Analysis Of Flavor Compounds

The analysis of food flavors is complicated because of several factors. The flavor-active compounds are found in very low concentrations, ranging from a few 100 ppm for strongly flavored food products to less than 10 ppm for weakly flavored foods. In addition, they represent a wide range of functional groups with widely differing physical and chemical properties. Moreover, some of the components are thermally labile whereas others are highly reactive and may be lost if great care is not exercised during their isolation and concentration as well as in the subsequent analysis. A further complication that puts great demands on the analytical methodology is that the trace components, even when present at levels of ppm or less, may sometimes make a greater contribution to the flavor than components present in vastly greater amounts. The economic importance of such a low threshold value flavor compound lies in the reduced cost of production of highly flavored food products. Despite impressive data available on the chemical composition of food flavors, there is still lack of complete understanding of what determines the flavor of many foods. This may be due, for some products at least, to the inability of the analytical techniques to identify important trace components. However, major advances both in GC and MS technology—such as the introduction of inert thermostable capillary columns; gentle non vaporizing on-column injection methods; improved interfaces between GC and MS; and sophisticated facilities for spectral acquisition, storage, data manipulation, and library search— make the modern GC/MS an analytical tool of outstanding powers, capable of acquisition of useful mass spectral data from the narrowest of GC peaks.

Establishing the chemical composition and the structure of flavor compounds found in food is important for correlating structures to sensory properties and for understanding the mechanisms by which flavors are formed from their precursors. Although analysis of food flavors is similar in many respects to analysis of any other mixtures, there are special problems associated with it, such as the low concentration of flavor components and their extreme complexity, frequently containing several hundred components of different functional groups. In some cases, trace components in an aroma have far greater sensory importance than other components present in larger amounts; hence, sample preparation and concentration is specially important for analysis of flavor compounds.

Sample Preparation

There are two different approaches to prepare samples for flavor analysis. The first approach is total volatile analysis, which attempts to isolate and concentrate from target food products all the chemicals that could possibly contribute to the flavor. This can be achieved by different distillation and extraction methods. The most appropriate method is largely dictated by the nature of the product under study and by the extent to which its flavors are affected adversely by heat. Fresh fruits and other heat-sensitive products, for example, can be distilled at low temperatures under vacuum, whereas heat-stable products can be steam distilled and simultaneously extracted into an organic solvent by using Likens- and Nickerson-type apparatuses. The second approach is headspace analysis, which aims at analysis of volatile compounds found at equilibrium in the vapor phase above the food. The second approach is faster and simpler and requires small quantities of the food product for analysis. In addition, the headspace contains volatiles in the same relative concentrations as one actually inhales. The headspace volatiles can be analyzed by injecting a small volume of the volatiles directly into a gas chromatograph (GC) or, after its concentration, by cryogenic trapping or by the use of porous polymers such as Porapak and Chromosorb or by activated charcoal (23).

Chemical Methods of Analysis

The identification of the flavor components present in a sample can be established rapidly by GC/MS analysis. However, in complex mixtures, important trace components are often either poorly separated from or totally masked by other components. Consequently, interpretation of their mass spectra may be difficult to perform. To overcome this problem the sample can be modified prior to, during, or after GC/MS analysis to simplify its complexity. Modification of the sample prior to GC/MS analysis can be achieved by preliminary fractionation of its components into polar, nonpolar, and weakly polar fractions or into acidic, basic, and neutral fractions by column chromatography using silica gel. Carbonyl compounds as a group are of special significance since they occur in a wide range of food products of plant and animal origin. As a group they can be isolated by treating the sample with an acidified solution of 2,4-dinitrophenylhydrazine and analyzed as their 2,4-dinitrophenylhydrazone derivatives by GC, HPLC, TLC, and so on. Modification of the sample injected onto a GC/MS can be performed on-line by means of chemical abstractors deposited on deactivated solid supports contained in a coiled tubing and placed before the analytical column of the GC. The function of the abstractors is to remove components bearing specific functional groups by reacting with them. The absence of a particular peak is an indication of the presence of the functional group when the resulting chromatograms are compared with those obtained in the absence of the abstractor. Alternatively, to achieve greater resolution for incompletely separated peaks, components of each chromatographic peak can be recovered by different trapping techniques and analyzed again using different columns. Trapping techniques may be very simple, such as collection of peaks in cooled glass capillary tubes inserted into the GC column or by use of porous-layer open-tubular (PLOT) glass capillaries containing a layer of diatomacous earth support permanently fused to the wall of the capillary tube (23).

Gas Chromatography /Mass Spectrometry

The combined GC/MS remains the most powerful technique available to the chemist for the separation and identification of complex mixtures, because it generates the maximum amount of structural information for the smallest amount of sample in the shortest time. For more than 20 years it has been the mainstay technique in the analysis of flavor components in food and still is, despite recent advances in complementary techniques such as GC/IR (24).

The separation of the complex mixture into its components is achieved on the gas chromatographic column, which can be divided into two types: packed and open tubular (or capillary). The separated components are then introduced through the GC/MS interface into the ionization chamber of the mass spectrometer, where the molecules are bombarded with a beam of energetic electrons (electron impact mode), causing them to ionize and fragment in a way that is characteristic of the molecule. The resulting mixture of ions are then separated on the basis of their mass/charge ratio (m/z) and their relative abundances are recorded. The results are then displayed as a plot of ion abundance versus m/z, which is called the mass spectrum. This represents the characteristic fingerprint of the molecule. The mass spectrum of an unknown compound then can be used to identify its structure by comparison to other known mass spectra; this can be done conveniently by computers.

Gas Chromatography-Olfactometry (GCO)

GC is used mainly in the quantitative analysis of flavor mixtures. However, when the instrumental detector is replaced with a human nose—through the use of a sniffing port—GC can also be used to perform sensory analysis. The technique is known as GCO (25). The main application of GCO is to establish and identify the odor-active components and odor quality of individual components present in a mixture and separated on a GC column. The most common techniques that utilize GCO include Charm-Analysis® and Aroma Extract Dilution Analysis (AEDA). Both techniques are based on dilution analysis and produce quantitative estimates of relative potency of compounds eluting from a GC column. In dilution analysis, the aroma extracts are serially diluted, and each dilution is analyzed until no significant odor can be detected. In AEDA, the number of dilutions required to eliminate the odor is used to estimate the potency of that particular compound. In CharmAnalysis®, retention times are included in the calculation of the potency factors that are known as "charm values."

Purge and Trap-Thermal Desorption

Low concentration of aroma volatiles can be isolated and concentrated on porous polymer traps such as poly[2,6-diphenyl-p-phenylene oxide] (Tenax-GC) using purge and trap (P&T) apparatus (26). Subsequently, the trapped volatiles can be thermally desorbed into a GC column using thermal desorption (TD) devices mounted on a GC. Both solid and liquid samples can be analyzed, if proper sampling apparatus is used. In both cases, samples are purged with purified inert carrier gas for a specific period of time (20-90 min), at a flow rate in the range of 10 to 80 mL/ min. The temperature, which can range from subambient to few hundred degrees Centigrade, depends on the nature of the sample. The adsorbant trap is then removed and fitted with a syringe needle and attached to the thermal desorber. The traps are thermally desorbed for a period of 3 to 10 min at temperatures depending on the nature of the sample (50-350°C). During the desorption period, the temperature of the GC column is maintained at subambient values to cryofocus the aroma volatiles. After the desorption phase, the volatiles can be separated and eventually identified by initiating the temperature programming of the column.

Solid-Phase Microextraction

Solid-phase microextraction (SPME) is a simple adsorption/desorption technique (26) that eliminates some of the problems associated with solvent extraction of aroma volatiles. The device used to perform SPME consists of a 1-cm fused silica fiber, coated with a specific stationary phase (such as polydimethylsiloxane, carboxene, etc) and bonded to a stainless steel plunger and encased in a holder. The fused silica fiber can be drawn into a hollow needle attached to the holder by using the plunger. The device can be used to adsorb aroma compounds either from a solution or from the headspace above the solution, by inserting the needle through the septum that seals the sample vial. After sample adsorption, the needle can be introduced into the GC injector where the adsorbed volatiles are thermally desorbed into the GC column and analyzed. The adsorption equilibrium is usually attained in 2 to 30 min, depending on the concentration of the sample and the thickness and the type of the coating of the fiber.

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