4 Gas Chromatographic Analysis of Plant and Insect Surface Compounds : Cuticular Waxes and Terpenoids

The surfaces of all higher plants are covered by a layer of cuticular waxes. These are composed mainly of long-chain aliphatic components but also of cyclic compounds. The primary role of the waxes is to prevent uncontrolled water loss. The chemical composition of plant cuticular waxes can affect the resistance of plants to herbivores and herbivore behaviour. Cuticular waxes and their separate components enhance or deter insect oviposition, movement or feeding.


Introduction
The surfaces of all higher plants are covered by a layer of cuticular waxes.These are composed mainly of long-chain aliphatic components but also of cyclic compounds.The primary role of the waxes is to prevent uncontrolled water loss.The chemical composition of plant cuticular waxes can affect the resistance of plants to herbivores and herbivore behaviour.Cuticular waxes and their separate components enhance or deter insect oviposition, movement or feeding.
Most plants have trichomes on their aerial surfaces.The trichomes may be simple hairs or more specialized glandular trichomes, whose main function may be the production and accumulation of chemicals such as essential oils.The vast majority of these consists of monoterpenoids, sesquiterpenoids and diterpenoids with a high vapour pressure.They may be absorbed on the cuticular wax layer.The trichome secretions are closely related to plantinsect or plant-microbe interactions.Terpenoids can attract, repel or initiate defence reactions in insects.Apart from their ecological roles, plant terpenoids are widely used in the pharmaceutical and fragrance industries.The properties of essential oils are correlated with their qualitative and quantitative compositions.
The surfaces of insects are also covered by a layer of wax.Insect cuticular waxes are also involved in various types of chemical communication between individuals of a species and reduce the penetration of chemicals and toxins as well as infectious microorganisms.Analyses and identification of insect waxes is the first step towards developing methods of insect control.
Improvements in analytical techniques have led to the characterization of plant and insect surface compounds and provided new insights in chemical ecology.Moreover, an enormous number of plant terpenoid analyses are associated with their pharmaceutical and fragrance applications.Qualitative and quantitative analyses of cuticular waxes and terpenoids are usually achieved by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS).Peak identification is based primarily on retention times, retention indices and comparison of recorded spectra with an MS library.This review will describe gas between species.Hydrocarbons, e.g.n-alkanes, n-alkenes and methyl-branched hydrocarbons, often constitute a major fraction of insect cuticular waxes: they were found to be the major compounds in the cuticular waxes of adults, nymphs and eggs of Zygogramma exclemationis (Nelson and Charlet, 2003).79.4% of cuticular waxes isolated from Calliphora vicina larvae were fatty acids (Gołębiowski et al., 2008a).In contrast, the fatty acids in D. pini exuviae extracts made up only 2.0% of all waxes (Gołębiowski et al., 2010).Alcohols ranging from C 24:0 to C 34:0 constituted the major lipid fraction of the cuticular waxes from Heliothis virescens pupae (Buckner et al., 1996).Wax esters consisting of even-numbered-carbon compounds from C 38 to C 64 were the major compounds (86%) present in the cuticle of Bemisia argentifolii nymphs and exuviae (Buckner et al., 1999).Cuticular waxes of insects also contain aldehydes as minor constituents.This group of waxes were identified in Heliothis virescens pupae, Helicoverpa zea pupae (Buckner et al., 1996), Aleurodes singularis adults and exuviae (Nelson et al., 1998), Bemisia argentifolii nymphs and exuviae (Buckner et al., 1999), Bemisia tabaci and Trialeurodes vaporariorum adults (Buckner et al., 1994).Triacylglycerols are compounds of cuticular waxes but they could also be contaminants derived from body lipids.For example, triacylglycerols were identified in the waxes of Melanoplus sanguinipes and M. packardii adults (Soliday et al., 1974).Fatty acid methyl esters are not common constituents of insect cuticular waxes.Both methyl and ethyl esters were identified in the cuticular waxes of Acanthoscelides obtectus Say (Gołębiowski et al., 2008b).Like fatty acid methyl esters, ketones are minor constituents of cuticular waxes.The cuticular lipid of Ceutorrhynchus assimilis contains 98% of C 29 , smaller amounts of C 26 (<1%) and traces of C 28 , C 30 and C 31 (Richter & Krain, 1980).The ketones of C. assimilis are very similar to those in the surface waxes of Brassica napus, which suggests that they could be of host plant origin.Sterols are also minor constituents of cuticular lipids.Cholesterol is a cell membrane component in insects and a precursor of steroid hormones such as ecdysone.Insects lack the capacity to synthesize sterols, so they convert phytosterols to cholesterol via dealkylation.Cholesterol is the most abundant sterol in the cuticular waxes of insects, occurring, for example, in the waxes of M. sanguinipes and M. packardii (Soliday et al., 1974).

Isolation and class separation of plant cuticular waxes
Plant cuticular waxes are usually extracted by dipping the plant material in an organic solvent of intermediate polarity for 10 -60 sec (Bakker et al., 1998;Hamilton, 1995b;Jetter et al., 2006;Stammitti et al., 1996).The solvents used include chloroform, dichloromethane, hexane, petroleum ether and toluene.A two-step extraction with the use of two portions of solvent may improve recovery.The classical extraction method is usually carried out at room temperature but occasionally at the boiling point of the solvent for the complete extraction of insoluble polymeric forms of aldehydes (Haas et al., 2001;Szafranek et al., 2008a).The different sides of the same plant organ (the adaxial and abaxial sides of leaves) may be characterized by cuticular waxes with different compositions, so their extraction is more complicated (Jetter et al., 2000;Buschhaus et al., 2007a).Epicuticular waxes can also be isolated by mechanical methods (Ensikat et al., 2000;Jetter et al., 2000;Riedel et al., 2007), which distinguish the epicuticular waxes outside the plant cuticle from the cuticular waxes embedded in the cuticle matrix.The extraction of 1 to 10 cm 2 of the plant surface area should yield a sufficient amount of wax for a single GC-FID or GC-MS analysis (Jetter et al., 2006).
Gas Chromatography in Plant Science, Wine Technology, Toxicology and Some Specific Applications 42 GC is the most commonly used method to selectively identify and quantify the components of cuticular waxes.A schematic diagram of the procedures used in the analyses of plant cuticular waxes is given in Figure 1.Because of the complex nature of plant and insect cuticular waxes, fractionation according to chemical class is usually required before identification of individual components can be achieved.Special care has to be taken in the case of quantitative analyses.Internal standards representing all compound classes should be added early during the wax extraction before fractionation.Traditionally, separation schemes have been developed by using preparative column chromatography (LC) or thin layer chromatography (TLC) (Hamilton, 1995b).A typical example might involve LC separation on silica gel or alumina and gradient elution with increasing concentrations of ethyl acetate in petroleum ether.The separation can then be monitored by TLC.A rapid form of LC is flash chromatography where solvent is forced down the column by air pressure (Still et al., 1978), a procedure that has recently been used for the fractionation of eggplant cuticular waxes (Haliński et al., 2009).High-performance liquid chromatography (HPLC) has not been widely used in the analysis of cuticular waxes since these have no useful UV chromophores and because of the difficulties of separating components with different polarities (Hamilton, 1995b).Hwang et al. (2002) developed an HPLC method using a silica gel column and an evaporative light scattering detector (ELSD) to analyse sorghum wax.Szafranek & Synak (2006) and Szafranek et al. (2008b) used HPLC-ELSD for the preparative fractionation of potato cuticular waxes.HPLC separation of cuticular waxes has a better resolution and is more efficient than column chromatography.
Solid-phase extraction (SPE) is today the most popular sample preparation method but still hardly used in the fractionation of plant cuticular waxes.Nass et al. (1998) has developed a method involving the separation of the wax sample into two fractions using simple SPE in order to avoid artefacts in trimethylsilyl derivatization; the fraction containing aldehydes could be GC analysed without silylation.Perez-Camino et al. (2003) have described a procedure for the simultaneous determination of long-chain aliphatic aldehydes, and aliphatic and triterpenic waxes in olive oils.A fraction containing these compounds was isolated from the oil using SPE on silica-gel cartridges.Commercial SPE cartridges are convenient for the isolation of lipid classes, so they may be used more extensively in the Analytical TLC

Wax extract
Derivatization Preparative LC, TLC, HPLC or SPE www.intechopen.comfuture.Solid phase microextraction (SPME) is a promising new technique for sample extraction/isolation before GC analysis, but as far as we know, it has been applied to the cuticular waxes of insects, but not yet to those of plants.

Isolation and separation of insect cuticular waxes
Extraction procedures aim to remove all of an insect's cuticular waxes.For this, a non-polar solvent is usually used, for example, pentane, hexane and petroleum ether for 10 seconds or chloroform for 1 min (Gołębiowski et al., 2011;Nelson & Blomquist, 1995).The use of a nonpolar solvent minimizes the possible extraction of internal lipids, which are mostly free fatty acids and glycerides.Non-polar solvents are used for extracting waxes rich in hydrocarbons, whereas chloroform extracts the oxygenated compounds in cuticular waxes.Chloroformmethanol mixtures are used for short extractions to avoid the possible extraction of internal waxes.The choice of extraction solvent and extraction time depend on the quantity and quality of the waxes.For example, the cuticular waxes of the larvae and adults of khapra beetles Trogoderma granarium were obtained by immersing the insects in dichloromethane for 20 seconds (Maliński et al., 1986).A longer extraction was used to separate the cuticular waxes of four Periplaneta species (Said et al., 2005); in this case, insects anaesthetized in CO 2 were immersed in dichloromethane for 2 min.A 30-second immersion in chloroform was sufficient to obtain the cuticular waxes from Tribolium destructor larvae and beetles (Hebanowska et al., 1990).One immersion does not extract all of the cuticular waxes, however: two short extractions are more effective for the complete removal of the cuticular waxes without extraction of internal waxes.Sometimes, internal waxes are extracted because the cuticle is damaged or the extraction time is too long.Triacylglycerols are major components of internal waxes, but they are present in the cuticular waxes of insects, too.The presence of triacylglycerols in cuticular wax extracts can be a useful indicator that internal waxes have been extracted.A two-step extraction was used for the cuticular waxes of adults of Frankliniella occidentalis (Gołębiowski et al., 2007).The insects were first extracted in petroleum ether for 30 s, after which they were immersed in dichloromethane for 2 min.
Wax extracts are separated into classes of waxes by TLC, LC or HPLC.Table 1 gives examples of the methods for separating insect cuticular waxes.Recently, SPME and solid injection (SI) have been used for extracting cuticular waxes of insects.SPME and SI can be used in combination with GC or GC-MS.SPME is a sample preparation technique using a fused-silica fibre coated on the outside with an appropriate stationary phase.The compound in the sample is directly extracted and concentrated on the fibre coating.The method saves preparation time and solvents, and it can improve detection limits.SPME can be used routinely in combination with GC, GC-MS, HPLC and liquid chromatography-mass spectrometry.SPME-GC and SPME-GC-MS are applicable to the extraction of volatile and semi-volatile organic compounds from biological samples.The following types of stationary phase can be used: non-polar polydimethylsiloxane (PDMS), polar polyacrylate (PA), Carboxen (Car)/divinylbenzene (DVB), Carbowax/templated resin (polar), mixed-polarity PDMS/DVB, Car/PDMS and DVB/Car/PDMS (Gołębiowski et al., 2011).Compounds of different volatility and polarity can be sampled simultaneously by carefully selection of the polarity and thickness of the fibre coating.The application of SPME to the analysis of insect cuticular hydrocarbons is based mainly on direct contact (DC-SPME) between the fibre and the cuticular surface.SPME adsorption from heated samples (headspace SPME, HS-SPME) is also used; in this technique the fibre is exposed to the vapour phase above a gaseous, liquid or solid sample.The cuticular fatty acid methyl esters from Calliphora vicina were analysed by HS-SPME (Gołębiowski, data unpublished).The photo of the sampling procedure of insect volatiles is given in Figure 2. The cuticular hydrocarbons from Bagrada hilaris were analysed by SPME-GC-MS (Pasquale et al., 2007) having been sampled using fibres coated with polydimethylsiloxane (PDMS, 100 μm), polyacrylate (PA, 80 μm) and Carbowax-divinylbenzene (CW-DVB, 65 μm).The fibres were conditioned in the gas chromatograph injector port: PDMS at 250 °C for 30 min, PA at 300 °C for 2 h, and CW-DVB at 220 °C for 30 min.Sealed vials with the insects were thermostatted at temperatures of 130 and 150 °C for 10 min.The headspace volatiles were absorbed on the exposed fibre for 2 min.The loaded fibre was then desorbed in the gas chromatograph inlet port for 2 min.12 hydrocarbons were identified in the cuticular waxes of B. hilaris: they consisted of a homologous series of n-alkanes ranging from C 17 to C 29 .Although the hydrocarbon profiles of males and females were qualitatively similar, quantitative differences were observed.Two sampling techniques for the GC-MS analysis of free fatty acids from exocrine glands were used (Maile et al., 1998).Solvent-free sampling, either by SPME or SI, are the ideal conditions for the analysis of insect samples.The insect waxes are analysed without any danger of contamination.Small insects or their parts are first sealed in a soft soda glass capillary tube.This is inserted in the gas chromatograph injector port, then heated for 3 min, after which GC analysis commences.The SPME sampling of fatty acids from solutions has been examined with different fibres; the stationary phases used as coatings were polydimethylsiloxane (PDMS, 7 μm), polyacrylate (PA, 85 μm), and Carbowax-divinylbenzene (CW-DVB, 65 μm).The best results for saturated fatty acids were obtained with PDMS-coated fibres, whereas PA fibres are suitable for palmitoleic, oleic and linoleic acids.Solid injection was used to analyse the hydrocarbons of the small ant Cardiocondyla wroughtonii (Turillazzi et al., 2002) and can be used to analyse solid samples, including body parts, glands or even small size insects.The solid injector technique involves inserting sealed glass capillaries into the GC-MS injector.Samples are inserted into the groove, which is then moved to align with two holes that allow passage of the carrier gas over the sample in the GC once injected.44 hydrocarbons were identified on the mass spectra obtained with solid injection.This method proved to be a superior means of extracting hydrocarbons from insects.

GC separation and detection of cuticular waxes
Gas chromatography was first applied to analyse cuticular waxes in the early 1960s, and its use has progressed with the introduction of capillary columns.Many of the more recently published papers deal with gas chromatography analysis (GC-FID or GC-MS) rather than with liquid chromatography (LC, TLC or HPLC).The aim of GC is to resolve the individual constituents of the complex wax mixture in order to obtain information on their qualitative and quantitative composition.This can be done in the following ways: a. GC analyses of intact cuticular wax extracts or fractions; b. chemical derivatization of functional groups containing active hydrogen and then GC analyses; c. chemical hydrolysis of high-molecular-weight esters and then GC analyses of the liberated moieties.
The detectors most frequently used for GC analysis of cuticular waxes are the flame ionization detector (FID) and mass spectrometer (MS) (Evershed, 1992a;Hamilton, 1995b;Riederer & Müller, 2006).Occasionally, other detectors can be applied.For example, Grossi & Raphel (2003) used GC coupled to an atomic emission detector (AED) for the identification of a series of 1-chloro-n-alkanes in the leaf waxes of three halophytes.
The direct analysis of wax extracts or, alternatively after the conversion of all hydroxylcontaining compounds into the corresponding trimethylsilyl (TMSi) derivatives, is often used to obtain GC profiles of plant cuticular waxes.For example, Buschhaus et al. (2007b) reported alkanes, primary alcohols, alkyl esters, triterpenoids and minor components in the waxes of rose leaves.GC analysis was carried out with the temperature programmed to rise from 50 °C to 320 °C and held for 30 min at 320 °C.Generally speaking, gas chromatographic elution with the oven temperature programmed to rise from 40 or 50 °C to 320 or 340 °C is the usual procedure in the analysis of plant cuticular waxes containing typical constituents such as hydrocarbons, fatty acids, alcohols, aldehydes, ketones, diols, esters and triterpenoids (Buschhaus et al., 2007a;Jetter, 2000;Jetter et al., 1996;Jetter & Riederer, 2000;Szafranek & Synak, 2006).Figure 3 shows the typical GC pattern of intact cuticular wax extract.Insect cuticular waxes, consisting mainly of hydrocarbons, fatty acids, alcohols, aldehydes, and wax esters, have been analysed with the oven temperature programmed to rise from about 150 °C to 300 or 320 °C (Buckner et al., 1996;Nelson et al., 1994;Pasquale et al., 2007;Turillazzi et al., 2002) or from about 100 °C to 300 or 320 °C (Gołębiowski et al., 2007;Said et al., 2005).The initial temperature was significantly lower for the analysis of cuticular hydrocarbons, aldehydes, alcohols and wax esters in Bemisia argentifolii (50 °C to 320 °C) (Buckner et al., 1999), the fatty acids in Dendrolimus pini (50 °C to 310 °C) (Gołębiowski et al., 2010) and the surface waxes in Acanthoscelides obtectus (30 °C to 320 °C) (Gołębiowski et al., 2008b).The initial temperature depends on the molecular weights of the compounds to be analysed.For example, the fatty acids of D. pini (C 8:0 -C 34:0 ) were analysed by GC as methyl esters (Gołębiowski et al., 2010).Owing to their volatility even at relatively low temperatures, short-chain fatty acids (C 4 to C 10 ) should be GC analysed with special care (Evershed, 1992a): temperature-programming from the lowest possible temperature (ca.30 °C) is necessary to prevent the shorter chain compounds co-eluting with the solvent.
Wax esters, steryl esters, triterpenoid esters or triacylglycerols are analysed at slightly higher temperatures.For example, wax esters from Salix leaves were GC-FID analysed and the oven temperature was programmed to increase from 200 °C to 380 °C, then held for 20 min at 380 °C (Szafranek et. al., 2008a).It should be noted that the high temperatures (generally above 300 °C) required for the elution of intact high-molecular-weight compounds can cause the loss of some thermally labile compounds (e.g.unsaturated compounds) (Evershed, 1992a).Wax esters can be GC analysed on non-polar capillary columns.Recently, Stránský et al. (2006) analysed a series of more than 200 wax ester standards using a temperature gradient programme from 240 to 340 °C.The GC analysis of n-alkanes up to C 54 and esters up to C 52 -C 53 could be performed under these chromatographic conditions.The problem with wax ester analysis, however, is that separation largely occurs according to carbon number; hence, the chromatographic peaks represent mixtures of non-separated wax esters.Some ester isomers elute at nearly the same time: for example 18:0-20:0 (an 18:0 acid moiety with a 20:0 alcohol moiety) and 20:0-18:0.
The study shows the influence of the number of double bonds in the ester molecule on chromatographic behaviour in comparison with the corresponding saturated ester.Saturated esters have the longest retention times.Esters with three double bonds are eluted before esters with one or two double bonds.
High-temperature gas chromatography (HTGC) and HTGC coupled to mass spectrometry has great potential for the determination of a great number of high-molecular-weight compounds, e.g.sucrose fatty acid esters, ethoxylated fatty alcohols, triglycerides and wax esters (Pereira & Neto, 1999;Pereira et al., 2004).HTGC non-polar and medium polar capillary columns can be used up to 400°C and in some cases 480 °C.On the other hand, high-resolution GC commercial capillary columns can be used up to 370 °C in the routine analysis of compounds of 1200 Da and more.This enables the direct analysis of extracts and fractions of natural products, in some cases without prior derivatization or clean-up.The injection method in HTGC is very important.Cold on-column or programmable temperature vaporizing (PTV) injection are the preferred techniques, as they eliminate discrimination against high boiling analytes.For example, high molecular weight alkanoates (three series of -tocopherol, -tocopherol and phytol esterified to fatty acids) in Amazonian plants were analysed by HTGC (Pereira et al., 2002).The cuticular waxes from ivy leaves containing ester waxes (alkyl alkanoates and alkyl coumarates) were analysed directly by GC-MS, HTGC-MS and ESI-MS/MS by Santos et al. (2007).However, with ESI-MS/MS analysis the detection of a wider range of ester waxes was possible than with HTGC-MS.
Fatty acids are GC analysed after their conversion to non-polar derivatives such as methyl or silyl esters (Evershed, 1992a).Often, esters are prepared as derivatives of the fatty acids liberated from wax esters or sterol esters by hydrolysis (Sümmchen et al., 1995;Szafranek & Synak, 2006).The details and other important considerations of fatty acid analysis have been well summarized by Christie (1994).Generally speaking, fatty acid methyl esters (FAME) can be GC analysed employing non-polar stationary phases like other cuticular wax components.Unsaturated compounds should be analysed on polar stationary phases (e.g.Carbowax).The determination of FAME often requires high resolution for the separation of positional and configurational isomers.The elution order is as follows (Evershed, 1992a): a. branched-chain FAME: increasing alkyl-chain branching reduces the retention time compared to a linear saturated counterpart and the elution order is independent of the polarity of the stationary phase; b. positional isomers of unsaturated FAME: the ECL values increase with increasing distance of the double bond from the carboxyl group on both polar and non-polar phases; c. geometric isomers of unsaturated FAME: the E-isomer elutes before the Z-isomer on a polar column.The order is reversed on a non-polar column.
Hydrocarbons are easily analysed by GC because of their chemical and thermal stability (Evershed, 1992a).The elution order of n-alkanes reflects the differences in their molecular weights.The effects of chain-branching and isomerism follow the same trends as for fatty acid methyl esters.Insects contain a variety of methyl-branched alkanes that have been widely investigated since the 1970s (Nelson & Blomquist, 1995).With certain combinations of the number and position of methyl branches, methyl-branched alkanes may have GC similar retention times to those of n-alkanes with one or two carbon atoms fewer in the chain.Elution times may overlap for methyl-branched alkanes that differ by one or even two carbons in the chain (Carlson et al., 1998).Since separation of branched-chain alkanes is difficult, it is usually necessary to perform GC-MS.
Fatty alcohols can be GC analysed as native compounds, but their separation is improved by preparing silyl or acetate derivatives (Szafranek & Synak, 2006;Shepherd et al., 1999;Stammitti et al., 1996).Ketones and aldehydes are analysed without derivatization (Szafranek & Synak, 2006;Nass et al. 1998), although aldehydes can be converted to more stable dimethyl acetal derivatives (Drozd, 1981) or other derivatives.See Table 2 for examples of derivatives used in the GC analysis of less common cuticular wax components.

Derivatives of cuticular wax components for GC analyses
The most common derivatives used in the GG analysis of cuticular waxes containing hydroxy-compounds are trimethylsilyl derivatives (TMSi ethers and esters).They are volatile but not stable, particularly in acidic or wet conditions.Analytes are derivatized with common silylation reagents such as BSA [N,O-bis(trimethyl-silyl)acetamide], BSTFA [N,Obis(trimethyl-silyl)trifluoroacetamide] and others (Blau & Halket, 1995;Drozd, 1981).TMCS (trimethylchlorosilane) can be used as catalyst.TMSi derivatives should be prepared immediately prior to the analysis because of their possible sensitivity to moisture.In the case of GC-FID analyses, silicone dioxide, produced by the decomposition of silyl derivatives in a flame, is deposited on the electrodes and slowly decreases the sensitivity of the detector.The reaction is simply carried out in a septum-closed vial by dissolving the sample of cuticular waxes in the reagent mixture and leaving it in the heating block for some time.For example, the reagent mixture of BSTFA-pyridine at 70 °C for 30 min (Jetter et al. 2000;Jetter & Riederer, 2000;Vermeer et al., 2003); BSTFA-TMCS (99:1, v/v) at 90 °C for 15 min (Haliński et al., 2009); BSA-TMCS (85:15, v/v) at 70 °C for 30 min (Szafranek & Synak, 2006); BSA-TMCS (85:15, v/v) at 100 °C for 60 min (Gołębiowski et al., 2008b;2010).Little (1999) described the artefacts in TMSi derivatization reactions and the formation of several artefacts in the silylation of aldehydes and ketones.Aldehydes have been SPE separated and GC analysed without silyl derivatization (Nass et al., 1998).
Dimethyl acetals can be prepared by heating the aldehydes under reflux with 2% anhydrous methanolic HCl for 2 h, after which the methanolic solution is cooled and neutralized with a small excess of sodium carbonate.Finally, the acetals are extracted from methanol with light petroleum (Drozd, 1981); alternatively, an aldehyde mixture with 1% sulphuric acid in methanol is left overnight in a vial at 50 ºC (Christie, 1994).
High-molecular-weight esters, such as wax esters, steryl esters, triterpenoid esters and triacylglycerols, are often hydrolysed, after which the liberated moieties are GC analysed; however, this procedure provides only limited compositional information.The very convenient one-pot small-scale hydrolysis-silylation procedure, applicable to esteremulsifiers, oils, fats, wax esters and other hydrolysable lipids, has been proposed by the IUPAC (Brüschweiler and Hautfeune, 1990).The procedure involves the saponification of esters (ca. 10 mg) in 0.5 N ethanolic KOH solution (0.25 mL) at 80 °C for 3 h, evaporation to dryness in a stream of nitrogen and then silylation of the hydrolysis products.This enables the components of very small samples to be determined (< 0.1 mg).This procedure was used for the hydrolysis of wax esters and triterpenoid esters in potato and eggplant cuticular waxes (Szafranek & Synak, 2006;Haliński et al., 2009).A solution of 1-2% (v/v) concentrated sulphuric acid in methanol can be easily used for the transesterification of acyl lipids and the esterification of free fatty acids (Christie, 1994).More procedures for ester hydrolysis and transesterification and for fatty acid esterification are described in detail by Christie (1994).
The free hydroxyl groups of long-chain alcohols and other compounds are frequently acetylated prior to GC analysis.Acetic anhydride in pyridine (5:1, v/v) is a mild reagent for acetylation: the sample (up to 50 mg) is dissolved in acetic anhydride in pyridine (2 mL) and left at room temperature overnight, following which the excess reagents are removed in a stream of nitrogen (Christie, 1994).Another procedure was applied for alcohols in potato waxes (Szafranek & Synak, (2006) after Blau & Halket (1995)): the sample (ca.0.1 mg) was dissolved in chloroform (0.5 mL), and acetic anhydride (0.1 mL) in acetic acid (0.2 mL) was added.The sample was left overnight in a glass vial at 50 °C, and the excess reagents were removed in a stream of nitrogen.
Dimethyl disulphide adducts are easily prepared derivatives for the GC-MS location of double bonds in alkenes and fatty acid methyl esters (Evershed, 1992b).The procedure is as follows: a small (ca.0.5 mg) sample is dissolved in 50 L dimethyl disulphide, and 50 L carbon disulphide is added with 300 g iodine; the mixture is kept at 60 °C for 40 h in a sealed vial, after which the reaction is quenched with aqueous Na 2 S 2 O 3 ; the organic phase is extracted and evaporated in a stream of nitrogen (Vincenti et al., 1987).

GC identification of cuticular waxes
Gas chromatography (GC-FID and GC-MS) can be used for both qualitative and quantitative analyses.Complete identification can be effected if GC retention data and mass spectral data are together taken into consideration.GC-FID identification is based on the comparison of retention times of analytes and authentic standards determined under identical GC conditions or by their co-chromatography.Unfortunately, standards of cuticular wax constituents are rarely commercially available.Some standards may be produced in the laboratory from pure components or natural extracts.For example, the carbon number in the analyses of wax esters can be assigned by comparison of their retention times with that of a synthetic wax ester of known structure (Evershed, 1992a).
Beeswax is well-characterized so it can be used as a standard mixture.In beeswax wax esters, the predominant fatty acid moiety is hexadecanoic acid and the chain lengths of the alcohol moieties range from C 26 to C 36 .
For other compounds, the literature should be consulted for retention data.These are presented in a number of ways, usually as relative retention times, as Kovats retention indices (KI) or programmed-temperature retention indices (RI), or as equivalent chain length values (ECL).Kovats retention indices are based on a linear relationship between the logarithms of the adjusted retention times (log t' R ) of a homologous series of compounds (saturated straight-chain alkanes) and their carbon chain lengths (Castello, 1999).
where t' R.X is the adjusted retention time of unknown compound X; t' R.Z is the adjusted retention time of an n-alkane with z carbon atoms; t' R.Z+1 is the adjusted retention time of an n-alkane with (z+1) carbon atoms; compound X is eluted between both n-alkanes; t' R.Z < t' R.X < t' R.Z+1 .
The programmed-temperature retention index is used when linear temperature programmes are carried out and defined as: '' 100 100 '' The retention times of the n-alkanes should behave linearly as a function of the carbon number, i.e. the difference between the retention time of two adjacent homologues is the same in the whole programmed analysis (Castello, 1999).
Insect cuticular hydrocarbons are commonly identified on the basis of retention indices (Nelson & Blomquist, 1995).Pomonis et al. (1989) determined the Kovats retention indices of monomethyl-pentacosanes, some internally branched dimethylalkanes and 2,x-dimethylheptacosanes on a cross-linked methyl silicone fused silica capillary column (Hewlett-Packard).Carlson et al. (1998) described a protocol for the identification of methyl-branched hydrocarbons in insect cuticular waxes.In this protocol, programmedtemperature retention indices are assigned to peaks, then the patterns in GC peaks that probably contain homologues are marked to assist subsequent GC-MS interpretation.
The authors also included data from the literature covering most of the insect methylalkanes.

GC-MS identification of cuticular waxes
Combined GC-MS is the standard technique used for identifying cuticular waxes, and electron ionization (EI) is the most widely used MS ionization technique in their analysis.
The most frequent and simple method of identification involves comparison of the recorded mass spectra with those in standard mass spectral libraries or with those of authentic standards.Unfortunately, the spectra of wax constituents are rarely published in MS libraries (e.g.NIST Chemistry WebBook, Wiley Registry of Mass Spectral Data, MSDC).For compounds that remain unidentified in the above MS libraries, the mass spectra found in the literature can be consulted.Table 2 gives the references of published mass spectra or fragmentation patterns for some less common cuticular wax components.The interpretation of mass spectra of common wax components are described elsewhere (Hamilton, 1995b;Evershed, 1992b;Christie, 1994).The mass spectra of fatty acids, alcohols, wax esters and other lipids can be found in an open access website (The AOCS Lipid Library, 2011).Briefly, the identification is performed on the basis of the characteristic fragment and molecular ions.For example, the mass spectra of saturated fatty acid methyl www.intechopen.com

Gas Chromatographic Analysis of Plant and Insect
Surface Compounds: Cuticular Waxes and Terpenoids 53 esters generally exhibit characteristic fragment ions at m/z 74 and 87 (Evershed, 1992b).The relative abundance of m/z 74 decreases with increasing unsaturation, thereby complicating the interpretation of mass spectrum even more.The molecular ion M + · confirms the number of carbons and the degree of fatty acid unsaturation.Christie (1998) summarized the use of picolinyl ester and dimethyloxazoline derivatives for the analysis of unsaturated fatty acids and the interpretations of their mass spectra.
The ions at m/z 73 and 75 are very common in the mass spectra of trimethylsilyl derivatives (TMSi) (Evershed, 1992b).(Christie, 1994).In our opinion, acetate derivatives are less informative than TMSi derivatives, so it is more convenient to prepare the same TMSi derivatives for every component of the wax extract.Mass spectra of underivatized long-chain alcohols contain little structural information and are easily confused with those of monoalkenes (Christie, 1994).The characteristic ions are 55, 57, 69, 83, 97, 111 and weak [M-18] The mass of spectrum of an underivatized long-chain aldehyde is characterized by the ions m/z 82, 96, [M-18] + · and a weak M + · (Hamilton, 1995b;Christiansen et al., 1969).
Recently, the mass spectral characteristics of three classes of wax esters were presented by Zhang et al. (2010).The authors developed a system from GC/MS data for the identification of wax esters in complex samples.

Terpenoids
Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers.These compounds belong to the isoprenoid group.Even though isoprene itself has not been found in nature, its polymers, terpenic hydrocarbons and their oxygen derivatives are very often present in large quantities in different species.The single isoprene unit, therefore, represents the most basic class of terpenes, the hemiterpenes.An isoprene unit bound to a second isoprene is the defining characteristic of terpene, which is also a monoterpene (C 10 ).Sesquiterpenes contain three isoprene units (C 15 ), while diterpenes (C 20 ) and triterpenes (C 30 ) contain two and three terpene units respectively.Tetraterpenes consist of four terpene units, polyterpenes more than four such units.In nature, terpenes occur predominantly in the form of hydrocarbons, alcohols and their glycosides, ethers, aldehydes, ketones, carboxylic acids and esters.The chemical diversity of plant terpenoids originates from often complex terpenoid biosynthetic pathways.McGarvey & Croteau (1995) reviewed terpene biosynthesis.Mapping strategies were used to determine the variance and composition of amino acids at terpene synthase active sites.In a more recent review of terpene synthase genes, Zwenger & Basu (2007) performed an in silico analysis of publicly available microarray data using Genevesitgator software (Zimmerman et al., 2004).
Apart from broad structural diversity, terpenes also exhibit a wide array of biological actions.Essential oils have antimicrobial, antiparasitic, insecticidal and antioxidant/prooxidant activities that often represent the combined bioactivity of multiple components.Many plant terpenoids are cytotoxic towards tumour cells and are applied as chemotherapeutic or chemopreventive compounds (Bhalla, 2003;Bifulco, 2005).Terpenes play an important role as signal compounds and growth regulators (phytohormones) of plants.Many insects metabolize the terpenes they have obtained with their plant food to growth hormones and pheromones.

Terpene isolation from plants
Organic solvent extraction has long been used for isolating essential oils from natural products (Taylor, 1993).To isolate terpenes from plant material dichloromethane extraction (Bowman et al., 1997) and methanol extraction can be applied (Verma et al., 1990).A twoday extraction with a mixture containing pentane and dichloromethane was used to isolate guajava essential oil (Vernin et al., 1991).Water extraction has proved to be an effective technique for isolating essential oil from citrus fruits (Lancas & Cavicchioli, 1990).Also, sequential extraction with toluene, citric acid and benzene has been proposed as a step prior to the determination of vinblastine in rose essential oil (Volkov & Grodnitskaya, 1994).The most common technique used to obtain essential oils from plant materials is steam or hydrodistillation.Analysis of the terpene hydrocarbon fraction of the essential oil obtained by the steam distillation of the fruit of Schinus molle L. was described by Bernhard & Wrolstad (1963).This technique has been applied extensively as a step prior to GC-MS for compositional studies of essential oils, as in the case of valerian (Bos et al., 1997), curcuma (Zwaving & Bos, 1992), cinnamon (Jayaprakasha et al., 1997) and rosemary, sage and lavender (Guilleèn et al., 1996).Steam distillation was also applied to obtain potato alcohols (Szafranek et al., 2006) and sesquiterpenes from potato varieties (Szafranek et al., 2005).Kawamura et al. (1999) reported using pressurized-liquid extraction (PLE) methods for extracting taxol from the bark of Taxus cuspidate.An interesting result from this study was that although in conventional extraction methods the taxol content of the water extract was very low, this was improved by use of the elevated temperature and pressure conditions of PLE.The influence of some experimental parameters on the PLE of P. gaudichaudianum Kunth leaves was elucidated (Peres et al., 2006).The optimization of the main variables involved in the PLE process (extraction temperature and time) was done using the extraction yield and the GC-MS profile of the extracts.The best results were obtained for the following parameters: 10 min and one extraction cycle at 85 °C.
Currently, supercritical fluid extraction (SFE) represents a new separation technique that is very suitable for the isolation of volatile compounds from plant matrices (Sedlakova et al., 2011).-Cellulose has been used as a model plant matrix for investigating the conditions required to optimize the SFE of typical plant constituents -limonene, caryophyllene, carvone, eugenol and santonin -using CO 2 as the extraction medium (Smith & Burford, 1992); 250 bar and 40 °C were chosen as the optimum conditions.The effects of adding modifiers to the supercritical fluid were also examined.The chemical composition and antimicrobial activity of essential oil-rich fractions obtained by supercritical CO 2 extraction from Rosmarinus officinalis L. were investigated (Santoyo et al., 2005).The most active fraction was obtained using 4% ethanol as modifier (extraction pressure -25 MPa; extraction temperature -60 °C).SFE with CO 2 was used for natural essential oil extraction from a Portuguese-grown rose geranium (Pelargonium sp.) (Gomes et al., 2007); the best extraction conditions were: extraction time 15-30 min, temperature 40 °C, pressure 90-100 bar.The CO 2 geranium extract had a superior organoleptic quality, with a very fresh natural floralfruity character and a pale yellow colour, most suitable for use in perfumery.
New approaches such as stir bar sorptive extraction (SBSE) have recently been used with interesting applications.SBSE is one of the most prominent techniques recently employed by some authors to determine volatiles from grapes (Caven-Quantrill & Buglass, 2006;Moreno et al., 2008;Zalacain et al., 2007;Pedroza et al., 2010).

Headspace methods
Headspace (HS) methods are used for the separation of volatile compounds from complex solid matrices such as plant materials.Reproducible and rapid identification of volatile compounds in aromatic plants can be achieved when static HS sampling is coupled to GC-MS (Esteban et al., 1996).More recently, the capabilities of automated HS sampling in the analysis of volatile compounds from Origanum vulgare was developed (Garciá & Sanz, 2001).The application of automatic HS-GC to the determination of the safrole content in different Asarum species from China and Europe was also described (Stuppner & Ganzera, 1998).
Solid-phase microextraction is a more recent solvent-free extraction method eliminating most of the drawbacks of other extractive methods, such as solvent use, thermal decomposition of compounds, excessive time preparation and high cost.To detect terpenes using SPME fibres, one of three sampling techniques is generally applied: (i) exposing fibres to the headspace of vials containing liquid or solid samples (Isidorov et al., 2003;Adam et al., 2005;Kos et al., 2006;Santos et al., 2006;Vichi et al., 2006), (ii) exposing fibres to air circulating over the sample (Zini et al., 2001;Isidorov et al., 2005), or (iii) inserting fibres into a vial containing the sample (Lopez et al., 2006).Because of the small dimensions of the sampling device and the simplicity and speed of the extraction procedure, HS-SPME is able to collect fragrances from live plants with minimum disturbance of the specimen, under both laboratory and field conditions.So far, there have been many qualitative experiments reporting the relative composition of individual compounds based on chromatogram peak areas (Isidorov et al., 2003;Santos et al., 2006;Vichi et al., 2006).The usefulness of eight available SPME fibres was elucidated to evaluate the recoveries of some terpene components with different polarities and structures present in the headspace of four aromatic and medicinal plants: rosemary (Rosmarinus officinalis L.), sage (Salvia officinalis L.), thyme (Thymus vulgaris L.) and valerian (Valeriana officinalis L.) (Bicchi et al., 2000).The results showed that the most effective fibres were those consisting of two components, i.e. a liquid phase (polydimethylsiloxane) and a porous solid (carboxen or divinylbenzene, or both).PDMS-SPME was used to study volatile compounds released by intact and mechanically damaged leaves of Abies fraseri (Vereen et al., 2000).After 5 min of extraction, monoterpenes such as 3-carene predominate; after 3 h, the major component in the chromatograms is bornyl acetate, with minor amounts of heavier compounds, e.g.camphor and borneol.PDMS/DVB fibres were used to quantify emissions of methyl chavicol, an oxygenated terpene from live branches (Pinus ponderosa) (Bouvier-Brown et al., 2007).In order to check the intra-and interpopulational variability of the terpene pattern, a total of 74 www.intechopen.comGas Chromatography in Plant Science, Wine Technology, Toxicology and Some Specific Applications 56 Juniperus communis samples were investigated by HS-SPME, GC-MS and GC-FID (Filipowicz et al., 2009).SPME−GC-MS was also used to determine monoterpene compounds in Mentha piperita (Rohloff, 1999).Author also compared the results obtained using SPME and steam distillation.Compared to solvent-based samples from essential oil distillation, relatively higher amounts of high-volatile monoterpenes and smaller quantities of less volatile compounds such as menthol and menthone were detected in SPME sampling.

Separation and detection of terpenes
Gas chromatography possesses inherent advantages that make it particularly attractive for the characterization and quantitative analysis of terpene mixtures.These include high separation efficiencies, short residence times in the chromatographic column, and the use of an inert atmosphere during analysis, the lack of azeotropes, and applicability to very small samples.In most cases capillary columns with dimethyl polysiloxane (methyl silicone) nonpolar and Carbowax 20M polar phases are used.Carbowax 20M phases include DB Wax, BP-20, PEG 20M and HP 20, while methyl silicone phases include SE-30, SF-96, OV-1, OV 101, BP 1, CPSIL 5CB, SP 2100, DB 1, DB 5 and HP 1 (Davies, 1990).Among these fusedsilica capillary GC columns, DB 1 or DB 5 and CPSil 5 are usually preferred.
One of the most important developments in gas chromatography was the introduction of enantioselective capillary columns with high separation efficiency in the mid-1960s by Gil-Av et al. (1966).At first, phases based exclusively on chiral diamide structures were used.The first optimum performance was achieved with the use of Chirasil-Val, a methylpolysiloxane phase containing about 6% branched aliphatic side chains with L-valine in a diamide linkage, and similar polymeric chiral diamide stationary phases (König, 1987) possessing excellent thermal stability.Finally, capillary columns using different hydrophobic cyclodextrin derivatives were introduced.The first GC separations of enantiomers using CDs were obtained by Koscielski & Sibilska in 1983; they separatedand -pinene, and the corresponding pinanes and δ-3-carene with a column packed with underivatized CDs.CDs are generally carried in non-polar to moderately polar polysiloxanes, as first proposed by Schurig & Novotny (1990).The main reasons for this are the wider range of operating temperatures, the inertness and efficiency of columns prepared by high temperature silylation, the possibilities of tuning column polarity by using different diluting phases, the small amounts of CD necessary to prepare columns, the shorter analysis times, and the possibility of measuring the thermodynamic parameters involved in enantiomer discrimination.Papers published over the period 1989-94 concerning applications of cyclodextrin derivatives (CDDs) to the GC separation of volatile racemates in the essential oil, extract, flavour and aroma fields were reviewed by Bicchi et al. (1995).
The most common detection method used in gas chromatography is FID.The nitrogenphosphorus detector (NPD) can be used to identify nitrogen-containing compounds (Stashenko et al., 1996).Another possibility is the use of oxygen flame ionization detection (O-FID) for the selective determination of oxygenates (Betts, 1994;Schneider et al., 1982).Mass spectrometry is a very useful tool for detecting complex terpene mixtures.

GC identification of terpenes
Retention indices are fundamental to making retention a reliable identification tool for GC.Identification is based on the direct comparison of retention times with standards or a precise knowledge of retention indices (Davies, 1990;Stashenko et al., 1993;Stashenko et al., 1996).Some 900 Kovats indices of 400 individual compounds were summarized from the general literature (Davies, 1990).A compilation such as that of Adams (1995) reveals an enormous number of compounds that are present in essential oils.The programmedtemperature retention indices listed in Adams' monograph were obtained on a DB-5 column.Identification is hampered, because the retention indices of many related components are similar.The presence of unsaturated bonds, various branched and cyclic compounds, and oxygenated analogues (e.g.alcohols and ketones) further complicate the issue.Problems still surround the variation of stationary-phase polarity and mobile-phase characteristics as a function of temperature in programmed analysis.An effective approach is to combine the retention data from stationary phases of different polarity.Apart from comparing obtained retention indices with values published in the literature, terpenes can also be identified using co-injection with standards.

GC-MS identification of terpenes
Nowadays the combination of GC-MS in electron impact mode is a well-established technique for the routine analysis of essential oils.With this technique, additional information can be obtained from mass spectra.However, it has to be emphasized that identification of terpenes based only on mass spectra is also virtually impossible.Molecular rearrangement and isomerization processes in unsaturated hydrocarbons result in very similar mass spectra lacking characteristic fragmentation patterns.There are several ways of solving this problem.One is to use GC-MS-MS (tandem mass spectrometry), which analyses each component of such complex peaks separately.Another is single ion monitoring (SIM), a very selective method that has turned out to be the most reliable procedure for quantitative analysis.Apart from these techniques, combined data of retention times, Kovats indices (Richmond and Pombo-Villar, 1997) and mass spectral data enable the unambiguous identification of sesquiterpenoid constituents.Numerous papers have recently been published on GC-MS techniques used to analyse essential oils and volatile compounds (MacLeod & Ames, 1991;Elias et al., 1997;Szafranek et al., 1998;Szafranek et al., 2005;Szafranek & Szafranek, 2008;Gołębiowski et al., 2008c;Gołębiowski et al., 2009).
The mass fragmentation of terpenes was widely discussed by Budzikiewicz et al. (1963), and the features of the mass spectra of mono-, bi-and tri-cyclic terpenoids were investigated Gas Chromatography in Plant Science, Wine Technology, Toxicology and Some Specific Applications 58 (Yermakov et al., 2010).It was established that the mass spectra of these compounds are absolutely identical in the mass values of fragment ion peaks, although there are minor differences in their relative intensities.The characteristic ions in the spectra of all compounds were [M-CH(CH 3 ) 2 ] + and [M-CH(CH 3 ) 2 -H 2 ] + .Eleven terpenoid derivatives from the extract including three steroids and eight pentacyclic triterpenes were identified by GC-MS in an extract of the stem bark of Ficus mucuso (Djemgou et al., 2009).HS volatiles from flowers, as well as green and ripe mango fruit of cv.Ataulfo from Soconusco, Chiapas, were identified by GC-MS (Sandoval et al., 2007).Chemical identification was confirmed by comparison of the mass spectra pattern with the NIST 2002 computer library and the retention times of synthetics.A new cis-sabinene hydrate chemotype was detected in large thyme (Thymus pulegioides L.) by Groendahl & Ehlers (2008).Analyses were done using an GC coupled to an inert mass selective detector with an ion source of 70.0 eV at 230 °C.Two columns of different polarities were used: one was an EC-Wax capillary column with oven conditions that included an isothermal hold at 60 °C for 5 min, followed by a ramp of 10 °C/min to 250 °C.The other was a HP-5MS capillary column with an initial oven temperature of 60 °C, followed by a ramp of 3 °C/min to 246 °C.Terpenes were identified using retention times of standards, the NIST 2005 mass spectral library and/or programmed-temperature retention indices.Five chemotypes were detected: carvacrol, linalool, geraniol and thymol, all of which are known to occur in large thyme.In addition, essential oils containing the monoterpene cis-sabinene hydrate as the dominant component were found.No sabinene hydrate chemotype has previously been detected in large thyme, although it does occur in other thyme species.
Nevertheless, sometimes mass spectra measured in EI mode are problematic, because they miss the molecular ions, especially esters.In this case, the application of GC-MS in chemical ionization (CI) mode using various reagent gases often yields valuable additional information.Negative ion chemical ionization (NCI) with OH as the reactant ion (Hendriks et al., 1985;Cazaussus et al., 1988) is an additional method.However, there is no characteristic fragmentation pattern in the mass spectra of terpenes obtained by NCI.For sesquiterpenes with one or two non-conjugated double bonds this problem can be solved by using trioxo(tert-butylimido)osmium(VIII) (Rücker et al., 1990).This reagent forms cyclic osmate ester amides as intermediates which, after reduction, yield vicinal mono-amino alcohols, bis-amino alcohols and aminotriols.The derivatives formed in such a reaction have characteristic mass spectra that can be used as a fingerprint for the identification of the respective parent compound.

Two-dimensional GC
While GC-FID is the traditional method for essential oil quantification, GC-MS is the most common analytical method for component identification.However, the wide concentration range of the analytes (from ppb to percentage levels), as well as the presence of numerous isomers (terpenes and oxygenated terpene structures), make qualitative analysis difficult.In addition, the mass spectra of these compounds are usually very similar, so peak identification often becomes very difficult and sometimes impossible.
Recently, comprehensive, two-dimensional gas chromatography (GC×GC) has been extensively used in studies of essential oils.Comprehensive GCxGC enhances the peak capacity for a chromatographic run, allowing better separation in complex sample analysis.GCxGC technology allows the use of two different separation mechanisms in order to increase the www.intechopen.comGas Chromatographic Analysis of Plant and Insect Surface Compounds: Cuticular Waxes and Terpenoids 59 separation power of the chromatographic system.The two directly coupled columns in GCxGC provide orthogonal separation of compounds, and importantly, enable the simultaneous twocolumn separation of the whole sample.Thus a combination of a non-polar column with a polar column may be a good first choice for a suitable dual column set for essential oils.The principles, practical and theoretical aspects, and the most significant developments of GC×GC were reviewed by Mondello et al. (2008).There are several reports of GCxGC used for determining essential oils (Dimandja et al., 2000;Shellie et al., 2003;Shellie et al., 2001).
The high resolution GC×GC separation of an essential oil and the identification of selected separated components by time-of-flight mass spectrometry (TOF-MS) was reported by Shellie et al., (2001).These authors showed that GC×GC allows orthogonal separation mechanisms on the two columns to achieve separation of components (e.g.borneol and terpinen-4-ol, and cis-caryophyllene and -farnesene) that would otherwise be unresolved on a single column.The authors concluded that peak compression led to the generation of fast second-dimension GC peaks and a ca 25 times better detection sensitivity than with conventional GC elution.This allows many more compounds to be detected when using the GC×GC approach.GC-MS and GC×GC with FID were used in the analysis of peppermint (Mentha piperita) and spearmint (Mentha spicata) essential oils.Components including acetates, alcohols, furans, ketones, sesquiterpenes and terpenes were detected.The GC×GC chromatogram of peppermint essential oil displays 89 peaks compared to the 30 peaks in the GC-MS chromatogram; likewise, 68 peaks were detected in the GC×GC chromatogram of spearmint compared to 28 in GC-MS.This technique has been successfully used in the industrial analysis of plant materials to improve component separation and identification.In addition, analysis of Artemisia annua L. volatile oils using multi-dimensional gas chromatography has indicated that this technique can achieve the complete separation of a wide range of terpenes (Ma et al., 2007).These authors found nearly 700 components, the majority of which were terpenes.The investigation of the volatile compounds of dried rhizomes of Coptis chinensis was carried out by Gao et al. (2011).Volatile profiles were established and compared after headspace solid-phase microextraction using a PDMS/DVB fibre coupled to comprehensive 2D GC×GC-TOFMS.Analyses were performed and compared on two column-phase combinations (non-polar/polar and polar/non-polar).Terpenoids represented the most numerous group of compounds identified.

Conclusion
Modern analytical techniques can provide accurate and precise profiles of plant and insect cuticular waxes as well as terpenoids.For structural studies the most effective procedures are those based on a combination of mass spectra and GC retention index data.

Acknowledgment
The work was financed by Grant DS/8200-4-0085-1 from the University of Gdańsk and by Grant N N303 504238 from the Polish Ministry of Science and Higher Education (2010-2013).

Fig. 1 .
Fig. 1.Summary of typical procedures used in the analysis of plant cuticular waxes.