Aroma SPME Extraction

Because SPME extracts compounds selectively, the response to each compound must be calibrated for quantification. A specific compound can be quantified by using three GC peak area values from solvent injection, static headspace (gas-tight syringe), and SPME. The solvent injection is used to quantify the GC peak area response of a compound. This is used to quantify the amount of the compound in the headspace. The SPME response is then compared to the quantified static headspace extraction. These three stages are necessary because a known gas-phase concentration of most aroma compounds at low levels is not readily produced. A headspace of unknown concentration is thus produced and quantified with the solvent injection. Calibration must be conducted independently for each fiber and must include each compound to be quantified. 

Gas chromatography/olfactometry (GC/O) based on dilution analysis (e.g., CharmAna-lysis or Aroma Extraction Dilution Analysis) gives an indication of what compounds are most potent in the aroma of foods. The application of SPME to GC/O dilution analysis can be achieved by varying the thickness of the fiber phase and the length of exposure, resulting in various absorbant volumes. 

For a compound to contribute to the aroma of a food, the compound must have odor activity and volatilize from the food into the head-space at a concentration above its detection threshold. Since aroma compounds are usually present in a headspace at levels too low to be detected by GC, headspace extraction also requires concentration. SPME headspace extraction lends itself to aroma analysis, since it selectively extracts and concentrates compounds in the headspace. Some other methods used for sample preparation for aroma analysis include purge-and-trap or porous polymer extraction, static headspace extraction, and solvent extraction. A comparison of these methods is summarized in Table Gl.6.2. 

Sampling volatile analytes from samples having complex matrices usually takes place in the HS-SPME mode. This variant yields decidedly better results in the determination of aroma compounds59 and other volatile components.60 Moreover, HS-SPME prolongs the life of the fiber because it is not in direct contact with the sample. On the other hand, the direct extraction of less volatile compounds from solution is possible using DI-SPME. But in this case, the fiber deteriorates more quickly, increasing the cost of analysis. Headspace sampling is therefore employed whenever possible. 

Aroma compounds from vanilla beans have been extracted using several extraction procedures, using alcohols and organic solvents (Galletto and Hoffman, 1978 Dignum et al., 2002), direct thermal desorption (Hartman et al., 1992 Adedeji et al., 1993) and solid-phase microextraction (SPME) (Sostaric etal., 2000), followed by identification of the compounds by gas chromatography-mass spectrometry (GC-MS). 

In the early 90s, a new technique called solid-phase-micro extraction (SPME), was developed (Arthur and Pawliszyn, 1990). The key-part component of the SPME device is a fused silica fiber coated with an adsorbent material such as polydimethylsiloxane (PDMS), polyacrylate (PA) and carbowax (CW), or mixed phases such as polydimethylsiloxane-divinylbenzene (PDMS-DVB), carboxen-polydimethylsiloxane (CAR-PDMS) and carboxen-polydimethyl-siloxane-divinylbenzene (CAR-PDMS-DVB). The sampling can be made either in the headspace (Vas et al., 1998) or in the liquid phase (De la Calle et al., 1996) of the samples. The headspace sampling in wine analyses is mainly useful for quantifying trace compounds with a particular affinity to the fiber phase, not easily measurable with other techniques. Exhaustive overviews on materials used for the extraction-concentration of aroma compounds were published by Ferreira et al. (1996), Eberler (2001), Cabredo-Pinillos et al. (2004) and Nongonierma et al. (2006). Analysis of the volatile compounds is usually performed by gas chromatography (GC) coupled with either a flame ionization (FID) or mass spectrometry (MS) detector. 

Table 5.2 Linear relationships for some wine aroma components between GC-peak areas ratios (Ax/Ai.s.) for Kaltron liquid-liquid or HS-SPME aroma extraction vs SPE/XAD-2 (mg/L) extraction as reference. y =[Area component]/[Area i.s.] by Kaltron or HS-SPME. X=[Area component]/[Area i.s.] x RF by XAD-2 (mg/L). a=angular coefficient. In italics are presented the relationships involving HS-SPME.

Figure 5.3 Linear relationships between concentrations (mg/L) of some aroma compounds evaluated by GC-FID after XAD-2 enrichment technique in different wines and the relevant ratios of the compounds areas to that of internal standard (2-octanol evaluated on the fragment at m/z 45) determined by GC-MS after HS-SPME (PDMS fiber) extraction (some scores could be hidden)... 

Instead by solvent extraction [207], aroma compounds from aqueous media, e.g. fruit juices, can even be separated and enriched by techniques of solid phase micro extraction (SPME), preferably from the headspace [208] , corresponding devices can often be directly connected to GC systems. These techniques provide the complete spec-tmm of the individual compounds of an aroma. As it will normally not be possible and even not necessary to analyse all components of the complex mixture, the separation of its main compounds may demand a multi-dimensional (MD) gas chromatographic system [209[ as displayed in Fig. 6.14 [210[. Examples for the multi-ele-ment/multi-compound isotope analysis by such systems will be given later (6.2.2.4.4, [211[) they can even integrate the identification of the compounds by molecular mass spectrometry and a simultaneous determination of the enantiomer ratios of isomers [210, 211 [. The importance of enantiomer analysis as a tool for authenticity assessment is extensively treated in chapter 6.2.3. 

Recently, rotundone was identified as a pepper aroma impact compound in Shiraz grapes (Siebert et al.,2008). Identification was achieved by performing GC-MS analysis of grape juice after purification by solid-phase extraction (SPE) using a styrene-divinylbenzene 500-mg cartridge and elution with n-pentane/ethyl acetate 9 1, followed by solid-phase microextraction (SPME) using a 65-pm polydimethylsilox-ane-divinylbenzene (PDMS/DVB) fiber immersed in the sample for 60 min at 35 °C. J5-Rotundone was used as an internal standard. The structure of the compound is reported in Fig. 4.5. 

More recently, headspace and HS-SPME-GC/MS approaches for analysis of aroma in must and grape extracts were also proposed (Lopez et al., 2004 Prosen et al., 2007 Sanchez-Palomo et al., 2005 Rosillo et al., 1999). 

Solid-phase microextraction (SPME) of wine was developed by both headspace (HS) (Vas et al., 1998) and liquid-phase sampling (De la Calle et al., 1996). Exhaustive overviews on materials used for the extraction-concentration of aroma compounds in wines were published from Ferreira et al. (1996), Cabredo-Pinillos et al. (2004), and Nongonierma et al. (2006). 

Solid Phase Microextraction (SPME) has become one of the preferred techniques in aroma analysis, offering solvent fi ee, rapid sampling with low cost and easy preparation. Also, it is sensitive, selective and compatible with low detection limits [18]. Placed in the sample headspace, SPME is a non-destructive and non-invasive method to evaluate volatile and semi-volatile compounds. In this sense, the extraction of volatile compounds released from a great number of foods has been carried out by using HS-SPME technique [29, 33],... 

Sorptive extraction (Solid Phase Micro Extraction [SPME] and Stir Bar extraction) are relatively new techniques for the isolation of food aromas. Pawliszyn s group [41] was the first to develop the SPME method, and they applied it in environmental analysis. Since then, it has become a widely used technique for the analysis of volatiles in foods. Harmon [42] and Marsili [43] have provided a comprehensive review and a critical review, respectively, of this technique. 

Three commonly used extraction techniques for the analysis of aroma are simultaneous distillation/extraction (SDE), dynamic headspace adsorption on Tenax TA (Buchem N.V., Apeldoorn, The Netherlands), and solid-phase microextraction (SPME) [1,2]. All of these techniques have positive aspects and drawbacks, and these are described. In SDE the sample is boiled for 1 to 2 hr and so precooking may not be necessary, although the meat is usually minced to maximize surface area for the extraction process. The other techniques can be used to examine either a chopped or a whole piece of cooked meat. 

Figure 7 Relative amounts of selected compounds from 5.5 g of freshly popped popcorn extracted by SFE DI and SFE SPME methods. B, Sulfurol (4-methyl-5-thiazolethanol), a compound with meaty aroma, was most abundantly observed by the SFE DI method. SFE DI, supercritical fluid extraction direct injection SFE SPME, SFE solid-phase microextraction DDMP, 2,3-dihydro 3,5-dimethyl-4(H)-pyran-4-one.

Figure 15 A comparison of the total ion chromatograms of the volatile aroma components of (a) a ripe Bartlett pear and (b) a pear-flavored jelly bean isolated by headspace SPME. Small plugs of the pear were removed with the blunt end of a disposable pipet and placed into a 20-mL headspace vial for extraction. The jelly bean was forced into a smaller 4-mL vial. Headspace extraction was performed on each sample for 10 minutes at room temperature using a 100-(xm PDMS fiber. Peak identities are as follows (1) butyl acetate, (2) hexyl acetate, (3) methyl cis-4-decenoate, (4) ethyl cis-4-decenoate, (5) methyl frawi-2-cw-4-decadienoate, (6) ethyl frani-2-ds-4-decadienoate, (7) a-famesene, (8) isoamyl acetate, (9) ds-3-hexenyl acetate, and (10) carveol propionate.

Figure 17 represents the analysis of a sample of curry powder that was thought to be lacking one of its spice components. Because the spice was known to contain a unique aroma chemical, it was an easy matter to transfer a small amount of the curry to a vial, perform a headspace extraction, and determine whether the spice had been added. The complete analysis required less than one hour from the time the sample was received in the laboratory. As a quality control measure, SPME can have a significant impact on the analysis of raw materials and finished products. 

The chromatograms of tomato volatiles obtained by the three methods are shown in Fig. 3. SPME was unable to detect the highly volatile flavor components l-penten-3-one and 3-methyl butanol. All important aroma volatiles were detected by liquid-liquid extraction. Phenylacetaldehyde and 2-phenylethanol were... 

It is not uncommon to hear some flavor chemists report that SPME doesn t work as well as other extraction/concentration techniques. Unfortunately, many haven t tried the newer fibers and are basing their assessment of SPME on previous work done only with PDMS. Examples of successful aroma analyses of polar and/or highly volatile analytes using some of these newer types of fibers follow. 

Tenax and would be able to collect aroma chemicals with a wide range of polarities. Figure 9 shows the effect of collection time on the aroma chemicals collected. The Zenith trap is capable of collecting sufficient quantities of headspace material in around 5 minutes for quantitative GC/MS analysis. The Zenith trap combines the advantages of SPME and dynamic headspace. It overcomes the problems of fiber fragility, long extraction times, and the need to analyze and combine the results of several SPME fibers. 

SPME has become a valuable alternative to solvent extraction, purge-and-trap (dynamic), and static headspace methods (1 ). This is true for the analysis of flavors, fragrances, food aromas, and biological systems as is evidenced by... 

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