution process of the solute molecules
inside and outside the stationary liquid phase. When
considering the average gas linear velocity as a constant
throughout an analysis, the solutes should spend an
identical period of time in the mobile phase to elute from
the column. To this extent, only the differences in the
time spent in the stationary phase are responsible for the
solutes distinct retention time values.
The time that any solute spends in the mobile phase,
that is the unretained peak time (tM), summed with the so-
called adjusted retention time ( t′R ), which corresponds to the
time the solute spends distributed into the stationary phase,
is equivalent to the retention time (tR) [see equation (1)]:
tR = tM + t′R (1)
The magnitude of retention depends on the partition
coefficient, or distribution constant (K), which is defined
as the ratio of the equilibrium concentrations of a solute
in the stationary (cs) and mobile phases (cm) during parti-
tioning in the column [refer to equation (2)]:
cs
KD = (2)
cm
Hence the greater is the K value for a sample component,
the higher is its solubility and the longer its retention in
the stationary phase.
In general, the affinity of a solute for the stationary phase
depends on its vapour pressure and the activity coefficient
of the solute in that phase. Differences between those two
properties result in the column’s ability to differentiate
two solutes, eluting them with different retention times.
Solute retention can also be expressed in terms of
retention factor, k, which quantifies the ratio of the time
spent in the stationary phase and that spent in the mobile
phase, as presented in equation (3). In practice, the latter
parameter is more utilized in association to column
efficiency calculations:
t′R
k= (3)
tM
A further parameter is the relative retention (r), which
expresses the degree of separation between two peaks,
not necessarily in adjacent positions; one represents a
standard (st) and the other the solute of interest. Relative
retention can be expressed using standard solute retention
factors or adjusted retention times. The latter are used in
equation (4), where i refers to an individual solute. The
relative retention of solutes eluting after the standard will
be >1, while that of compounds eluting prior to the
standard <1 (see Figure 1):
t′Ri
ri = (4)
t′R(st )
Also worthy of mention is the retention volume (VR),
which can be measured experimentally from the mobile
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 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 299
Figure 1. Relative retentions of multiple solutes
phase volumetric flow rate (Fc) and the retention time, as
shown in equation (5):
VR = t R ⋅ FC (5)
However, since tR can be measured directly, its application
is preferred over VR.
Retention Index Theories
A compound’s retention behaviour on a specific column
is characterized by the three parameters presented in the
previous section: retention time, retention factor and
relative retention. In general, the retention time is influ-
enced by the applied linear velocity, temperature, phase
ratio and column length. As a consequence, retention
time is not useful for peak identification. Compared to
retention time, retention factor presents an advantage due
to the inclusion of the unretained peak time, contouring
changes caused by linear velocity discrepancies and
column length differences. On the other hand, relative
retention depends also on the phase ratio, so that the
results are comparable as long as an identical reference
peak is chosen, and the same column temperature is
applied. However, the accuracy of this information can be
degraded for peaks eluting far from the reference peak.
In order to overcome this limit, É. Kováts3 introduced
a retention index (I) system in which a homologous
series of paraffins arranged in straight chains (normal
paraffins) were applied as reference peaks. As is well-
known, this homologous series elutes, when isothermal GC
conditions are applied, with retention times increasing
exponentially. Under such conditions a semilogarithmic
relationship exists between the adjusted retention times
( t′Ri ) of the n-paraffins and their carbon numbers (cn); a
and b are constants [see equation (6)]:
log t′Ri = a ⋅ cn ⋅ b (6)
Copyright © 2008 John Wiley & Sons, Ltd.
This system was based on the fact that each analyte is
referenced in terms of its position between the two n-
paraffins that bracket its retention time. Furthermore, the
index calculation is based on a linear interpolation of the
carbon chain length of the two bracketing paraffins. In
the original definition, the retention index of a particular
substance was calculated using only n-paraffins with even
carbon atoms as references [as represented in equation
(7)]:
Isst. ph. (T ) = 200(log Xs − log Xz /
(7)
log X( z +2) − log Xz ) + 100 z
where I is the isothermal retention index at temperature
T, s is the compound of interest, st.ph. is the stationary
phase, and X is, for instance, the retention time used for
the calculation, while z and z + 2 are n-alkanes with z and
z + 2 carbon numbers, respectively. By definition, the
retention index of the n-paraffins is equal to 100 times
their carbon number for any given stationary phase and at
any given column temperature, e.g. n-C6 has an index of
600.
At that time, the use of even-number n-paraffins was
justified by the possible occurrence of an oscillation in
the chromatographic properties of successive numbers of
the complete n-paraffin series. However, this statement
was experimentally confirmed to be erroneous and
Kováts redefined the first proposed fundamental equation
to:
Isst. ph. (T ) = 100[z + (log Xs − log Xz / log X( z +1) − log Xz )]
(8)
where, as for equation (7), z and z + 1 are n-alkanes with
z and z + 1 carbon numbers, respectively. Moreover, the
closer setting of the reference paraffins enables a more
accurate evaluation of a compound’s behaviour in the
uniform scale.
In general, the retention index system is a combination
of two chromatographic parameters, relative retention
[equation (4)] and retention volume [equation (5)]. However,
several drawbacks of this system could be observed. A
mere comparison between retention indices calculated on
columns of distinct stationary phases does not allow to
distinguish whether the difference in the values is related
to an increased retention of the substance of interest or to
a decreased retention of the members of the reference
series on the stationary phase in question, or vice versa.14
In addition, the retention indices, as proposed by Kováts,
refer solely to data obtained under isothermal elution
conditions, while by applying a column temperature pro-
gramme the series of n-paraffins elutes in a linear mode.
To each successive peak, a constant increment is added
to the retention time of its predecessor, instead of a non-
linear increment, as could be observed under isothermal
conditions. A similar relationship was observed between
the programmed-temperature retention times ( t RT ) of the
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DOI: 10.1002/ffj
300 B. D’ACAMPORA ZELLNER ET AL.
Figure 2. Scheme of the calculation of isothermal
retention indices
n-paraffins and their carbon numbers (cn); it can be
expressed as in equation (9):
log t RT = a′ ⋅ cn ⋅ b′ (9)
where a′ and b′ are proportionality constants.
The programmed-temperature retention index calculation
is based on the following equation proposed by H. van
den Dool and D. J. Kratz,4 which does not use the loga-
rithmic form, as presented in equation (10):
I T = 100 [z + (t Ri
T − t T /t T
Rz R( z +1) − t Rz )]
T (10)
As previously cited, when the latter equation is applied
for the calculation of indices, these are commonly
denominated as linear retention indices (LRIs). Figure 2
illustrates schematically the retention index calculation
procedure. Moreover, when series of n-paraffins containing
only even or odd carbon atoms as references are used,
equation (11) must be applied, where n represents the
difference in carbon atom number of the two n-paraffins
that bracket a solute’s retention time:
I T = 100n(t Ri
T
− t Rz
T
/ t RT( z +1) − t Rz
T
) + 100 z (11)
It has to be pointed out that the retention indices calculated
for isothermal and temperature-programmed conditions
commonly present proximate values, but are not identical.
Furthermore, there is the possibility that IT values vary
according to the applied temperature rate and the initial
temperature. Moreover, since the reproducibility of
temperature-programmed retention indices is dependent
on working variables, such as carrier gas flow rate,
stationary phase film thickness and linear temperature
programming rate, those parameters should be standardized.
In 1974 van den Dool described the g-pack value,15 a
method for the characterization of GC columns using
relative retention indices, as proposed by Kováts. The
Copyright © 2008 John Wiley & Sons, Ltd.
indices were determined for six standard compounds
(limonene, linalool, linalyl acetate, acetophenone, naph-
thalene and cinnamyl alcohol) considered as representative
for the substances commonly detected in essential oils,
which were co-injected with even carbon-numbered n-
alkanes from C8 to C24. The g-pack value depends on the
type of stationary phase and, in general, alterations of the
carrier gas flow rate, temperature-programming rate and
initial temperature do not affect g-pack values. On the
other hand, modifications in the selectivity of a stationary
phase, such as those deriving from column bleeding, may
decrease the g-pack value, while the modifications of the
stationary phase due to oxidation may increase a g-pack
value. The latter values were calculated for two stationary
phases, a polyethylene glycol and a 100% dimethyl
polysiloxane, representative of polar and non-polar
columns, respectively. With polar columns, a satisfactory
upper temperature was considered to be 225 °C, so that
the elution temperature of n-tetracosane was adjusted
to 225 °C. However, it was observed that the elution of
homologous series under temperature-programmed con-
ditions was not linear, leading van den Dool to suggest
the use of both even and odd carbon-numbered n-alkanes
to reduce possible errors. The above-mentioned report was
published in 1980, followed by a second one released in
1981, also based on the reproducibility of chromatographic
retention data of essential oils analysed by temperature-
programmed GC, on methyl silicone stationary phases.16
Both publications present an appendix comprising methods
recommended for the control of the reproducibility of the
system, from the establishment of column performance to
the standardized tabulation of the results.
Retention index systems are not only represented by
the well-known proposals made by Kováts and, later on,
by van den Dool and Kratz; the continuous interest and
study on retention mechanisms, and also retention index
systems, led to the introduction of several other concepts,
some of which are briefly reported here.
Several concepts were derived from the equation pro-
posed by Kováts, such as the molecular retention index,
developed by M. B. Evans and J. F. Smith in 1961,10,11
which was defined as the molecular weight of a hypothetical
n-alkane presenting the identical retention of the compound
being investigated. The formula of that hypothetical n-
alkane equivalent to the target compound is CIH2I′+2 and
will possess an effective molecular weight according to
the following relation:
Me = 14.026 I ′ + 2.016 = 0.1406 I + 2.016 (12)
where I = 100I′, whereas I′ is the number of carbon
atoms and Me is the molecular weight of the hypothetical
n-alkane. The difference between that hypothetical and
the real molecular weight may be a useful parameter to
correlate retention and chemical structure. A further system,
defined as the theoretical nonane value, and developed
Flavour Fragr. J. 2008; 23: 297–314
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 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 301
by the aforementioned researchers, was propounded, also
in 1961.17 According to that system, the retention value
was first determined as proposed by Kováts and then
transferred to a system in which n-nonane is the standard.
However, the accuracy of the determination decreased
when compounds with retention values far from that of
the reference standard were analysed.
Another system based on the equation proposed by
Kováts is the standard retention index developed by P. G.
Robinson and A. L. Odell in 1971.8 In that system the
reference parameter is neither the retention time nor the
adjusted retention time, but the boiling points of the target
analyte and the reference standards. Moreover, the
generalized retention index, developed by J. Novák and
J. Ružiková in 1974,18 also consisted of a modification of
the equation proposed by Kováts, which may be applied
also when homologous series other than n-alkanes are used
as reference. The identical name, generalized retention
index, was given to a further system created by I. G.
Zenkevich and B. V. Ioffe in the 1980s19,20 that combines
the logarithmic system of Kováts with the retention indices
based on the linear relationship between I and (t′R + qlog t′R ),
where t′R is the adjusted retention time and q a coefficient
related to the analytical conditions.
Based on the fact that the retention indices should be
independent of the effects of the stationary phase, V. G.
Berezkin reported, in 1974, the invariant retention index
(I0), proposing the following relationships between the
retention indices and the invariant retention index:21
I = I0 + a/ PL (13)
I = I0 + b/kS (14)
I = I0 + c/VL (15)
where a, b and c are constants, PL is the percentage of
stationary phase present in the column, VL is the volume
of stationary phase in the column, and kS is the capacity
factor of the reference substance.
A further system, the homologous index, was intro-
duced in 1985 and may be defined as the retention index
obtained using a homologous series, consisting of com-
pounds similar to those to be characterized.22 Rasanen
et al.23 measured the homologous retention indices of
alkaline drugs through software able to automatically
assign the so-called secondary retention index standard
series (I*) by a pattern recognition algorithm; the I*
standards were then identified as members of a pattern
rather than individual peaks. Afterwards the same software
calculated the I* values of other peaks through linear
interpolation using absolute retention times, compared
the obtained values to those contained in libraries and
reported the identified compounds.
Moreover, the investigation on the variation in the
retention index of hydrocarbons on squalene, which are
Copyright © 2008 John Wiley & Sons, Ltd.
largely attributed to random errors, led to the develop-
ment of the unified retention index concept by N. Dimov.24
Considering that the function dI/dT, which represents the
variation of the indices according to the temperature, is a
hyperbolic function, the unified retention index (UIT) was
proposed, which may be defined as a statistical treatment
of data by means of linear regression [see equation (16)]:
UIT = UI0 + (dUi/dT )T (16)
where UI0 is the value of UIT at 0 °C and dUi/dT is the
index increment with the analysis temperature (the slope
of the curve).
The index itself and the temperature increment of the
unified retention index have the advantage of being
statistical values and, thus, more reliable than individual
experimental retention values. Furthermore, it is charac-
terized by a standard deviation (SD), and the calculation
of the confidence interval at any desired level is possible.
The concept was proposed not only as an easy and con-
venient mode of determining the retention index value at
any temperature within the investigated range, but also as an
evaluation method of the proper column temperature for
the analysis of complex mixtures, demonstrating that the
elution sequence of the sample components can change.25
The virtual carbon number was a further proposal studied
for the identification of compounds without applying any
index calculation, but using solely the retention behaviour.9
According to this system, an homologous series is not
necessary for the calculation of retention indices, since the
analysed matrix certainly contains a series of compounds
with well-known retention indices; the latter, which do not
necessarily belong to a homologous series, could be used
as index references. The index of the other compounds
shall then be calculated as with n-alkanes, although instead
of the carbon numbers of the n-alkanes, virtual carbon
numbers of the reference compounds should be used.
A further method for compound identification using
only retention behaviour without index calculation is
retention time locking (RTL) for programmed tempera-
ture analysis.26 In the RTL method, the inlet pressure is
adjusted to provide an identical retention time for the same
compound in any system equipped with the same nominal
column. This method applies constant pressure and only
small deviations in column size and stationary phase loading
parameters are tolerated; moreover, it cannot be used
under constant flow-rate conditions.
The reduced Kováts index (RKI) was also proposed for
retention data determination of wax esters. According to
Stránský et al.27 this index system should get round the
drawbacks arising from the large KI values generated by
the multiplying factor of 100. RKIs are calculated based
on equation (17), where NCA is the number of carbon
atoms:
RKI = I − 100 ⋅ NCA (17)
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302 B. D’ACAMPORA ZELLNER ET AL.
These values should express the influence of the presence
and position of the ester functionality or position of
double bond on the stationary phase. However, it has to
be pointed out that, although used for temperature-
programmed GC analyses, retention index calculations
were logarithmic-based.
A further index system, the molecular topological
index, has been shown to be a very important structural
parameter for describing the chromatographic behaviour
of a compound.28 The determination of these indices and
their correlation with retention indices of linear alkylben-
zene isomers with C10 to C14 linear alkyl chains were
investigated.29
Moreover, there has also been a great deal of interest in
the prediction of retention indices. The development of
predictive relationships would represent a key for the
reliability of retention data, the prediction of the retention
index of an analyte that was unavailable for study, as also
the extrapolation of values of an analyte’s specific physical
properties that could be correlated through its retention
index. More general approaches to retention index predic-
tion are based on the generation of topological, geometric
and electronic molecular descriptors, which are subse-
quently fitted to the retention index using multiple linear
regression or artificial neural networks. Developments in
artificial intelligence were represented by Bruchmann,
Zinn and Haffer30 with neural networks which were
explored to predict GC retention index data based on
electrotopographic indexes of monofunctional compounds,
such as acyclic and cyclic monoterpenes and a mixed set
of monosubstituted compounds and terpenes. According
to the authors, predictions by neural networks are gener-
ally in good agreement with predictions done by multiple
linear regression techniques. In addition, the prediction
of retention indices of homologous series was proved to
be effective.
Alternatives to n-Alkanes as Reference
Series
In general, retention index systems are based on the
incremental structure–retention relationship, viz. that any
regular increment in a series of chemical structures should
provide a regular increment in corresponding retention
times. This means that the retention index concept is not
restricted to the use of n-alkanes as standards, as is well
reported in the exhaustive review written by Castello.31
In practice, any homologous series presenting a linear
relationship between the logarithm of the adjusted reten-
tion time and the carbon number may be used.
In general, n-alkanes present fluctuating behaviour on
polar stationary phases. In consideration of the fact that
retention index values are correlated to retention mecha-
nisms, alternative standard series of intermediate polarity
were introduced, such as 2-alkanones, alkyl ethers, alkyl
Copyright © 2008 John Wiley & Sons, Ltd.
halides, alkyl acetates, alkanoic acid methyl esters13 and
para-substituted n-alkylphenols.32 These compounds are
less likely to be retained by interfacial adsorption on polar
stationary phases than the n-alkanes, resulting in more
reliable retention index values, and so more suitable
qualitative identification. On the other hand, Shibamoto33
suggested the use of polar series, such as ethyl esters, as
an alternative. In the characterization of volatiles, the most
commonly applied reference series are the n-alkanes and
the fatty acid ethyl esters (FAEEs), which are chosen
according to the stationary phase used.
Standard series may also be selected according to the
characteristics of the matrix, so that the chosen standards
are normally comprised in the sample under investigation,
or present a chemical structure and a behaviour with the
chromatographic system, similar to the compounds to be
determined. Several series have already been reported in
the literature, such as the linear fatty acid methyl esters
(FAMEs), which provide an equivalent chain length of
unsaturated and branched fatty acid esters in lipid
analysis,34,35 or polychlorobiphenyl (PCB) congeners
possessing distinct numbers of chlorine atoms to identify
the highly toxic PCB isomers.14 A further example is the
polynuclear aromatic hydrocarbons (PAH) retention index
system, which uses the numbers of rings in unsubstituted
PAHs to characterize the retention behaviour of their
homologues and related substituted compounds. The
determination of over 200 PAH structures by using naph-
thalene, phenanthrene, chrysene and picene as reference
standards has been reported by Lee et al.36
Further homologous series are related to drug screening gas
chromatographic analysis, such as the 1,4-benzodiazepine
index standard series for benzodiazepine screening37 and the
four series FA (N,N-dialkyl-4-fluoroanilines), FB (N,N-dialkyl-
4-fluorobenzylamines), BA (N,N-dialkyl-4-benzylamines)
and TA (trialkylamines), applied in the screening of
alkaline drugs in blood samples.38 A similar approach was
also performed using MH (3-alkyl-1-methylhydantoins)
and H (3-alkylhydantoins) series for the screening of
nitrogenous acidic and neutral drugs.23
The introduction into the market of element-specific
detectors is also a consistent reason for the selection
of most suitable standard series, as is the case of the
electron-capture detector (ECD) and the flame photometric
detector (FPD), which are not sensitive to n-alkanes.13,31
Homologous series of iodoalkanes, chloroalkanes,
bromoalkanes, n-alkanols or, as mentioned above, PCB
congeners can be exploited when an ECD is used;36 a
standard series of sulphur-containing molecules, such as
dialkylsulphides, can be used with a FPD, and trialkylamines
with a nitrogen phosphorus detector (NPD).39 Further
series, such as alkylbis(trifluoromethyl)phosphine sulfites
(M series), are employed for several detectors – ECD,
FID, FPD, alkali flame ionization detector (AFID),
photoionization detector (PID)40 and mass spectrometry
(MS).41
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 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 303
Application of Retention Index Values for
Phase Constant Determinations
The selectivity of a stationary phase can be estimated by
determining the degree to which polar compounds are
retained by that phase in relation to their retention on a
non-polar phase. The system of stationary phase constants
based on retention index differences (ΔI), introduced by
Rohrschneider,42 was the first widely adopted approach
for the systematic classification of stationary phases. The
system of phase constants is formulated on the assumption
that intermolecular forces are additive, and provide their
individual contributions on retention. The relative ΔI
values are obtained by measuring the degree to which
the stationary phase to be characterized retains several
probe compounds (benzene, ethanol, methyl ethyl ketone,
nitromethane and pyridine), relative to their retention on
a non-polar hydrocarbon phase (squalane), both at 120 °C
[equation (18)]:
ΔI = I polarphase − Isqualane (18)
where ΔI represents the retention index difference; the
latter is equivalent to the contribution from polar interactions.
The Rohrschneider constant is commonly represented by
the ratio of the index difference and 100:
ΔI/100 = Rohrschneider constant (19)
In order to evaluate the phase constants numerically, the
McReynolds constants,43 derived from the Rohrschneider
method, are commonly used. Those constants enable to
compare polarities of stationary phases based on their
selectivity for specific solute interactions; for instance,
benzene has a constant of zero for squalane, while on a
100% dimethyl polysiloxane stationary phase the con-
stant equals 0.16, and on a polyethylene glycol column it
has a value of 3.22. The larger the value of the McReynolds’
phase constant, the stronger is the interaction expressed
by that constant. The McReynolds values are used to
evaluate the degree of similarity between stationary phases,
to rank the phases by degree of polarity and to predict the
elution order of the analytes.
The Rohrschneider concept and related calculation
methods represent a very important research area,6,7,13 and
both the aforementioned approaches are widely applied
to characterize and classify stationary phases. However,
the McReynolds system may often contain erroneously
measured data; an additional drawback is that the constants
are determined only at 120 °C. The combination of the
Rohrschneider and McReynolds systems supplemented
with data measured at 80 °C and 140 °C would ensure a
greater development of this research area. The field of
structure-driven prediction of retention in gas chromato-
graphy is not well developed at present and new approaches
should be faced.
Copyright © 2008 John Wiley & Sons, Ltd.
Retention Index Compilations
A myriad of retention index databases have been published
and, since the majority of such retention index data have
not been generated under conditions in which stationary
film polarity and thickness, carrier gas flow rate and
temperature-programming rate have been standardized,
their precision, comparability, reliability and usefulness
may be limited.
Worthy of note, among the several retention index
datasets, are the compilations also published as books and
widely used as references, as those authored by Jennings
and Shibamoto44 and by Adams45 (the 4th edition was
published in 2007). The latter book includes mass spectra,
chemical structures and retention indices of 2205 compounds
obtained on a 5% diphenyl:95% dimethyl polysiloxane
column (30 m × 0.25 mm i.d., 0.25 μm film thickness).
The use of KI and AI made by Adams is noteworthy, with
AI standing for arithmetic index, the calculation of which
is based on the equation proposed by van den Dool and
Kratz.4 The former book reports the spectra of over 1150
flavour and fragrance volatiles and their respective KIs
on methyl silicone and on polyethylene glycol stationary
phases. Another retention index dataset is the Sadtler
Standard Gas Chromatography Retention Index Library.46
The latter comprises a four-volume reference set that
identifies over 2000 compounds by their retention indices
on three different stationary phases. Further books are
also available, such as the compilation of sesquiterpene
hydrocarbons authored by Joulain and König47, compris-
ing the mass spectra of 307 compounds along with their
retention indices determined on a 25 m fused silica capil-
lary column coated with 100% dimethyl polysiloxane. A
further interesting issue is the toxicological database
published by Maurer et al.,48 containing 7840 datasets of
drugs, poisons, pesticides, pollutants and their metabolites.
Notwithstanding these publications, a considerable
problem may be still observed in the inter-laboratory
comparison of retention index values, since the applied
chromatographic conditions are frequently not strictly
comparable. Richmond49 made an attempt to circumvent
this problem by merging data contained in different
datasets using a so-called GC calibration approach,50
which is just empirical and has no thermodynamic or
quantitative structure–retention relationship adjustment.
The merging of compilations led to the creation of the
Pseudo-Sadtler database, which comprises data obtained
from Sadtler46 and Jennings and Shibamoto.44 One successful
example of the calibration approach is 2-octene, whose
retention index of 811, as reported by Jennings and
Shibamoto, was equivocal due to ambiguous cis–trans
geometry. The cis and trans isomers are separable and
present retention indices of 803 and 811, respectively.51
Compilations of compounds and their respective retention
indices can be widely seen in several publications, e.g.
Lee et al.36 published the retention indices of over 200
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304 B. D’ACAMPORA ZELLNER ET AL.

Table 1. Some flavour and fragrance compounds whose spectra and linear retention indices are included in the
FFNSC ver. 1.3 MS Library (Chromaleont, Messina, Italy)

LRIa Common name CAS name CAS No. MW Formula

608 Vinyl dimethyl carbinol 3-Buten-2-ol, 2-methyl- 115-18-4 86 C5H10O

864 α-Angelicalactone 2(3H)-Furanone, 5-methyl- 591-12-8 98 C5H6O2
972 Pyromucic acid methyl ester 2-Furancarboxylic acid, methyl ester 611-13-2 126 C6H6O3
1030 Limonene Cyclohexene, 1-methyl-4-(1-methylethenyl)- 138-86-3 136 C10H16
1076 n-Octanol 1-Octanol 111-87-5 130 C8H18O
1101 Linalool 1,6-Octadien-3-ol, 3,7-dimethyl- 78-70-6 154 C10H18O
1115 Sotolone 2(5H)-Furanone, 3-hydroxy-4,5-dimethyl- 28664-35-9 128 C6H8O3
1152 Nona-(2E,6E)-dienal 2,6-Nonadienal, (2E,6E)- 17587-33-6 138 C9H14O
1153 Nona-(2E,6Z)-dienal 2,6-Nonadienal, (2E,6Z)- 557-48-2 138 C9H14O
1163 Furfurylthiol acetate Hexanoic acid, 2-furanylmethyl ester 13678-68-7 156 C4H8O2S
1180 Abhexone 2(5H)-Furanone, 5-ethyl-3-hydroxy-4-methyl- 698-10-2 142 C7H10O3
1200 Florosa 2H-Pyran-4-ol, tetrahydro-4-methyl-2-(2-methylpropyl)- 63500-71-0 172 C10H20O2
1250 Linalyl acetate 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate 115-95-7 196 C12H20O2
1250 Thymoquinone 2,5-Cyclohexadiene-1,4-dione, 2-methyl-5-(1-methylethyl)- 490-91-5 164 C10H12O2
1368 4-tert-Butyl-cyclohexanol acetate Cyclohexanol, 4-(1,1-dimethylethyl)-, acetate 32210-23-4 198 C12H22O2
1375 α-Copaene Tricyclo[4.4.0.02,7]dec-3-ene, 1,3-dimethyl-8-(1-methylethyl)- 138874-68-7 204 C15H24
1651 α-Muurolol 1-Naphthalenol, 1,2,3,4,4a,7,8,8a-octahydro-1,6-dimethyl-4- 19435-97-3 222 C15H26O
(1-methylethyl)-, (1R,4S,4aR, 8aS)-
1651 Vanillyl butyl ether Phenol, 4-(butoxymethyl)-2-methoxy- 82654-98-6 210 C12H18O3
1767 Amberlyn Naphtho[2,1-b]furan, dodecahydro-3a,6,6,9a-tetramethyl- 3738-00-9 236 C16H28O
1960 Neocembrene 1,5,9-Cyclotetradecatriene, 1,5,9-trimethyl-12- 31570-39-5 272 C20H32
(1-methylethenyl)-, (1E,5E,9E,12R)-

a
Linear retention index calculated on a 5% diphenyl:95% dimethyl polysiloxane column (30 m × 0.25 mm i.d., 0.25 μm df) programmed from 50 °C to 250 °C
(10 min) at 3.0 °C/min.

PAHs, and Stránský et al.27 reported Kováts indices of 205
wax ester standards. With regards to flavour and fragrance
materials, Davies52 published an extended compilation
reporting a summary of retention index values obtained on
methyl silicone and on Carbowax 20M stationary phases,
and Bianchi et al.53 presented the retention indices of 250
food volatile chemicals on a polar stationary phase. A
further collection of n-alkanes and ethyl ester retention
indices (IE) of 183 common constituents of essential oils
was published by Shibamoto.33
The World Wide Web is also a widely used source of
retention index values; however, their reliability may be
doubtful. Among the trustworthy and well-accepted
references are the Flavornet54 and the LRI & Odour
Database.55 The latter contains the retention indices of over
5000 volatile compounds identified in different food
matrices. The database is a compilation of LRI data, using
n-alkanes as reference series, and extracted from the
scientific literature on food flavour. The former database
comprises odour-active compounds detected by means of
GC–olfactometry (GC–O) applying quantitative odour
measurement techniques. The compilation reports the
retention indices of 738 compounds, calculated by applying
the equation proposed by Kováts using n-paraffins and
ethyl esters as reference series on four different stationary
phases. The first-mentioned database also reports the
compound’s odour description, while the second only
reports this for about 1500 compounds. A further reference
is the Pherobase56 developed by the Chemical Ecology
Group at the Hort Research in Lincoln, New Zealand.
This database contains about 3000 molecules including
their Kováts retention index on distinct stationary phases
extracted from publications in that research field.
With regard to GC–MS databases, several mass spectral
library software are available on the market; however,
only a few of them comprise retention index information
to facilitate peak assignment;57,58 this is the case of the
FFNSC MS Library ver. 1.3 (Chromaleont, Messina, Italy),
the MS library developed by Adams (Allured Publishing,
Carol Stream, IL, USA), and the NIST 05—NIST/EPA/
NIH Mass Spectral Library (National Institute of Standards
and Technology, Gaithersburg, MD, USA). With respect
to the FFNSC MS Library, this commercially available
product contains 1827 compounds. For the sake of
demonstration, Table 1 lists 20 compounds whose spectra
and linear retention indices are contained in the FFNSC
MS Library ver. 1.3. The spectra were acquired on a
GCMS-QP2010 system (Shimadzu, Milan, Italy) equipped
with a 5% diphenyl:95% dimethyl polysiloxane column
(30 m × 0.25 mm i.d., 0.25 μm df; SLB-5MS, Supelco,
Bellefonte, PA, USA). The oven temperature was pro-
grammed from 50 °C to 250 °C at 3.0 °C/min and held for
10 min. This library, dedicated to Shimadzu GC–MS sys-
tems, possesses not only information on retention indices
but also is equipped with retention index interactive tools,
whose application is enabled when used in association with
the data-collecting software GCMS Solution (Shimadzu,
Milan, Italy),59 since the latter software possesses an
automated retention index calculation function. To per-
form the automatic retention index calculation, a series of

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 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 305

Figure 3. GC–MS chromatogram of the Cistus ladanifer L. essential oil with indicated linalool peak (A), qualitative
parameters window of the GCMS Solution software, illustrating the retention index allowance and column type
selection tools (B), and the similarity search result window attained for this peak (C)

homologous standards, commonly n-alkanes, has to be
injected prior to analysis, and the retention times, as well
as retention indices, obtained for each standard inserted
in the index table of standards. Based on this information
set, the software is able to calculate the retention index
for the detected compounds. The library search algorithms
incorporating LRIs as part of the match criteria assist in
the analyses and characterization of flavour and fragrance
compounds, enhancing the credibility of the identification
through MS. The GCMS Solution software also permits
the insertion of five distinct retention index values for each
compound whose mass spectra was appended to a mass
spectral library. Figure 3 illustrates the library search of a
component of Cistus ladanifer L. essential oil.60 Moreover,
as illustrated in Figure 3B, a column type selection tool is
available, so that only retention index values determined
on that stationary phase are displayed in the similarity
search result window (Figure 3C).
Also of great interest is the AMDIS (Automated Mass
Spectral Deconvolution and Identification System) program,
frequently used in combination with NIST Mass Spectral
Libraries (both developed by the National Institute of
Standards and Technology). This software not only
deconvolutes GC–MS files but also enables the use of
mass spectra libraries containing retention index information,
which were previously converted to the AMDIS format.61
A retention index-matching window is also available as
an optional setting of the software. An example of the

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DOI: 10.1002/ffj
306 B. D’ACAMPORA ZELLNER ET AL.
Figure 4. AMDIS analysis of a GC–MS chromatogram of Cistus ladanifer L. essential oil detecting linalool (calcu-
lated RI of 1099.4 and RI-RI(lib) of –1.6)
use of AMDIS on a Cistus ladanifer L. oil analysis
applying the FFNSC MS Library ver. 1.3 converted to
AMDIS format is presented in Figure 4.
It is certain that the above-cited retention index compi-
lations represent a very small part of the studies performed
on flavour and fragrance materials.
Retention Indices and Chromatographic
Systems
As is well-known, flavour and fragrance materials are
characterized by a wide diversity of components belonging
to several chemical classes, and are commonly added to
a great variety of products, such as foods (ice creams,
alcoholic and non-alcoholic beverages, baked goods, etc.),
cosmetics, toiletries, functional products, tobacco products
and pharmaceutical preparations among others.62 The
advent of GC marked a real turning point for flavour and
fragrance research. In the early stages of research in this
field, attention was devoted to the development of methods
to acquire deeper knowledge on the profiles of the volatiles;
Copyright © 2008 John Wiley & Sons, Ltd.
however, this analytical task was made troublesome
due to the complexity of those real-world samples. In this
respect, the utilization of retention indices, when com-
bined with other characterization methods, such as mass
spectrometry, was shown to be an effective tool for the
identification of flavour and fragrance compounds.
In general, apart from the GC hardware configuration
applied, the retention index determination may be per-
formed through the injection of the homologous series as
internal or external standard. When solutions of standard
compounds or simple extracts are analysed, the use of an
internal standard series is a valid approach, since both
analytes and references are subjected to identical factors
influencing retention time measurement. However, when
complex samples have to be analysed, the use of an internal
standard series is not feasible. The work developed by
A. Tóth and L. Praszna63 on retention index reproducibility
reports values obtained for analyses carried out during a
period of 2 months. These researchers observed that at
isothermal temperatures the changes in retention times
over the analysis period may be negligible, but in general
the measured retention times decreased according to the
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 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 307
use of the column. On the other hand, when applying
programmed temperatures, retention data presented a
deviation, which was attributed to the programming rate
or carrier gas velocity. To overcome these limits, one of
the n-alkanes contained in the series of standard alkanes
was used as internal standard. The difference of the
retention times of that n-alkane analysed as internal and
as external standards was then applied as a correction
factor, normalizing the retention times that were used in the
calculation of the so-called corrected retention indices.
Concerning the stationary phases, in the case of signifi-
cant interfacial adsorption of solutes or standards, wide
variations in retention index values can occur. Those
variations may be related to sample size effects, in particular
the concentration ratio of the analysed substance to the
n-alkane, and with the phase loading or the column’s film
thickness. A remark need to be made on the influence of
the stationary phase film thickness (df); index values
calculated on a thinner film tend to be higher than those
measured for a thicker one, since the residual surface
activity will be shielded by the thicker film. Moreover,
the dependence of the index is hyperbolic, so that the
application of linear extrapolation may lead to unreliable
retention values.64 The fact that retention index values are
specific to a stationary phase at a determined temperature
has also to be considered.65
The LRI reliability in identifying an essential oil
component at an intra-laboratory level was investigated
in depth by Bicchi et al.66 They analysed three essential
oils with different columns [polysiloxane (OV-1) and
polyethylene glycol (CW 20M)] that had distinct charac-
teristics (producer and inner diameter) and under differ-
ent analytical conditions (mainly temperature programme
and flow rate). The data obtained were used to evaluate
the influence of analytical conditions on retention index
variation, showing that retention index repeatability with
CW 20M was mainly conditioned by the temperature
programme.
The application of two sufficiently different temperature
gradients may cause the alteration in the elution of an
analyte on identical polar stationary phases, e.g. the
application of an oven temperature rate of 2 °C/min or
10 °C/min may alter the calculated retention index value. In
this respect, Hérent et al. proposed the double indexation
method,67 which consisted of the determination of two
retention index values for an identical compound analysed
with two distinct oven temperature rates, viz. 6 °C/min and
10 °C/min, on the same polar column. FAMEs from C5 to
C20 were chosen as reference series, due to the previously
mentioned low solubility of n-alkanes in several polar
stationary phases. According to the authors, the method
presented increased the reliability of identification and
might be very useful for a rapid identification of major
constituents of essential oils.
Noteworthy is the increased reliability of retention
times obtained under programmed temperature since the
Copyright © 2008 John Wiley & Sons, Ltd.
introduction of electronic pressure control devices and
improved column oven heat distribution in GC systems.
The use of autosamplers and autoinjectors also enhanced
retention time precision and, in consequence, the reliability
of calculated retention index values increased. Furthermore,
the improved quality of stationary phases has also played
a role in the increased column stability and long-term
reproducibility of analysis. Additionally, according to the
previously mentioned report published by the Analytical
Methods Committee,16 it is desirable to use two columns
with different stationary phases to attain two retention
index values and enhance confidence in assignments.16,52
It is accepted that the reproducibility of retention indices
between different laboratories is usually within a few index
units for non-polar stationary phases and within 10 –50
index units for polar phases.13 The large range of units
for polar columns could be related to the heterogeneity
between columns manufactured by different companies, the
misuse of the gas chromatographic technique, a possible
reactivity between the analyte and the stationary phase,
as well as the amount of sample injected, which can induce
deviation. A further aspect of great importance, which is
frequently overlooked, is the analytical reproducibility;
most publications do not report data on analysis performed
in duplicate or triplicate. Moreover, some publications
report the use of an initial isothermal hold in the GC
oven temperature programme, even though in practice it
was found not to give any additional resolution, as reported
in an investigation on the monoterpene hydrocarbon
contents of essential oils.5
Publications on flavour and fragrance materials which
report calculated retention indices and make reference to the
works published by Kováts3 and van den Dool and Kratz 4
are countless. However, publications that inappropriately
use the retention indices are also innumerable, e.g. apply-
ing the equation proposed by Kováts in temperature-
programmed GC analysis. Although there is general and
constant interest in the application of retention indices in
GC, their standardization is still a target to be achieved.
To better illustrate the divergences observed in the literature
and investigate the reliability of retention indices, the
values reported in the Flavour and Fragrance Journal
publications of the year 2006, for the monoterpene limonene
and the oxygenated monoterpene linalool on polar
(polyethylene glycol) and non-polar (5% diphenyl, 95%
dimethyl polysiloxane) stationary phases, were collected.
Tables 2 and 3 present an overview of the retention index
values of limonene and linalool, respectively, measured
using n-alkanes series throughout 2006. During that year,
the retention index of limonene was reported in 31 and
55 articles, on polar and non-polar phases, respectively,
while that of linalool was in 28 and 64 articles. The dis-
crepancies between the values demonstrate that published
retention indices, when originated from poor analytical
procedures, might not always be used as complementary
information for the reliable identification of unknown
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308 B. D’ACAMPORA ZELLNER ET AL.

Table 2. Retention index values of limonene and their frequency of citation in

Flavour and Fragrance Journal published throughout the year 2006

Polar stationary phase Non-polar stationary phase

LRIa Frequency of citation LRIb Frequency of citation

Limonene 1183 3 1010 1

1190 2 1013 1
1195 1 1022 5
1199 2 1023 1
1201 1 1025 3
1202 3 1026 2
1203 10 1029 3
1204 4 1030 10
1205 5 1031 17
1032 8
1033 1
1035 1
1038 2

a
Linear retention index obtained on polyethylene glycol column.
b
Linear retention index obtained on 5% diphenyl, 95% dimethyl polysiloxane column.

Table 3. Retention index values of linalool and their frequency of citation in

Flavour and Fragrance Journal published throughout the year 2006

Polar stationary phase Non-polar stationary phase

LRIa Frequency of citation LRIb Frequency of citation

Linalool 1509 2 1079 1

1521 4 1088 1
1522 1 1092 2
1539 1 1096 1
1542 3 1097 5
1543 2 1098 22
1544 3 1099 9
1546 1 1100 7
1547 2 1101 4
1549 1 1102 1
1551 2 1103 10
1553 4 1108 1
1556 2

a
Linear retention index obtained on polyethylene glycol column.
b
Linear retention index obtained on 5% diphenyl, 95% dimethyl polysiloxane column.

compounds. As previously mentioned, by convention,
calculated values which are comprised within a determined
range may be considered as acceptable, i.e. ±5 units for
methyl silicone stationary phases and ±10 units for
polyethylene glycol phases.
Specific Applications of Retention Indices
Usefulness of Retention Indices in GC and
GC–MS Analyses
Considering gas chromatographic analyses using flame
ionization (FID), thermal conductivity (TCD) or other
detectors which do not provide structural information of
the analysed molecules, retention times, and more precisely
retention indices, are used as the primary criterion for
peak assignment. The application of the retention index
system has been extended to other gas chromatographic
approaches, such as olfactometry. The latter technique
was a breakthrough in analytical aroma research, enabling
the differentiation of a multitude of volatiles, previously
separated by GC, into odour-active and non-odour-active
compounds, related to their concentrations existing in the
matrix under investigation. Especially when working with
GC–O systems equipped with detectors that do not provide
structural information, a compound’s retention index is
then commonly associated to its odour description, sup-
porting reliable peak assignment.
Moreover, with the growing popularity of GC coupled
to mass spectrometry (GC–MS), a large number of studies
has exploited mass spectral information for peak identifi-
cation; the most frequent and simple identification method
consisting in the comparison of the acquired unknown

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 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 309

Figure 5. Representation of the similarity between mass spectra of monoterpenes: (A) α-thujene (A) and α-
phellandrene (B), and sesquiterpenes: (E)-β-farnesene (C) and (E)-β-bergamotene (D)

mass spectra with those contained in a reference MS
library. However, as is well-known, isomers or similar
substances, when analysed by means of GC–MS, may
have their identity imprecisely or unreliably assigned, a
drawback which is often observed in essential oil analyses.
As is widely acknowledged, the composition of essential
oils is mainly represented by terpenes. The latter can be
considered as the most structurally varied class of natural
plant products, derived by repetitive fusion of branched
five-carbon units based on the isopentane skeleton
(isoprene units).68 As the mass spectra of these compounds
are usually very similar, peak assignment becomes a
difficult and sometimes impracticable task. In Figure 5A
and B the mass spectra similarity of monoterpenes is

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DOI: 10.1002/ffj
310 B. D’ACAMPORA ZELLNER ET AL.
represented through α-thujene and α-phellandrene. For the
former compound, a retention index of 927 is commonly
obtained on 5% diphenyl stationary phase, while for the
latter the retention index is 1007. The same can be
observed for the sesquiterpenes (E)-β-farnesene and (E)-
β-bergamotene, whose mass spectra are showed in Figure
5C and D, respectively, and whose retention indices of
1452 and 1483 are obtained on a 5% diphenyl stationary
phase. In order to increase the reliability of the analytical
results and to address the qualitative determination of
compositions of complex samples by GC–MS, retention
indices can be an effective tool. The use of retention indices
in conjunction with the structural information provided
by GC–MS is widely accepted and routinely used to con-
firm the identity of compounds.
It is worthwhile to mention the series of collaborative
trials performed by the Working Group on Methods of
Analysis of the International Organization of the Flavor
Industry (IOFI), aiming to investigate the reproducibility
of retention indices of some flavour compounds and
to establish quality control parameters.69 As is widely
acknowledged, most of the methods applied to identify
components of flavour and fragrance samples rely on
chromatographic techniques, although the use of an
additional confirmatory method is of extreme importance.
The trials consisted of the study of 21 compounds com-
monly detected in flavours or essential oils, or whose
concentration in food products has been limited by
regulatory legislation. These compounds were analysed
on polar and non-polar stationary phases, under identical
operating parameters, using n-alkanes as reference series
for the retention index values calculation. For all com-
pounds investigated the relative standard deviation (RSD)
was <1%, except for coumarins, which presented an
RSD of 1.3% on the polar column. In general, the RSD
values varied from 0.2% to 1.3% and 0.2% to 0.9% for
polar and non-polar columns, respectively. According to
the IOFI, the use of retention index data obtained in two
analytical columns coated with stationary phases of distinct
polarities is highly suggested, and identifications made
on a single column may only be accepted if used in com-
bination with mass spectral information. Furthermore, it
is outlined that the most accurate identifications are made
by using authentic standard compounds, and that neither
GC nor MS are satisfactory when applied as stand-alone
techniques. In some cases, even when using GC–MS, a
further method may still be required to satisfactorily
characterize a compound, such as nuclear magnetic reso-
nance (NMR) or infra-red (IR) spectroscopy.
The above-mentioned considerations may be exemplified
by the identification of sesquiterpene hydrocarbons, as
highlighted by Joulain and König.47 As can be widely
observed, a few of the large number of published works
report a reliable identification of known sesquiterpenes.
It could be observed that when associated with its non-
polar GC retention index, the mass spectrum of a given
Copyright © 2008 John Wiley & Sons, Ltd.
sesquiterpene is usually sufficient to ensure its identification,
provided the reference data have been recorded using
authentic samples. Indeed, for this class of compounds
there would be no need to adopt a polar stationary phase,
which could even lead to misinterpretations caused by
possible changes in the retention behaviour of sesquiter-
pene hydrocarbons as a result of column ageing or deteri-
oration, and depending on the manufacturer. Moreover,
in agreement with the authors’ experience, attention should
be paid to the retention index and the mass spectrum
registration of each individual sesquiterpene, since many
compounds with rather similar mass spectra may be eluted
in a narrow range; more than 160 compounds can be
eluted within 100 KI units on a non-polar column, e.g.
1400 –1500.
In addition, it has to be brought to light that in 1997,
the Analytical Methods Committee reported a method
which has been developed from a collaborative study
performed by 16 laboratories that applied temperature-
programmed GC using capillary columns coated with
non-polar stationary phases.70 In this study the previously
described g-pack value, proposed by van den Dool15 for
packed columns, has been specified for each investigated
capillary column, and reproducible relative retention
indices have been obtained for the six standard com-
pounds (limonene, linalool, linalyl acetate, acetophenone,
naphthalene and cinnamyl alcohol). The study consisted
of the analysis of the standards mixture, an even carbon-
numbered n-alkanes solution (C8–C24), a Spanish rosemary
oil sample, and the latter oil co-injected with the afore-
mentioned n-alkanes solution, on different 100% dimethyl
polysiloxane columns with dimensions in the ranges
25 – 60 m length, 0.20 –0.32 mm i.d. and 0.25–1.05 μm
stationary phase film thickness, produced by different
manufacturers. In all analyses the oven temperature pro-
gramme was set from 50 °C to 250 °C at a rate of 4 °C/min,
and the carrier gas control could be either constant flow
or pressure. Initial results demonstrated that there were
differences between different column manufacturers, but
similar results were obtained between laboratories using
similar columns purchased from the same manufacturer.
The recommended method should be applied to columns
in the ranges 25–30 m length, 0.22–0.32 mm i.d. and
0.25 μm film thickness, using the identical above-described
oven temperature programme. The standards mixture is
co-injected with the n-alkanes solution, and the retention
indices and the g-pack values are calculated. After the
column’s performance within a chromatographic system
has been satisfactorily established, it can be used under
identical conditions to analyse the essential oil, and the
latter co-injected with the aforementioned n-alkanes solution.
The two chromatograms obtained are compared to check
the reproducibility of the system, as also to confirm whether
there are interferences due to co-elutions of an essential
oil’s component with an n-alkane peak. It is worthy of
note that, according to the committee, the operating
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 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 311
conditions and the chromatographic system may be con-
sidered as satisfactory when the g-pack value is 1.0 ± 0.005.
Moreover, a much wider variation in elution tempera-
tures was observed on capillary columns than on packed
ones.
Gas Chromatographic Enantiomer
Characterization Supported by Retention Indices
Enantioselective GC (Es-GC) became an essential tool
for stereochemical analysis mainly after the introduction
of cyclodextrin (CD) derivatives as chiral stationary
phases about 25 years ago.71,72 The main application of
Es-GC to flavour and fragrance materials concerns the
precise determination of enantiomeric compositions and
ratios, which characterize the matrix, determining its quality
and, in the case of natural materials, its geographic origin,
biogenesis, authenticity and extraction technique applied.73
The calculation of linear retention indices of optically
active compounds can be considered troublesome due to
complex retention mechanisms on chiral stationary phases
(CSPs). In this case the latter are made up of CD derivatives
and the mechanism of retention is based on the host–guest
interaction, and if a homologous series, such as the n-
alkanes, is used, the hydrocarbons randomly occupy positions
in the chiral cavity. As a consequence, n-alkanes can be
considered as unsuitable for retention index determina-
tions on CSPs. Nevertheless, other reference series can
be utilized on CD stationary phases, such as linear chain
FAMEs and FAEEs. However, it can be affirmed that
retention indices are seldom reported for optically active
compounds, and publications refer to retention times
rather than indices.
For many years, GC–MS has been the benchmark tech-
nique for the qualitative analysis of flavour and fragrance
volatiles. However, when considering Es-GC–MS, the
identification of a target compound can be hindered due
to the similar fragmentation pattern of enantiomers. The
automation of library search algorithms incorporating
linear retention indices as part of the match criteria,
briefly described in the section on Retention Index
Compilations above, can also be extended to enantiose-
lective GC–MS analysis, assisting in the discrimination of
enantiomers.74–76
An enantioselective MS library is characterized by two
features, the mass spectrum, which could be useful to
determine the position of enantiomers in a chromatogram,
and the retention index, which can be considered as a
tool to discriminate these isomers. Moreover, considering
that a universal chiral selector with widespread capability
for enantiomer separation is not available, and thus effective
optical separation of all chiral compounds present in a
matrix may be unachievable on a single chiral column, the
use of a set of columns coated with distinct CD derivatives
as chiral selectors is necessary. The same is true for the
Copyright © 2008 John Wiley & Sons, Ltd.
homologous series to be applied as references; the use
of FAMEs and FAEEs series is advisable rather than
n-alkanes.74 –76
Retention Indices Applied to Multidimensional
Gas Chromatographic Analysis
As is well known, flavour and fragrance matrices are
complex multi-component mixtures with many overlapping
peaks in the gas chromatograms. The determination of
the structure of a component directly from such mixtures
is difficult, if not impossible. The analysis is then more
reliable when multidimensional gas chromatography
(MDGC) is applied.
The performance of MDGC analysis enables higher
resolution and better characterization of the components
of flavour and fragrance matrices. Briefly, in MDGC key
fractions of a sample are selected from the first column
and re-injected onto the second one, where, ideally, they
should be fully resolved. Two chromatographic columns
of differing polarities, but generally of identical dimen-
sions, are employed. Furthermore, when the heart-cut
operations are not carried out, the primary column elutes
normally in the first dimension GC system, while the pre-
viously re-injected fractions are analysed in the second
dimension GC system, equipped with a detector.77
A heart-cut MDGC approach based on retention index
theory was demonstrated for the analysis of FAMEs
mixtures.78 The reported method used predetermined
retention index data of the target compound to estimate
the retention time range in which this compound would
elute and so precisely define the linear retention window
for the heart-cut to be set. In this experiment the equation
proposed by van den Dool and Kratz was applied in the
following mode:
I ( x ) − Iref 2
t R ( x ) = t R,ref 2 + (t R,ref 2 − t R,ref 1) ⋅ (20)
Iref 2 − Iref 1
A similar concept was adopted for the screening of
allergens in flavour and fragrance materials.79 The proposed
method consisted, firstly, of the determination of the linear
retention index of each allergen in the first dimension
(5% diphenyl stationary phase) of the MDGC system in
stand-by mode. Secondly, cut windows were derived from
the application of ±10 LRI units with respect to the
experimental LRI value of each compound. The ±10 LRI
unit values were applied in equation (20) and in heart-cut
time windows for the target compounds were established.
Through such an approach, the injection of an allergen
solution prior to each MDGC application would not be
required; it would only be necessary to derive the LRIs
through preliminary analysis with n-hydrocarbons. The
allergens standard solution would have to be injected
only periodically for calibration purposes.
Flavour Fragr. J. 2008; 23: 297–314
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312 B. D’ACAMPORA ZELLNER ET AL.
A further MDGC technique is comprehensive two-
dimensional gas chromatography (GC × GC), which
promotes an orthogonal two-column separation, with
complete sample transfer executed by means of a modulator;
the latter entraps, refocuses and releases fractions of the
GC effluent from the first dimension (1D, column 1), onto
the second dimension (2D, column 2), in a continuous
mode; this method enables an accurate screening of
complex matrices, offering very high resolution and enhanced
detection sensitivity.80,81
The use of a retention index system in GC × GC analysis
has also been widely discussed in the literature and, although
several investigations have been focused on the develop-
ment of calculation methods, a standardized method has
not yet been introduced. The establishment of accurate
retention indices from GC × GC retention data is still an
emerging topic of research.
Dallüge et al82 used 1D retention indices and information
obtained from 2D retention times to improve the identifi-
cation of volatiles detected in cigarette smoke by means
of GC × GC coupled to a time-of-flight mass spectrometer
(GC × GC–TOF–MS). Out of a total of 2500 volatiles,
reference retention indices were found for only 238 com-
pounds, and out of these a group of 152 volatiles had
their experimentally determined index in good agreement
with the literature values.
Retention index calculation using data derived from
unconverted comprehensive GC chromatograms is also
among the proposed retention index calculation methods.83
In a GC × GC–qMS analysis on a highly complex perfume,
the assignment of peak identifications was based on the
injection of pure standard compounds, MS library matching
and 1D linear retention index data. The latter was used as a
filter during MS library searches, with the elimination of
matches outside a predefined index window. As described
by the authors, retention indices were calculated through
data derived from unconverted GC × GC chromatograms,
in which the retention time of the central component was
considered in the case of an odd number of modulated
analyte peaks, while in the case of an even number of
modulated peaks the central retention time between the
two most internal peaks was considered. Furthermore, by
using a column set consisting of a non-polar 30 m × 0.25 mm
i.d. primary column and a polar 1 m × 0.25 mm i.d.
secondary capillary, the authors considered the influence
of the polar 2D column negligible for the less polar
analytes, in terms of retention, but relevant for the more
polar ones, which were strongly retained.
A different approach was used in the analysis of nine
lavender oils (Lavandula angustifolia);84 the correlation
of the linear retention indices from GC–MS analysis and
the GC × GC retention data was successfully used to identify
unknown compounds in the oils. About 70% of the
total number of components in each sample was reliably
characterized and 85 components were identified. The latter
compounds accounted for more than 95% of the oil’s
Copyright © 2008 John Wiley & Sons, Ltd.
composition. Furthermore, ordered structures were observed
within a 2D contour plot; a monoterpene hydrocarbons
region could be observed, as was also a similar region at
higher 1tR (1D retention time) corresponding to sesquiter-
pene hydrocarbons. The oxygenated derivatives of both
these groups are generally encountered closely after the
elution of the main groups in the 1D.
In order to improve peak identification in GC × GC,
retention index databases can be used for optimization
and provisional peak identification.85 At present, only 1D
retention index databases derived from monodimensional
GC are available and several papers report their use in
GC × GC. Western et al.86,87 proposed a method to generate
a retention index database also for the 2D separation. The
repeated analyses during a GC × GC run of reference
compounds, such as n-alkane, 2-methyl ketones and
FAMEs, were carried out by timed sequential solution
injection, and the 2D retention indices were further
interpreted by the authors as pseudo-isothermal retention
indices, based on the isovolatility curves discussed by
Beens et al.88,89
Concluding Remarks
Retention data can be considered a robust tool for
qualitative gas chromatographic analysis. In many cases,
the identification of an unknown compound is supported
by correlation between retention times, or retention tem-
peratures, for that unknown and a series of closely related
standard substances on two different stationary phases
presenting distinct polarities. Besides, the narrow peaks
inherent to capillary gas chromatographic applications are
well suited to qualitative analysis through retention data.
It is certainly true that the utilization of selective or spectro-
scopic detectors provides additional and indispensable
information for positive identity confirmation.
Obviously, it is not feasible to report all the research
work carried out on retention index systems, as also all the
results that have been obtained by individual research
groups. The widespread use of retention indices in flavour
and fragrance materials analysis is a known fact, although
the need is towards a uniform expression through the
normalization of those indices. Particular attention should
be paid not only to repeatability, but also to reproducibility,
which is far from acceptable in many cases.
As a consequence, Flavour and Fragrance Journal
enforces in the instructions for authors the need for replicated
sample preparation and analysis, as well as the use of
appropriate statistical treatment to the resultant data. The
demand for reliable compound identification and quantifi-
cation is also of utmost importance. Compound identification
must be validated by at least two different methods, with
the most commonly applied being GC and MS. Moreover,
assuming the use of GC data for identification, calculated
and reference retention indices of each identified compound
Flavour Fragr. J. 2008; 23: 297–314
DOI: 10.1002/ffj
 LINEAR RETENTION INDICES IN GAS CHROMATOGRAPHIC ANALYSIS 313

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Copyright © 2008 John Wiley & Sons, Ltd. Flavour Fragr. J. 2008; 23: 297–314
DOI: 10.1002/ffj