Info

CH4 He

Mass spectrometer

CH4 He

Pyrolysis reactor T=1450°C

Mass spectrometer

Water separator He -

Open split Faraday cups | 2 ;3 P and 1 I

amplifiers

Water separator He -

Main column

Open split Faraday cups | 2 ;3 P and 1 I

amplifiers m

FIGURE 2.7 Scheme of an MDGC-C/P-IRMS device. (From Sewenig, S., et al., 2005. J. Agric. Chem, 53: 838-844. With permission.)

of the solvent vapor can be removed through the solvent vapor exit (SVE). After closing this valve and increasing the GC-oven temperature, the sample components are volatilized and separated in the main column reaching the detector. The main drawback of this technique, however, may be the loss of highly volatile compounds that are vented together with the solvent. As an example of an HPLC-GC investigation, the preseparation of lemon oil with gradient elution into four fractions is quoted (Munari et al., 1990). The respective gas chromatograms of the individual fractions exhibit good separation into hydrocarbons, esters, carbonyls, and alcohols, facilitating gas chromato-graphic separation and identification. Due to automation of all analytical steps involved, the manual

FIGURE 2.8 Basic arrangement of an HPLC-GC device with a sample loop interface. RG: retention gap; C1: retaining column; C2: analytical column; LVI: large volume injector; and SVE: solvent vapor exit.

operations are significantly reduced and very good reproducibility was obtained. In three excellent review articles, the different kinds of HPLC-GC couplings are discussed in detail, describing their advantages and limitations with numerous references cited therein (Mondello et al., 1996, 1999; Dugo et al., 2003).

2.2.3.7 HPLC-MS, HPLC-NMR Spectroscopy

The on-line couplings of HPLC with MS and NMR spectroscopy are further important techniques combining high-performance separation with structurally informative spectroscopic techniques, but they are mainly applied to nonvolatile mixtures and shall not be discussed in more detail here, although they are very useful for investigating plant extracts.

Some details concerning the different ionization techniques used in HPLC-MS have been presented among other things by Dugo et al. (2005).

2.2.3.8 Supercritical Fluid Extraction-Gas Chromatography

Although SFE is not a chromatographic technique, separation of mixtures can be obtained during the extraction process by varying the physical properties such as temperature and pressure to obtain fractions of different composition. Detailed reviews on the physical background of SFE and its application to natural products analysis inclusive of numerous applications have been published by Modey et al. (1995), and more recently by Pourmortazavi et al. (2007). The different types of couplings (off-line and on-line) have been presented by several authors. Houben et al. (1990) described an on-line coupling of SFE with capillary GC using a programmed temperature vaporizer as an interface. Similar approaches have been used by Blanch et al. (1994) in their investigations of rosemary leaves and by Ibanez et al. (1997) studying Spanish raspberries. In both the last two papers an off-line procedure was applied. A different device has been used by Hartonen et al. (1992) in a study of the essential oil of Thymus vulgaris using a cooled stainless steel capillary for trapping the volatiles connected via a six-port valve to the extraction vessel and the GC column. After sampling of the volatiles within the trap they have been quickly vaporized and flushed into the GC column by switching the walve. The recoveries of thyme components by SFE-GC were compared with those obtained from hydrodistilled thyme oil by GC exhibiting a good agreement. The SFE-GC analyses of several flavor and fragrance compounds of natural products by transferring the extracted compounds from a small SFE cell directly into a GC capillary has already been presented by Hawthorne et al. (1988). By inserting the extraction cell outlet restrictor (a 20 pm I.D. capillary) into the GC column through a standard on-column injection port, the volatiles were transferred and focused within the column at 40°C, followed by rapid heating to 70°C (30°C/min) and successive usual temperature programming. The suitability of that approach has been demonstrated with a variety of samples including rosemary, thyme, cinnamon, spruce needles, orange peel, and cedar wood. In a review article from Greibrokk, published in 1995, numerous applications of SFE connected on-line with gas chromatography and other techniques, the different instruments, and interfaces have been discussed, including the main parameters responsible for the quality of the obtained analytical results. In addition, the instrumental setups for SFE-LC and SFE-SFC couplings are given.

2.2.3.9 Supercritical Fluid Chromatography-Gas Chromatography

On-line coupling of SFC with gas chromatography has sporadically been used for the investigation of volatiles from aromatic herbs and spices. The requirements for instrumentation regarding the pumps, the restrictors, and the detectors are similar to those of SFE-GC. Additional parts of the device are the separation column and the injector, to introduce the sample into the mobile phase and successively into the column. The most common injector type in SFC is the high-pressure valve injector, similar to those used in HPLC. With this valve, the sample is loaded at ambient pressure into a sample loop of defined size and can be swept into the column after switching the valve to the injection position. The separation columns used in SFC may be either packed or open tubular columns with their respective advantages and disadvantages. The latter mentioned open tubular columns for SFC can be compared with the respective GC columns; however, they must have smaller internal diameter. With regard to the detectors used in SFC, the FID is the most common applied detector, presuming that no organic modifiers have been admixed to the mobile phase. In that case, for example, a UV detector with a high-pressure flow cell has to be taken into consideration.

In a paper, presented by Yamauchi et al. (1990), cold-pressed lemon-peel oil has been separated by semipreparative SFC into three fractions, namely hydrocarbons, aldehydes and alcohols, and esters together with other oil constituents. The obtained fractions were afterward analyzed by capillary GC. SFC has also often been combined with SFE prior to chromatographic separation in plant volatile oil analysis, since in both techniques the same solvents are used, facilitating an on-line coupling. SFE and on-line-coupled SFC have been applied to the analysis of turmeric, the rhizomes of Curcuma longa L., using modified carbon dioxide as the extractant, yielding fractionation of turmerones curcuminoids in a single run (Sanagi et al., 1993). A multidimensional SFC-GC system was developed by Yarita et al. (1994) to separate on-line the constituents of citrus essential oils by stepwise pressure programming. The eluting fractions were introduced into a split/splitless injector of a gas chromatograph and analyzed after cryofocusing prior to GC separation. An SFC-GC investigation of cloudberry seed oil extracted with supercritical carbon dioxide was described by Manninen et al. (1997), in which SFC was mainly used for the separation of the volatile constituents from the low-boiling compounds, such as triacylglycerols. The volatiles were collected in a trap column and refocused before being separated by GC. Finally, an on-line technique shall be mentioned by which the compounds eluting from the SFC column can be completely transferred to GC, but also for selective or multistep heart-cutting of various sample peaks as they elute from the SFC column (Levy et al., 2005).

2.2.3.10 Couplings of SFC-MS and SFC-FTIR Spectroscopy

Both coupling techniques such as SFC-MS and SFC-FTIR have nearly exclusively been used for the investigation of low-volatile more polar compounds. Arpino published in 1990 a comprehensive article on the different coupling techniques in SFC-MS, which have been presented up to 1990 including 247 references. A short overview of applications using SFC combined with benchtop mass spectrometers was published by Ramsey and Raynor (1996). However, the only paper concerning the application of SFC-MS in essential oil research was published by Blum et al. (1997). With the aid of a newly developed interface and an injection technique using a retention gap, investigations of thyme extracts have been successfully performed.

The application of SFC-FTIR spectroscopy for the analysis of volatile compounds has also rarely been reported. One publication found in the literature refers to the characterization of varietal differences in essential oil components of hops (Auerbach et al., 2000). In that paper, the IR spectra of the main constituents were taken as films deposited on AgCl disks and compared with spectra obtained after chromatographic separation in a flow cell with IR transparent windows, exhibiting a good correlation.

2.2.4 Identification of Multicomponent Samples without Previous Separation

In addition to chromatographic separation techniques including hyphenated techniques, several spectroscopic techniques have been applied to investigate the composition of essential oils without previous separation.

2.2.4.1 UV Spectroscopy

UV spectroscopy has only little significance for the direct analysis of essential oils due to the tnability to provide uniform information on individual oil components. However, for testing the presence of furano-coumarins in various citrus oils, which can cause photodermatosis when applied externally, UV spectroscopy is the method of choice. The presence of those components can be easily determined due to their characteristic UV absorption. In the European Pharmacopoeia for example, quality assessment of lemon oil, which has to be produced by cold pressing, is therefore performed by UV spectroscopy in order to exclude cheaper distilled oils.

2.2.4.2 IR Spectroscopy

Several attempts have also been made to obtain information about the composition of essential oils using IR spectroscopy. One of the first comprehensive investigations of essential oils was published by Bellanato and Hidalgo (1971) in the book Infrared Analysis of Essential Oils in which the IR spectra of approximately 200 essential oils and additionally of more than 50 pure reference components have been presented. However, the main disadvantage of this method is the low sensitivity and selectivity of the method in the case of mixtures with a large number of components and secondly the unsolvable problem when attempting to quantitatively measure individual component concentrations.

New approaches to analyze essential oils by vibrational spectroscopy using attenuated reflection (ATR) IR spectroscopy and NIR-FT-Raman spectroscopy have recently been published by Baranska et al. (2005) and numerous papers cited therein. The main components of an essential oil can be identified by both spectroscopic techniques using the spectra of pure oil constituents as references. The spectroscopic analysis is based on characteristic key bands of the individual constituents and made it, for example, possible to discriminate the oil profiles of several eucalyptus species. As can be taken from this paper, valuable information can be obtained as a result of the combined application of ATR-IR and NIR-FT-Raman spectroscopy. Based on reference GC measurements, valuable calibration equations have been developed for numerous essential oil plants and related essential oils in order to quantify the amount of individual oil constituents applying different suitable chemo-metric algorithms. Main advantages of those techniques are their ability to control the quality of essential oils very fast and easily and above all, to quantify and analyze the main constituents of essential oils in situ, that means in living plant tissues without any isolation process, since both techniques are not destructive.

2.2.4.3 Mass Spectrometry

MS and proton NMR spectroscopy have mainly been used for structure elucidation of isolated compounds. However, there are some reports on mass spectrometric analyses of essential oils. One example has been presented by Grutzmacher (1982). The depicted mass spectrum (Figure 2.9) of an essential oil exhibits some characteristic molecular ions of terpenoids with masses at m/z 136, 148, 152, and 154. By the application of a double focusing mass spectrometer and special techniques analyzing the decay products of metastable ions, the components anethole, fenchone, borneol, and cineole could be identified, while the assignment of the mass 136 proved to be problematic.

A different approach has been used by Schultze et al. (1986), investigating secondary metabolites in dried plant material by direct mass spectrometric measurement. The small samples (0.1-2 mg, depending on the kind of plant drug) were directly introduced into a mass spectrometer by means of a heatable direct probe. By heating the solid sample, stored in a small glass crucible, various substances are released depending on the applied temperature, and subsequently their mass spectra can be taken. With the aid of this technique, numerous medicinal plant drugs have been investigated and their main vaporizable components could be identified.

2.2.4.4 13C-NMR Spectroscopy

13C-NMR spectroscopy is generally used for the elucidation of molecular structures of isolated chemical species. The application of 13C-NMR spectroscopy to the investigation of complex mixtures is relatively rare. However, the application of 13C-NMR spectroscopy to the analysis of

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m/z 136

m/z 152

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