Isolation and Concentration of Aroma Compounds

Due to its very low odor threshold (Table 5.21), the trisulfide is very aroma active and is frequently found in dilution analyses as a companion substance of methanethiol. For the moment, it is unknown whether it is derived from food or whether it is an artifact obtained in the isolation and concentration of volatile compounds. 

Solvent extraction is an excellent choice for aroma-compound isolation from foods when applicable. Unfortunately, many foods contain some lipid material, which limits the use of this technique since the lipid components would be extracted along with the aroma compounds. Alcohol-containing foods also present a problem in that the choice solvents (e.g. dichloromethane and diethyl ether) would both extract alcohol from the product, so one obtains a dilute solution of recovered volatiles in ethanol. Ethanol is problematic since it has a high boiling point (relative to the isolated aroma compounds), and in concentration for analysis, a significant proportion of aroma compounds would be lost with the ethanol. As one would expect, the recovery of aroma compounds by solvent extraction is dependent upon the solvent being used, the extraction technique (batch or continuous), and the time and temperature of extraction. 

It should be no surprise that the methods used to produce most flavourings from plant sources are based on similar principles as those used in the isolation of aroma compounds from foods. However, economics and scale play major roles in dictating methods. Additionally, the physical characteristics of the plant material, and concentrations and properties of flavouring materials also... 

The world of aroma compounds is becoming more and more complex. In the early days people used aromatic products like fruit juices or fruit juice concentrates which were relatively weak and still close to the related foodstulf. Later, with more knowledge of separation techniques, infusions, extracts, oleoresins and absolutes ranging from weak to strong impact were used to impart aroma. Essential oils such as spice oils already had a very strong impact. Modern analytical technologies allowed the evaluation of the chemical compositions of extracts and essential oils, so that isolates either as powerful mixtures or even as single compounds could be obtained. 

Aroma compounds are present in minute levels in foods, often at the ppb level ( ig/liter). In order to analyze compounds at these levels, isolation and concentration techniques are needed. However, isolation of aroma compounds from a food matrix, which contains proteins, fats, and carbohydrates, is not always simple. For foods without fat, solvent extraction (unit gu) can be used. In foods containing fat, simultaneous distillation extraction (SDE see Basic Protocol 1) provides an excellent option. Concentration of headspace gases onto volatile traps allows sampling of the headspace in order to obtain sufficient material for identification of more volatile compounds. A separate protocol (see Basic Protocol 2) shows how volatile traps can be used and then desorbed thermally directly onto a GC column. For both protocols, the subsequent separation by GC and identification by appropriate detectors is described in unitgu. 

An Isolation and concentration scheme should be appropriate for the sample under Investigation. As an example of this, a distillation technique would not be satisfactory for separating caramel aroma compounds such as Maltol, Cyclotene, or Furaneol which possess an enolone structure and do not steam distil. 

As has been previously said, 2,3-butanodione (diacetyl) is an important aroma of alcoholic beverages, it has not been studied and measured extensively in the past because of analytical difficulties in the quantitation caused by its highly volatile nature, chemical instability, and interference of other compounds. Colorimetric methods to measure diacetyl have been widely used in the past. These methods involve steam distillation to isolate diacetyl from the matrix. However, distillation has the disadvantage of incomplete isolation of diacetyl from other closely related compounds that will result in an overestimation of its concentration. A fluorometric method was developed to improve upon the lengthy distillation methods that involve derivatization. Although acetaldehyde and its acetal can be determined by direct injection GC-FID in spirit drinks (EU reference method for spirits), most chromatographic methods for minor aldehydes implicate also derivatization. While a very sensitive and accurate method based on SMPE without derivatization and MS detection has been developed, it requires the use of... 

These established methods for isolating and concentrating flavor compounds, and their effects on the composition of the aroma concentrates, are described in comprehensive detail in the literature (1-27). 

In terms of specificity in isolation, one will also isolate food constituents that are not aroma compounds (e.g. pesticides, herbicides, PCBs, plasticisers, and some antioxidants). Since these compounds are typically present in foods at very low levels, they generally present few complications. The primary volatile that complicates the application of this methodology is water. In all cases, one obtains an aroma isolate that consists of volatiles in an aqueous solution . Thus, unless the amount of water is small and the subsequent analytical step is tolerant of some water, volatility-based techniques must include some water-removal process. This may be freeze-concentration, the addition of anhydrous salts, or solvent extraction. Distillation is often used to isolate aroma compounds from fat-containing foods. Since fat is not volatile (under isolation conditions), its presence does not prohibit the use of this methodology. 

A prerequisite for the calculation of OAVs are exact quantitative data. Aroma compounds, which are relatively stable and are present in food extracts in higher concentrations (>100 pg/kg food) are often quantified by using an internal standard containing a similar pattern of functional groups as the analyte. In a quantitative study on cherry odorants [63] it has been shown, that the results are significantly influenced by the isolation technique used and by the structure of the odorant. However, under appropriate conditions the values differed only between 7 % (benzaldehyde) and 26 % ((E,Z)-2,6-... 

Reaction of an aqueous solution of cystine with thiamin, glutamate, and ascorbic acid produces a complex mixture of compounds with an overall flavor resembling that of roasted meat. The reaction was carried out at 120 C for 0.5h at pH 5.0 in a closed system. The aroma compounds were isolated by means of the simultaneous steam distillation/solvent extraction method. The flavor concentrate was pre-separated by liquid chromatography on silica gel and subsequently analysed by GC and GC/MS. Unknown flavor components were... 

Chang et al. (42). They Isolated and identified 2,4,5-triinethyl-3-oxazoline in boiled beef. This compound was described as having a "characteristic boiled beef aroma". Mussinan et al. (35) identified oxazolines and no oxazoles in their beef system. Peterson et al. (43) reported on the volatiles of canned beef stew. Both 2,4,5-trimethyloxazole and 2,4,5-trimethyl-3-oxazoline were present. The relative concentration of 2,4,5-trimethyloxazole was medium while for 2,4,5-trlmethyl-3-oxazoline was extra high. Lee et al. (44) identified 2-methyl-3-oxazoline, 2,4-dimethyl-3-oxazoline and 2,4,5-trimethyl-3-oxazoline in the volatiles of roasted peanuts. The latter two 3-oxazollnes were also identified in the volatiles of fried chicken (19). 

Quantification of aroma-impact components by isotope dilution assays (IDA) was introduced in food flavor research by Schieberle and Grosch (1987), when trying to take into account losses of analytes due to isolation procedures. The labeled compounds have to be synthesized, the suitable fragments have to be chosen, and calibration has to be effected. A quantitative determination of ppb levels of 3-damascenone (Section 5,D.38) in foods, particularly in roasted coffee (powder and brew), was developed by Sen et al. (1991a). Semmelroch et al. (1995) quantified the potent odorants in roasted coffee by IDA. Hawthorne et al. (1992) directly determined caffeine concentration in coffee beverages with reproducibility of about 5 % using solid-phase microextraction combined with IDA. Blank et al. (1999) applied this combined method to potent coffee odorants and found it to be a rapid and accurate quantification method. They also concluded that the efficiency of IDA could be improved by optimizing the MS conditions. 

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