The Headspace Approach

The results passed on to the perfumer would include the name of the compound, its relative proportion in the headspace sample and an indication of the odour intensity of the material. The perfumer can then select the important odour materials and combine them in an accord to re-create the scent of the flower. 

Modern natural product analysis reveals both the chemical composition of new oils or flower scents and the identity of novel fragrant molecules that may become new perfumery ingredients in the future. It is chemical detective work to solve the mysteries of nature s fragrances that have evolved over the millennia. 

Steam distillation is the main commercial extraction procedure for the production of essential oils from almost any type of plant material. Solvent extraction is also used commercially and yields a resinoid, concrete or absolute according to the solvents and techniques used (see Chapter 4). Both steam distillation and solvent extraction are used on a laboratory scale to produce oils and extracts for analysis. Other methods of extraction, such as supercritical fluid extraction (SFE), which uses supercritical CO2 as the extraction solvent, are now being developed and used on both commercial and laboratory scales. The extracts produced by SFE may contain different materials from the steam-distilled oil because of the solvating power of C02 and the lower extraction temperature, which reduces thermal degradation. The C02 extract may therefore have an odour closer to that of the original material and may contain different fragrant compounds. The choice of extraction procedure depends on the nature and amount of material available, and the qualities desired in the extract. Solvent extraction is better suited to small sample amounts or volatile materi- 

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There are available a wide range of analytical tools for the extraction of volatile compounds. These methodologies are essentially based on the solubility of the analytes in organic solvents, based on the adsorptive capacity of polymeric phases and based on their sorptive capacity on polymeric phases or solvents. Moreover, since the techniques deal with volatile compounds, the headspace approach is typically associated whenever appropriate (Andujar-Ortiz et al., 2009 Ridgway et al., 2007). 

Clinical samples of urine, blood, expired air, and tissue have been examined using headspace sampling approaches. Thus, chlorinated organic compounds, methanol, acetone, methyl ethyl ketone, and phenols have been determined in urine. Volatile substances in urine have also been used as a guide to acute poisoning, and the determination of stimulants in urine has been proposed as screening test for field use. The determination of the concentration of blood alcohol is the most well-known application of headspace techniques to biological samples. Blood has also been examined for cyanide, methyl sulfide, and formaldehyde levels, the last as a measure of methanol intoxication. The headspace approach for blood samples overcomes the difficulties associated with the alternative direct injection of two-phase samples. 

With regard to the solution approach, it is imperative that the solvent used be of the highest possible purity. Solution headspace is applicable to a much wider range of samples than the solid approach. When working with... 

Fig. 21.9. Flame ionization gas chromatograms of the headspace of the acceptable and unacceptable flavor samples. (Reprinted/redrawn from J. Chromatogr., 351, R.A. Sanders, and T.R. Morsch, Ion profiling approach to detailed mixture comparison. Application to a polypropylene off-odor problem, 525-531, Copyright (1986) with permission from Elsevier.)...

Solid phase microextraction (SPME) is an ideal approach to monitor volatile flavor components. This approach has been used to identify the volatile compounds in the headspace of fresh fruit during maturation [92], Using SPME fibers and GC/MS, the key flavor components are hexanal, 2-isobutyl-3-methoxypyrazine, 2,3-butanedione, 3-carene, trans-2-hexenal, and linalool (Fig. 8.1). In this study, the principal aroma compounds whose abundance varied during fruit development were specifically identified. 

Figure 4.1 depicts the difference between these two modes. Since there is no longer any partitioning of the analyte between the condensed phase and the vapour phase, compounds of limited volatility may yield a higher vapour pressure in this mode than by the conventional headspace approach, where they predominantly remain in solution. InvolatUe materials (such as API usually) do not vaporise, but instead condense onto the inside of the vial. The headspace vial effectively becomes a disposable injector liner. Care must be taken to operate at an incubation temperature that will not cause degradation of API into volatile components, which might interfere with the analysis. 

SPME (Figure 2.48) can be conducted as a direct extraction in which the coated fiber is immersed in the aqueous sample in a headspace configuration for sampling air or the volatiles from the headspace above an aqueous sample in a vial (headspace SPME analyses are discussed elsewhere) or by a membrane protection approach, which protects the fiber coating, for analyses of analytes in very polluted samples [136]. The SPME process consists of two steps (Figure 2.49) (a) the sorbent, either an externally coated fiber or an internally coated tube, is exposed to the sample for a specified period of time (b) the sorbent is transferred to a device that interfaces with an ana-... 

The United States Pharmacopeia (USP) test (467) describes three different approaches to measuring organic volatile impurities in pharmaceuticals. Method I uses a wide-bore coated open tubular column (G-27, 5% phenyl-95 % methylpolysiloxane) with a silica guard column deactivated with phe-nylmethyl siloxane and a flame-ionization detector. The samples are dissolved in water and about 1 p is injected. Limits are set for benzene, chloroform, 1,4-dioxane, methylene chloride, and trichloroethylene. Methods V and VI are nearly identical to method I except for varying the chromatographic conditions. For the measurement of methylene chloride in coated tablets, the headspace techniques described above are recommended. 

Wils et al. (25,26) previously reported an entirely different approach to TDG analysis. TDG in urine was converted back to sulfur mustard by treatment with concentrated HC1. The sample treatment is less straightforward than the methods described above, but analysis as sulfur mustard is facile. Urine, plus 2H8-TDG as internal standard, was cleaned up by elution through two C18 cartridges. Concentrated HC1 was added and the sample stirred and heated at 120 °C. Nitrogen was blown over the solution and sulfur mustard isolated from the headspace by adsorption onto Tenax-TA. The method was used to detect TDG in urine from casualties of CW attacks (see below). A disadvantage of this method is that it may convert metabolites other than TDG to sulfur mustard. This is supported by the detection of relatively high levels of analytes in urine from control subjects. Vycudilik (27) used a similar procedure, but recovered the mustard by steam distillation and extraction. 

In an alternative approach, cyanide and thiocyanate can be converted to cyanogen chloride using the chlorinating agent chloramine T. Conversion can be performed in solution or in the headspace. Conversion in solution followed by headspace analysis gave a detection limit of 5 ng/ml by gas chromatography/electron capture detection (GC/ECD) (72). Conversion in the headspace above acidified blood, in a precolumn packed with chloramine T powder and attached to the injection port of the GC, gave a detection limit of 50 ng/ml (73). 

Headspace analysis and SPME methods produce a wealth of chromatographic data and the best approach is to use chemometric analysis of selected chromatographic peaks under which circumstances identification of the individual compounds is not usually necessary. These techniques have been applied with some success to characterize olive oils (Morales and Aparicio, 1993 Morales etal., 1994). 

Most of the static headspace methods determine the partition coefficient by quantifying volatile concentration above a sample by gas-chromatography. The vapour phase calibration method (VPC) uses an external vapour standard for calibration. One must assure that the pure component is completely vaporized before injection. A widely employed alternative is the Liquid calibration static headspace (LC-SH) method (YoiWey et al. 1991 Nedjma 1997). A third approach uses HS-SPME. SPME may be used to determine partition coefficients if short sampling times are applied the process must only sample the headspace and not disrupt the equilibrium (Jung and Ebeler 2003). This method has become very popular to study the effect of wine macromolecules on the liquid-vapor equilibrium, (Whiton and Zoecklein 2000 Escalona et al. 2002 Hartmann et al. 2002 Aronson and Ebeler 2004). 

The isolation and concentration of petroleum products can be performed in several ways. The most efficient method is passive adsorption. In this method, the sample along with a tube filled with Tenax TA adsorbent is placed in a thermostated (60-70 °C) tightly closed container, such as a glass jar, for over 10 h. Under these conditions, a balance between compounds present in the headspace of the sample and the sample adsorbed on the polymer adsorbent is established. Adsorbed compounds are subjected to thermodesorbtion then, the desorbed compounds together with the carrier gas are injected onto a GC column, where they are separated and then identified. This approach has enabled easy detection and identification of trace amounts of petroleum products. Headspace analysis with passive adsorption on Tenax TA is normally used for separation and concentration of analytes. Gas chromatography coupled with an autothermal desorber and a mass spectrometer (ATD-GC-MS) is the best technique for separation of multicomponent mixtures... 

Several temperature-catalyzed stability tests are used in evaluating the oxidative stability of oils and fats. The oldest method is the Schaal oven test (39). It is inexpensive but subjective, because it uses organoleptic and odor intensities in the procedure and still requires days to obtain the result. This approach has been standardized into a recommended practice (AOCS method Cg 5-97). In the active oxygen method (AOM) (39), the development of peroxide is measured with time. As the formation and decomposition of peroxides are dynamic processes, the results obtained by this method do not correlate well to the actual stability of the oils and fats observed under practical application conditions. Other methods that have been based on oxygen absorption are the gravimetric (59) and the headspace oxygen concentration measurement (60, 61). 

The use of pervaporation as an alternative to the headspace technique is worth separate discussion. This is, in fact, one of the most promising uses of this approach, as revealed by two existing methods for mercury speciation and VOC analysis in solid samples that exemplify the advantages of pervaporation over static and dynamic head-space modes. Both methods were developed by using the overall assembly depicted in Fig. 4.24A, by which the analytical process was developed in the following four steps ... 

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