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Current Analytical Chemistry, 2013, 9, 61-70 61

Recent Advances in Analysis of Essential Oils

Andrija Smelcerovic1,*, Aleksandra Djordjevic2, Jelena Lazarevic1 and Gordana Stojanovic2

1
Department of Pharmacy, Faculty of Medicine, University of Nis, 18000 Nis, Serbia; 2Department of Chemistry,
Faculty of Science and Mathematics, University of Nis, 18000 Nis, Serbia

Abstract: This review provides an overview of the emerging trends in analysis of essential oils and indicates future
trends. Since early human history the essential oils have been used in folk medicine, food and cosmetic industries in
various parts of the world. GC-FID is the traditional method for essential oils quantification while GC-MS is the most
common analytical method for qualitative analysis. Chiral GC allowed the identification of a great number of chiral
essential oil constituents. An alternative to GC analysis is HPLC chromatography. The use of hyphenated techniques, such
as LC-MS-MS provides important information about the structure of essential oils constituents. However, only a
comparatively small number of reports on essential oil analyses by HPLC can be found in the literature. Multidimensional
chromatography is an approach capable of providing greater resolution.13C-NMR spectroscopy is a complementary tool
for analysis of essential oils. Advantage of this method compared to mass spectrometry is identification of stereoisomers
and thermally unstable compounds while its disadvantage is inability to identify the minor oils constituents. 1H-NMR as
an online tool for GC analysis showed promising results but requires further research to be applied on the analysis of
essential oils. The range of information obtained from essential oils analysis enables the application of chemometrics.

Keywords: Essential oils analysis, GC, HPLC, Multidimensional chromatographic techniques, NMR, Chemometrics.

1. INTRODUCTION and elucidating thousands of new essential oil compounds

over the last few decades, we have learned to understand the
Since the middle ages, essential oils have been widely
complexity and enormous diversity in this group of natural
used for bactericidal, virucidal, fungicidal, antiparasitical,
products [2].
insecticidal, medicinal and cosmetic applications, especially
nowadays in pharmaceutical, sanitary, cosmetic, agricultural The quality control of essential oils is necessary to ensure
and food industries. A big advantage for the medical applica- the genuineness of the product, the shelf life, and the storage
tions of essential oils is the fact that they are usually devoid conditions [3]. Gas chromatography in combination with
of long-term genotoxic risks. Moreover, some of them show mass spectrometry is the most widely used technique for
a very clear antimutagenic capacity which could well be determination of the chemical composition of essential oils.
linked to an anticarcinogenic activity. In nature, essential A very large number of publications over recent decades
oils play an important role in the protection of the plants [1]. have dealt with the GC-FID and GC-MS analysis of essen-
tial oils from different plant species. The excellent review by
The composition of essential oils (the volatile fraction of
Marriott et al. [4] outlines the developmental nature of
a plant matrix), due to physicochemical constraints on vola-
instrumental approaches to essential oil analysis using gas
tility, is limited to low-molecular and largely lipophilic
chromatography until 2001. HPLC chromatography re-
products that belong to the classes of terpenes and non-
presents a supplementary or even alternative method for
terpene alyphatics, phenylpropanoids and benzenoids. Ter- analysis of some essential oils and/or some class of
penes can be considered as the most structurally diverse
compounds. For detection, a diode array detector or a mass
class of plant natural products, built up from the repetitive
spectrometer can be used. Multidimensional GC and LC as
fusion of isoprene subunits. It is important to point out that
well as LC-GC are novel techniques which have been al-
an essential oil profile is closely related to the extraction
ready demonstrated as very useful for analysis of complex
procedure employed. Essential oils are usually obtained by
matrices such as essential oils [5]. The NMR spectroscopy
hydro-, steam- or dry-distillation or by expression (mechani- provides important information about the structure of
cal process without heating used for Citrus fruits) of a plant
essential oils constituents and it is mainly used for
or some plant parts. At present, approximately 3,000 essen-
confirmations of the results of GC-MS analysis.
tial oils are known, 300 of which are commercially impor
tant especially for the pharmaceutical, agronomic, food, As noted above, our knowledge about essential oil
sanitary, cosmetic and perfume industries [1]. Discovering composition has markedly increased in the recent decade.
Moreover, a great deal of new information has become
available concerning new analytical techniques for essential
oil analysis. For those reasons, it seems to be appropriate to
*Address correspondence to this author at the Department of Pharmacy, review recent advances in analysis of essential oils. In gen-
Faculty of Medicine, University of Nis, 18000 Nis, Serbia; Tel: +381 18
4570029; Fax: +381 18 4238770. E-mail: [email protected] eral, the present survey focuses on the articles published in

1875-6727/13 $58.00+.00 © 2013 Bentham Science Publishers

62 Current Analytical Chemistry, 2013, Vol. 9, No. 1 Smelcerovic et al.

the last decade about the analysis of essential oils as well as means of a combustion interface has proved to be a valuable
to indicate perspectives for the use of some analytical tech- method to evaluate the genuineness of oils. By commercially
niques in the future. It will first focus on the mono and mul- available GC-C-IRMS the measurements of the ratios of the
tidimensional chromatographic techniques, followed by a stable isotopes 13C/12C have become accessible and respec-
description of application of NMR and chemometrics as tive investigations have been reported in several papers [10-
emerging trend in essential oil data elaboration. Finally, 12].
probable future trends will be discussed.
2.1.1. High-speed GC in Essential Oil Analysis
2. MONODIMENSIONAL CHROMATOGRAPHIC The high-speed GC separation is based on the reduction
TECHNIQUES of the analysis time, usually needed for conventional GC,
while maintaining the separation and providing reliable
2.1. Gas Chromatography
qualitative and quantitative results. The analytical technique
The complexity and the diversity of molecular structures can be accomplished by manipulating the number of parame-
that can be found as essential oil constituents demand ters, such as the length and the internal diameter of the GC
chromatographic procedures with high resolution separation column, stationary phase, film thickness, velocity of the mo-
in order to characterize the contributors. Gas bile phase, oven temperature and ramp rate. The most impor-
chromatography is the analytical technique that has been tant high-speed GC methods with various theoretical and
most frequently applied in essential oils analysis. The practical aspects have been reviewed by Cramers et al. [13]
volatility and polarity of essential oil constituents has made and by Korytar et al. [14]. According to Bicchi et al. [9] an
capillary GC the method of choice. For analytical purposes, important contribution to fast GC in routine analysis was the
volatiles are commonly separated on fused silica capillary method translation approach introduced by Klee and Blum-
columns with different stationary phases, such as the non- berg [15] which makes it possible to find the optimal separa-
polar methyl polysiloxanes (e.g. DB-1, OV-1, OV 101, HP- tion/speed trade-off for a conventional GC method and to
1) and methyl-phenyl-polysiloxanes (DB-5, HP-5, CPSil 5, derive fast GC conditions from it automatically. The defini-
etc.) and polar polyethyleneglycol (Carbowax®, DB-Wax, tions used in rapid GC analysis (fast, very fast, ultrafast)
HP-20M, etc.). Identification is based either relaying on have been defined in the literature by different authors and
chromatographic data (Kovats indices, Linear retention are based on parameters such as analysis time, column effi-
indices, relative retention time, retention time locking) ciency, speed enhancement factor and peak half-width
obtained from a detector (FID, TCD), or by spectral data measures [13, 14, 16, 17]. Over the years instruments and
mainly by mass spectrometry (GC-MS), or preferably by methods were developed to considerably increase the analy-
their combination [6]. Mass spectrometry (MS) detectors are sis speed of capillary GC, enabling the routine use of fast
the most popular type of detector for routine plant volatile GC with short narrow-bore columns and the introduction of
GC analysis. In the mass spectrometers compounds exiting modern GC instruments provided with automatic injectors,
the GC column are ionized by electron impact (EI) and/or electronic flow control, ovens with high precision tempera-
chemical ionization (CI) and the resulting charged molecules ture and temperature rate controls, detectors with high sensi-
and molecule fragments are selected according to their mass- tivity, electronic stability and frequency of signal acquisi-
to-charge (m/z) ratio by entering a quadrupole ion trap or a tion. The success of fast GC required the development of
quadrupole mass filter. Total ion chromatograms are mass detectors with quadrupole and time-of-flight analysers
obtained, which provide information on the retention time of capable of acquiring a number of reliable spectra per second
each compound and its mass spectrum consisting of a compatible with peak width deriving from fast GC to afford
characteristic ion fragmentation pattern. Detection limits of safe automatic analyte identifications and/or single monitor-
highly sensitive mass spectrometers are in the picogram ing-MS quantification [9]. For speeding up the analysis of
range for the full scan mode (scanning ions over a wide plant volatiles, two approaches are available. The most
molecular range) and may be as low as in the femtogram widely used one is the narrow-bore column approach [5, 18-
range (in quadrupole mass filters) in the selected ion 20]. The second one is the short capillary column approach
monitoring (SIM) mode scanning selected ions that are that has been proved to be useful in the rapid (routine)
representative of a compound [7]. Besides mass analysis of low to medium complexity samples [21]. An ex-
spectrometry, Fourier transform infrared spectroscopy (FT- cellent review by Donato et al. [22] and the references cited
IR) has also been proposed as a GC detector (GC-FT-IR) in therein) dealing with rapid analysis of food products along
analysis of volatiles. This spectroscopic method has been with essential oils has covered all the literature available
employed to differentiate closely related isomers with very published up to 2006. In the mean time, Rubiolo et al. [23]
similar EI mass spectra [8]. Fourier transform infrared spec- investigated in depth the compatibility of fast GC-qMS in
troscopy provides information on the intact molecular struc- essential oil analysis, in a study dealing with the separation,
ture and allows unique spectra to be obtained even for simi- identification and quantification of ten components of pep-
lar isomers. The utility of technique was limited by difficul- permint essential oils. Their results showed that, operating at
ties in quantification and time-consuming data interpretation, a suitable scan speed in the total ion current (TIC) mode
because of lacking of commercial available IR-spectra librar- (from 999 to 11,000 amu/s) or at suitable dwell time in the
ies [9]. The same was with the gas chromatography- single ion monitoring (SIM) mode (10 to 50 ms), the results
ultraviolet spectroscopy (GC-UV) and gas chromatography- obtained with fast-GC-qMS with a 10 m, 0.1mm i.d. column
atomic emission spectroscopy (GC-AES). Both of the meth- combined with temperature programmes from 20 to 60
ods have not gained much importance in the field of essen- ˚C/min are fully comparable, and in some cases better, than
tial oil research. Nevertheless, gas chromatography corre- those obtained by conventional GC-qMS, while the analysis
lated to isotope ratio mass spectrometry (GC-IRMS) by
Recent Advances in Analysis of Essential Oils Current Analytical Chemistry, 2013, Vol. 9, No. 1 63

time is reduced by a factor of about ten (from about 35 to 3- chromatogram and to generate an odor profile for the whole
4 min). essential oil sample [33].
2.1.2. Enantioselective Gas Chromatography (ES-GC) 2.2. High Performance Liquid Chromatography
The development of stable chiral phases for gas HPLC-UV may represent a supplementary or even
chromatography, mostly based on cyclodextrins, has allowed alternative method for analysis of volatile oils because of its
detailed study of enantiomeric composition of volatile com- versatility, sensitivity, and selectivity [40]. Usually, HPLC is
pounds [24, 25]. Analysis of such a type has become impor- the method of choice in the analysis of less volatile constitu-
tant for detection of adulterants, characterization of oil pro- ents of essential oils [41]. The use of HPLC-MS provides
files for quality control and what is more important, has fa- great information about the content and nature of constitu-
cilitated investigations into the underlying mechanisms asso- ents of natural complex matrices, such as essential oils [42].
ciated with the biosynthesis of the terpenoids [4]. A great Atmospheric pressure chemical ionisation (APCI) is suitable
number of essential oils have already been investigated by for less polar compounds and of lower molecular weight
means of ES-GC using distinct chiral stationary phases, but than electrospray ionisation (ESI) [42]. Despite the above
it has appeared that effective separation may not be achieved mentioned advantages of HPLC yet, only a comparatively
on a single chiral column. Bicchi et al. [26] developed col- small number of reports on essential oil analyses by HPLC
umns that addressed particular problem chiral separations, can be found in the literature [43].
noting that certain phases preferentially resolved certain
enantiomers. Even over 100 of stationary phases with im- Piry and Pribela [44] applicated the reversed-phase
mobilized chiral selectors were used so far, there is no uni- HPLC-UV for the analysis of the complex volatile mixture
versal chiral selector with widespread potential for enanti- of blackcurrant buds (Ribes nigrum L.). Bonaccorsi et al. [3]
omer separation [6]. Chiral recognition of a complex sample developed a fast HPLC method for the determination of the
components often need a two dimensional approach [9]. The oxygen heterocyclic compounds of citrus essential oils. Five
innovations in ES-GC are not related only with continuous different oils were analyzed by reversed-phase HPLC with
development and applications of diverse chiral stationary photodiode array detector. The method developed is good
phases, but also with the development of enantioselective for rapid screening or fingerprinting of these essential oils; a
analytical techniques. Some of the techniques recently used slightly slower method is recommended for higher resolution
for essential oil samples analysis are: ES-GC-mass spec- and better quantitative results. Hudaib et al. [45] analyzed
trometry (ES-GC-MS) and fast ES-GC-MS [27], ES-GC-O essential oils hydrodistilled from both healthy and naturally
as the technique that has proven to be a valid tool for the infected by broad bean wilt fabavirus, serotype I (BBWV-I)
simultaneous stereodifferentiation and olfactive evaluation Salvia sclarea plants by GC-MS and HPLC-UV systems.
of components present in oils [28]. ES-GC hyphenated to Although not marked, the chemical differences in the oil
isotopic ratio mass spectrometry is the most currently avail- compositions were well established and useful to character-
able approach to prove a sample authenticity as being the ize the product, whose pharmacological activity is frequently
most effective tool to detect adulterations of essential oils reported to correlate with its composition.
containing optically active components (ES-GC-IRMS) [12]. Dugo et al. [42, 46] analysed the oxygen heterocyclic
Advantages and currently used techniques in chiral recogni- compounds (coumarins, psoralens, and polymethoxylated
tion of volatiles have already been published and discussed flavones) present in citrus essential oils (lemon, mandarin,
in more details in two recent reviews by Rubiolo et al. [6] sweet orange, bitter orange, bergamot, grapefruit, and lime)
and Bicchi et al. [9]. by HPLC-MS system equipped with APCI source. Some
2.1.3. Gas Chromatography-olfactometry (GC-O) minor components were detected for the first time in some
oils [46]. Giuffrida et al. [47] reported the application of
There is no simple generalization about the key odorants liquid chromatography-atmospheric pressure chemical ioni-
in essential oil; the odor of some essential oils is due to a
zation mass spectrometry (LC-APCI-MS) with a C-30 re-
large amount of a single compound, while trace amounts
versed-phase column in the separation and identification of
could determine the odor of others, and in most cases the
the main carotenoids esters present in mandarin essential oil.
true odor is the manifestation of a complex mixture of com-
Good amounts of
-cryptoxanthin and its esters were deter-
pounds [29]. GC-O is a well-known standard technique
mined, indicating mandarin peel as a potential source of vi-
which enables the assessment of odor-active components in
complex mixtures, based on the correlation between the tamin A precursors. Turek and Stintzing [43] developed an
chromatographic peaks of the eluted substances perceived HPLC-DAD/APCI-MS method suitable to detect essential
simultaneously by two detectors, one of them being the hu- oil components of diverse chemical classes.
man olfactory system. Over the years, the GC-O has been Ogawa et al. [48] describes the evaluation of alternative
frequently employed in essential oil analysis [30-35], espe- techniques, for the determination of selected volatile com-
cially in Citrus sp. [36-38] by using different assessment pounds in cold-pressed orange oil. Two techniques were
methods [39]. Results of GC-O analysis can give informa- investigated: capillary-LC (CLC) and micellar electrokinetic
tion about the presence/absence of odor in a compound, chromatography (MEKC). Capillary-LC allowed the deter-
measure the duration of odor activity, describe the quality of mination of terpenes such as mircene, limonene,
-pinene,
the odor perceived, and quantify the intensity of specific and oxygenated terpenes including linalool, citral, and car-
odorant which may have an ultimate purpose in the applica- vone while, in MEKC, carvone, mircene, linalool, limonene,
tion of such compounds in industry of flavor and fragrances. and citral were determined.
GC-O is also used to locate the odor-active regions in the
64 Current Analytical Chemistry, 2013, Vol. 9, No. 1 Smelcerovic et al.

Smelcerovic et al. [49] used high resolution LC-MS and first to the second dimension was achieved through me-
LC-MS-MS, together with GC, GC-MS and 13C NMR, to chanical valves. This approach was soon abandoned and
determinate the chemical composition of the hydro-distilled replaced by a new interface based on a pressure balance be-
essential oils of Achillea millefolium and Achillea crithmifo- tween two columns, proposed by Deans [54]. Later, the sys-
lia. For the first time, the use of the combination of different tem was improved by Schomburg et al. [55] and largely ap-
chromatographic and spectral methods, primarily the advan- plied in the studies of enantiomeric volatile components in
tage of LC-Orbitrap over standard methods, enabled the de- citrus oils ([5] and references cited therein). A different and
tection of azulenes and their progenitors, in minute quanti- inexpensive approach for transferring individual GC peaks
ties, in previously believed proazulene free Achillea taxon onto a second column has been presented by Kubeczka [56],
(A. crithmifolia). Figure shows the high resolution LC-MS using a solid-phase microextraction (SPME) device while
spectral data of 1,4-dimethylazulene from A. crithmifolia Mondello et al. [57] developed a six rotary mechanical valve
essential oil [49]. Fillatre et al. [50] demonstrates the feasi- as interface, which was later improved by the same research
bility of developing a multiresidue multiclass method for group and used for the determination of enantiomeric pro-
pesticides in essential oils. The developed LC-MS/MS files in citrus essential oils ([5] and references cited therein).
method allows the determination of 67 pesticides with good A detailed study of column combinations and different
sensitivity compared to other published methods referring to modulators used in the analysis of plant extracts, essential
essential oils. oils and chiral compounds was given by Shellie [58].
Considerable improvement in two-dimensional gas
3. MULTIDIMENSIONAL CHROMATOGRAPHIC chromatography occurred when Liu and Phillips [59] intro-
TECHNIQUES duced and implemented an entirely new form of two-
Complex samples, such as essential oils, require analyti- dimensional comprehensive gas chromatography or GC-GC
cal methods characterized by an extremely high resolving in 1991. It is possible to construct a complete two-
power in order to provide thorough analysis of the sample dimensional chromatogram by continuously taking small
components. Multidimensional chromatography is an ap- volume heart-cuts from the first column and subjecting each
proach capable of providing greater resolution [51]. of these to the second-dimension separation. When first ap-
peared, GC-GC almost immediately found its place in petro-
3.1. Multidimensional Gas Chromatography chemical industry and in analyzing environmental pollutants
([53] and references cited therein, [58]). One of the first ap-
Despite significant instrumental technique progress, the plications of GC-GC to essential oils was performed by Di-
detection of all the constituents of an essential oil remains mandja et al. [60], who reported the analyses of spearmint
very difficult task. Due to the complexity of the sample and peppermint essential oils, illustrating a two-to threefold
compositions, a single capillary column is often unable to increase in the separation power of GC-GC over monodi-
resolve completely all the components of interest in an ac- mensional GC. After this one, following reports described
ceptable time. Widespread peak overlapping and the struc- the separation of vetiver [61], tea tree and lavender essential
tural similarity of many terpene compounds is often a hin- oils [62]. These first reports led to the steady development of
drance of reliable MS structural elucidation [5]. Unambigu- GC-GC for essential oils and fragrance analysis, and the
ous identification of minor compounds is extremely difficult approach can now be routinely employed.
to accomplish when high concentration matrix constituents
mask their presence in the sample [52]. Therefore, in many The current knowledge of essential oils is available due
cases two-dimensional GC (GC-GC) has to be performed. to the GC-MS technique, taking into account number of pub-
lished manuscripts, presenting the composition of essential
This technique (GC-GC) implies coupled columns of dif-
oils, determined by GC-MS. Some authors suggest that GC-
ferent polarities and subjects selected analytes to two inde-
MS can be considered as a two-dimensional analysis system
pendent separation steps, characterized by complementary
[53, 58], so hyphenation of GC-GC with mass spectrometry
(or orthogonal) selectivities ([5] and references cited
therein). Usually, the first column is a low-polarity station- therefore represents triple-dimensional analysis [58]. Due to
ary phase column, which affects displacement of the sample the fast second-dimension elution in GC-GC, time-of-flight
components along the x-axis of the two-dimensional separa- mass spectrometry (TOFMS) is the most viable technology.
tion plane, while the second column is a polar stationary This hyphenated approach was used for the characterization
phase column (although the column arrangement is sample of lavender essential oil [63] and has been further optimized
depending). Different compounds undergo different separa- to fully exploit the three analytical dimensions. Very high-
tion mechanisms, and there are no fixed rules to the combi- resolution separations with accurate full mass spectra are
nation of column phase types as long as they provide or- achievable using GC-GC-TOFMS. For example, Wu et al.
thogonal separation. The selected, unresolved effluent frac- [64] identified 394 components in Pogostemon cablin Benth
tions from a first column are transferred onto the second one (Cablin Patchouli) volatile oil, using this technique. A wide
and that process is commonly referred to as heart-cutting. range of plant derived samples was examined by GC-GC-
[52, 53]. The instrumentation involves transfer device TOFMS ([58] and references cited therein).
(modulator), situated between the two dimensions, which The expense of GC-GC-TOFMS, led to the investiga-
presents the key of the system and enables the entire proce- tions of quadrupole MS (qMS) suitability for GC-GC-qMS
dure. system. Relatively slow data acquisition speed may not be
In the first multidimensional gas chromatography suitable for quantitative analysis, but improved quality of
(MDGC) systems, the transfer of eluate fractions from the uncorrected spectra (due to the enhanced separation) over
Recent Advances in Analysis of Essential Oils Current Analytical Chemistry, 2013, Vol. 9, No. 1 65

single-column GC-qMS analysis, is apparent ([58] and refer- 100% optically pure. The results indicated that fractionation
ences cited therein). This system was used for the analysis of of davana oil with supercritical fluids at near room tempera-
Pelargonium graveolens essential oil [65]. ture had little effect on the optical integrity of the sample.
Xie et al., have reported successful resolution of seven ra-
3.1.1. Analysis of Chiral Compounds cemic -lactones (-C6--C12), important flavor and fragrance
The separation of enantiomers by GC using an enantiose- substances, via analytical scale pSFC using the polysaccha-
lective stationary phase was discovered in 1966 [66], while ride chiral stationary phases of Chiralpak AD and the or-
MDGC with an enantioselective separation step (enantio- ganic modifier of iso-propanol [77].
MDGC) was first described in 1984 [67]. The use of enan-
tio-MDGC was reported for the analysis of lavender and 3.2. Multidimensional Liquid Chromatography
peppermint essential oils [68]. The usage of enantioselective Comprehensive two-dimensional LC is a novel technique
MDGC online coupled with isotope ratio mass spectrometry coupling two independent LC separation processes with or-
(MDGC-IRMS) was described by Reichert et al. [69]. thogonal selectivities [78]. If compared to single-dimension
MDGC and enantio-MDGC were utilized in number of stud- chromatography, the separation power of comprehensive
ies refering to citrus essential oils ([58] and references cited two-dimensional chromatography is greatly increased. The
therein). The only suitable experimental arrangement for use of normal- and reversed-phase mode in the two dimen-
enantio-MDGC involves an achiral primary column and an sions can be helpful in the separation of a complex mixture
enantioselective stationary phase second-dimension analyti- of natural origin, which contains uncharged molecules of
cal column. comparable dimension, different in polarity and hydropho-
A very small number of publications have reported the bicity [51]. Paola Dugo and coworkers published several
potential application of GC-GC in analysis of chiral com- manuscripts about the application of comprehensive two-
pounds. The performance of enantioselective GC-GC ex- dimensional LC chromatography for analysis of essential
periment involves two distinctly different approaches. The oils [51, 79, 80]. They analyzed the oxygen heterocyclic
first approach uses the enantioselective column as the first- components of cold-pressed lemon oil using a comprehen-
dimension column and it shall be called "enantio-GC-GC", sive 2D-LC system, which is based on the use of a mi-
while the second uses a short enantioselective column in the crobore silica column operated in normal-phase mode in the
second-dimension position and shall be called "GC-enantio- first dimension and a monolithic type C18 column operated
GC". Both techniques should provide a valid result, and al- in reversed-phase mode in the second dimension [51].
though interpretation of the GC-enantio-GC chromatogram Dugo et al. [79] demonstrated the ability of a LC-LC-
is easier, the experimental set up and operation of the enan- DAD/APCIMS method to analyze and identify the native
tio-GC-GC experiment is technically less demanding ([58] carotenoid composition of an extremely complex matrix
and references cited therein). Enantio-GC-GC is useful as such as red orange essential oil. It was possible to identify 40
analternative to enantio-MDGC which may require multiple different carotenoids in the orange oil including 3 free caro-
heart-cuts and individual temperature programmed analysis tenoids, 16 carotenoid monoesters, and 21 carotenoid di-
of these heart-cuts and the main advantage of GC-GC here, esters, analyzed without any kind of sample pretreatment.
is the possibility of reducing the total analysis time. A num-
ber of enantiomeric monoterpenes (sabinene,
-pinene, li- The same authors [80] used two different comprehensive
monene, -phellandrene, etc.) in tea tree (Melaleuca alterni- HPLC methods to study the whole carotenoid composition
folia) essential oil were determined by enantio-GC-GC [70]. of mandarin essential oil. A silica microbore column was
A second GC-GC approach (GC-enantio-GC) employs a coupled to a C18 monolithic column to study the mandarin
short, fast enantioselective stationary phase column as the saponified extract, while the coupling of a cyano microbore
second-dimension column. The successful enantiomeric column to a C18 monolithic column was employed to study
separation of several monoterpene compounds in bergamot the intact mandarin essential oil sample in order to character-
essential oil was determined by GC-enantio-GC [71]. ize the native carotenoid esters composition. Detection was
performed by connecting a photodiode array detection
GC-GC approach cannot match MDGC performance for (DAD) system in parallel with a MS detection system oper-
enantioselective applications. At this time, conventional and ated with an atmospheric pressure chemical ionization
rapid enantio-MDGC are the most powerful techniques for (APCI) interface.
enantioselective analysis, in terms of speed and resolution
applications. Recent investigations, conducted by Mondello 3.3. Multidimensional Liquid–Gas Chromatography
et al. [72], on the rosemary essential oil composition, illus- (LC–GC)
trate all advantages of MDGC method.
The analysis of essential oils composition is often com-
Even though the on-line coupling of supercritical fluid plicated by the fact that sample components belong to a vari-
chromatography has sporadically been used for the investi- ety of chemical classes and are present in a wide range of
gation of volatiles from aromatic herbs and spices [73-75], concentrations. The multidimensional LC–GC approach
chiral packed column supercritical fluid chromatography combines the selectivity of the LC separation with the high
(pSFC) may be promising in enantioseparation of fraction- efficiency and sensitivity of GC separation, enabling the
ated essential oils. A semi preparative separation of natural separation of compounds with similar physicochemical
davana oil that yielded three fractions by pSFC with CO2 properties in samples characterized by a great number of
based mobile phase was consequently developed on a 2- chemical classes [5]. This technique was first applied to cit-
ethylpyridine phase [76]. The davanone fraction was nearly rus essential oil analysis and later on-line LC-GC coupled
66 Current Analytical Chemistry, 2013, Vol. 9, No. 1 Smelcerovic et al.

Fig. (1). High resolution LC-MS spectral data of 1,4-dimethylazulene from Achilea crithmifolia essential oil [49]

with Fourier transform infrared spectroscopy (FTIR) was the spectra of pure compounds recorded under identical con-
used for the determination of some components in bergamot ditions.
oil sample. The LC-GC technique was greatly exploited for
Karl-Heinz Kubeczka, a pioneer in the application of
the study of single classes of components, such as industrial 13
C-NMR in the analysis of essential oils, gave an overview
citrus oil mono- and sesquiterpene hydrocarbons, aldehyde
of analytical techniques applied in the analysis of oils in-
composition in sweet orange oil and the enantiomeric distri-
cluding 13C-NMR [82]. Previously, he published with For-
bution of monoterpene alcohols in lemon, mandarin, sweet
mácek [83] a book containing 13C-NMR spectra of 60 com-
orange, and bitter orange oils. The hyphenation of LC-GC
mercial oils important for perfumery, cosmetic, pharmaceu-
system to a mass spectrometer (LC-GC-MS) increases the
tical and food industry as well as spectra of 188 their most
potential of the technique; preliminary LC separation, which common components. These spectra provide the authentica-
reduces mutual component interference, greatly simplifies
tion of commercial oils and identification of individual con-
MS identification ([5] and references cited therein).
stituents of examined oils.
13
4. CARBON-13 AND PROTON NUCLEAR MAG- C-NMR spectra are used mostly in conjunction with
NETIC RESONANCE SPECTROSCOPY GC-MS data [49, 84, 85]. However, advantage of this
method compared to GC-MS is identification of thermally
Nuclear magnetic resonance (NMR) is an analytical unstable compounds [86], stereoisomers [87] and other com-
technique that provides the most information of all spectro- pounds which exhibit insufficiently resolved mass spectral
scopic methods for determination of the structure of pure patterns or which co-eluate on GC [85].
compounds [81]. In this sense, use of NMR in the analysis of
essential oils constituents is determined by the development Formácek and Kubeczka [88] developed a procedure for
of preparative chromatography in isolation of pure sub- quantification of the oils components using average signal
stances from oils. intensity per carbon atom as a measurement characteristic
after elimination of the 13C-NMR signals of nonprotonated
Application of NMR in the analysis of complex mixtures nuclei. The obtained results were in good agreement with
such as essential oils, prior separation of the individual gas chromatographic quantitative analyses. Cavalli et al.
components, is limited due to its low sensitivity, great [86], determined the actual content of a heat-sensitive com-
number of signals and their overlapping as well as pound, ascaridol by 13C-NMR spectroscopy. GC analysis
difficulties in coupling of GC to NMR. In general, 13C-NMR gave lower concentrations of ascaridol due to its partial
is used of line, while 1H-NMR is applied on line. thermal isomerisation to isoascaridole.
4.1. 13C-NMR Spectroscopy Different computer aided techniques have been presented
13
to facilitate the identification of chemical constituents pre-
The identification of essential oils constituents by C- sent in essential oils through analyses of 13C-NMR data.
NMR is based on comparison of oils 13C NMR spectra with Some of them based on chemical shifts and signal intensity
Recent Advances in Analysis of Essential Oils Current Analytical Chemistry, 2013, Vol. 9, No. 1 67

[85]. The others consider chemical shifts and multiplicities tial oils constituents. It is therefore suggested that HPLC
obtained from the 13C-NMR DEPT spectra [89]. should be more often considered as a versatile method for
essential oil analyses complementary to GC in the future
4.2. 1H-NMR Spectroscopy [43]. Furthermore, the analysis time is becoming an impor-
Powerful analytical technique is obtained by on line cou- tant issue today, and therefore fast GC and HPLC methods
are needed. If shorter columns are used, with smaller particle
pling of GC and NMR as the excellent separation character-
size, it is possible to achieve good efficiency in a shorter
istics of gas chromatography is combined with NMR data
analysis time [3]. Our recent study showed that capillary-LC
about the structure of oil constituents. Commercial available
coupled with a sensitive high-resolution spectrometer is able
NMR instruments with high magnetic field and microprobes
to provide a rapid and accurate (target) screening of small
are allowed recording of NMR spectra in the gaseous phase.
Albert and co-workers [81, 90] demonstrated the potential of amounts of essential oil constituents [49]. In many cases, the
amount of the essential oil sample is too small to be detected
the hyphenation of capillary GC to solenoidal-type micro-
with conventional LC instruments, and therefore the use of a
probe 1H-NMR detection as a technique for the analysis of
capillary-LC instrument that offers small injection volumes
volatile compounds. Grynbaum et al. [90] used a custom-
and lower limits of detection is a big advantage. Of line
built solenoidal NMR microprobe with an active volume of
NMR spectroscopy already has a wide application in identi-
2 L for the NMR detection of several volatile compounds
(diethyl ether, tetrahydrofuran, acetone, and dichloro- fication of essential oils constituents.13C-NMR spectroscopy
allowed the identification of stereoisomers and thermally
methane) at 400 MHz. The injected amounts of each analyte
unstable compounds. On line NMR spectroscopy is still in
in the hyphenated experiments were in the range of 15-50
development. In the future the hyphenation of GC and NMR
mol. Kühnle et al. [81] detected cis/trans 2-pentene and
may be evolved into a potent tool in the analysis of essential
cis/trans 2-hexene with the sample amount (for each com-
oils. It can be expected a greater use of multidimensional
pound) 100-300 g at 400 MHz.
(GC-GC, LC-LC and LC-GC) techniques for separation of
essential oils. Due to capability of multidimensional chroma-
5. CHEMOMETRICS AS EMERGING TREND IN ES- tography for discovery of new compounds it can be expected
SENTIAL OILS DATA ELABORATION
new technical innovations in this technology. Whatever the
The range of information that can be obtained from a future bring, we do not expect the dominance of GC-FID
"conventional" analysis of a plant volatile fraction has been and GC-MS like in the last decades. The use of the combina-
extended due to recent progress in chemometrics and soft- tion of different chromatographic and spectral methods will
wares for statistical elaboration [9]. Samples under investi- certainly be optimal for structure determination of the con-
gation are usually statistically preceded according to their stituents of essential oils. It is expected the increased use of
chromatographic profiles defined by the abundance of a se- chemometrics for statistical elaboration.
lected number of markers set as variables. The most widely
used approach is multivariate analysis, in particular principal CONFLICT OF INTEREST
component analysis (PCA), a method that can explain the
The author(s) confirm that this article has no conflicts of
differences within a set of samples characterized by a suit-
able number of components (variables) through the linear interest.
combination of those explaining most of the variability [9].
This method allows differentiating among groups of samples ACKNOWLEDGEMENTS
within a set, pointing at variables (individual components or This work was supported by the Ministry of Education
MS fragments) that have the highest statistical significance and Science of the Republic of Serbia (project 172044).
in sample discrimination, providing valuable information
upon origin and/or chemotypification [6, 9, 91], differentia- REFERENCES
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Received: October 10, 2011 Revised: December 21, 2011 Accepted: December 21, 2011