Qualitative Analysis by Gas Chromatography GC versus the Nose in Formulating Artificial Fruit Flavors P. W. Rasmussenl Universiti Kebangsaan Malaysia, Bangi, Malaysia I t is sometimes forgotten that, besides being a very powerful technique of quantitative analysis, GC can also be used aualitativelv ( I ) . This is esoeciallv true if, as is often the case, cbnsidrrabie information is already a\,aijablt>on the sample to I,(.identified. Kxamples would be in monitoring environmental samples fur a suspected nmtaminant, or thr idenritication ot onr or more of a restricted set uf coml~uundssuch a s drugs in a biological sample, or volatile comp&nds in an artifical flavorina. In these cases, a direct coupling of the GC with other instrumentation,espe( ially MS,gives good qualitative and auantirative results hut such expensire and dedicated instrimentation is often not necessiry for nearly unambiguous identification. At the very least, proper use of the GC alone can limit the use that needs to he made of other instrumentation. follow The - -~ ~ ~ in^ exoeriment is intended for the underzraduate laboratory as ill;stration of the use of GC retentionindexes for the identification of unknown compounds, specifically, the identification of the volatile compounds responsible for the odor of the hanana. This complex sample was chosen to show the limitations as well as the usefulness of identification by retention index onlv. .. and because of student interest in such a sample. In addition, other common laboratory procedures are illustrated: preparation of the sample illustrates steam distillation and solvent extraction, and the preparation of standards reauires an ester svnthesis. The experiment can be extended so that individual components of banana are isolated and their identity confirmed by infrared, nuclear magnetic resoname, and/or mass spectrometry. Finally, from the results of the GC analysis, an artificial hanana flavor can be prepared and compared (by GC and by nose) to the natural flavor and to a commercially available artificial banana flavor.
an
Extraction of the Volatlle Compounds from Banana
There are two main problems in the analysis of any fruit by GC: large amounts of non-volatile material are present and can clog the injector port and column; and the main volatile component is water, which obliterates the very small quantities of other volatile compounds present. The usual methods of dealing with these problems are distillation to separate the non-volatile material and solvent extraction to separate the water (2). These two methods can be carried out in either order. but the extraction-distillation seauence involves extracting a pulpy mass with the attendant difficulties of nons e ~ a r a t i n eimmiscible lavers and distillation of very small amounts i f volatile mate&. Thus, the distillation-extraction sequence is easier. A quantity of hanana pulp is liquefied in a blender with approximately twice its weight of distilled water and is steam distilled. (The slurry will rapidly turn to a greyish-brown color due to oxidation.) The best extract is obtained if the co-distillation is carried out a t reduced pressure t o avoid the possihlity of a "cooked" flavor developing. Under aspirator pressure, the banana slurry need only he heated t o 40-50°C and about % of it should he distilled. The distillate is saturated with NaCl and extracted four times with small portions of
CHzCIz. The combined CHzC12 extracts are dried over NaCl and distilled to a small volume. Although banana is a fruit that contains a relatively high oercentaae of volatile flavorina comoounds, still only about kl ppm iynormally present i3i:'l'his h e m s ;hat if a kilogram ot'fruit is usvd in thedistillation, thr final extract will cmtnin only about 'LOmg uf volatile material. This will hc sufficient if the volume of the extract is reduced to 100-200~1and if a CC with flamr ionization d r t r r t ( ~ rii s used. It will prtlhahly not he rnough if a thermal conductivity instrument ii to be usrd, and it may then be necessary mcombine the extractsof sereral students; in fact, it would be an advnntage to have a small b~oupof students do the rxperiment as a pruject and use a p w h d extract. The extract should haw an odor similar to the original bananas; the residual CH2C12does not contrihute significantly to the odor. Preparation of Standards
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Althoueh it is nossihle to look uo retention indices in the literature ( 4 ) , not very satisfactory results are obtained since the GC columns used will orohablv differ considerahlv from those in the literature. Therefore, i t is necessary to prepare standards. This is made easier bv the fact that by far the majority of volatile compounds present in banana are lowmolecular-weight alcohols. esters. and ketones (5).In fact, the C2-C6alcohol-: (including isohutyl and iauimyl), esters of thrie alcohols with - " acids rincludinr! isoh~~tvric and isovaleric~, and the C4-Cs 2-alkanones are those most commonly found in most fruits (6). In banana, 2-pentauol and esters of 2-pentanol are also common, while propanoates are rare and can be omitted. The propyl esters can be omitted as well, hut to include them requires no extra time, and i t helps in the assignment of GC peaks (vide infra). Later they may he deleted. Thus, 59 standards must he measured, hut they may he run as only nine samples by combining them in series that will give
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' Present address: Department of Chemishy. University of Calgary. 2500
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Universiry Dr ve. NW. Calgary. Aloena. Canaoa T2N Journal of Chemical Education
1N4.
1W
50 10
150 20
200 1
30
OC
min
Figure 1. Gas chromatagramson Apiezon L liquid phase. (a) Mixture of valerate esters. (b) Mixture of *alkanes.
separate peaks on the GC. All other things being equal, retention time on anv GC column will increase with molecular weight and decreaie with chain branching (7). Therefore, a mixture of the C& straight chain alcohols plus 2-methyl1-propanol (isobutyl alcohol) and 3-methyl-1-butanol (isoamyl alcohol) will give separate peaks on any GC column, and esters prepared from this mixture and a carboxylic acid will also give separate peaks. This is illustrated in Figure l(a), which shows a regular series of n-alkyl compounds, interspersed with a two-member isoalkyl series. The regularity of the series makes assignment of peaks easy, and it was for that reason that the propyl group was included. Figure l(a) actually shows the mixture of valerate (pentanoate) esters, which is prepared as follows: About 0.1 ml of the mixture of alcohols mentioned above, 0.2 ml of valeric (pentanoic) acid and 2 drops of concentrated sulfuric acid are mixed in a small test tube or vial and the test tube put in a water bath a t 75'C. After about 1min, when the tube contents have warmed up, the tube is stoppered with a cork to prevent evaporation of the more volatile alcohols and esters. Heating is continued. with occasional shaking. for about one hour, thenlthe tube is cooled and the contents s k e n with two 1-ml portions of 10%NaOH and then dried over a small quantity of Na2S04 or NaCI. The supernatant liquid can he decanted and used directlv or diluted with a volatile solvent such as acetone. The esters of the other acids can be prepared in the same way. The esters of 2-pentanol can be inciuded in the same mixtures by addine 2-pentanol to the mixture of alcohols, but it may be difficultto interpret the GC. I t is better to combine 0.1 ml of 2-pentanol with 0.2 ml of a mixture of acids (Cz, C4-C6, including isobutyric and isovaleric acids) and 2 drops of concentrated HzS04and react as ahove to get a mixture of 2-pentyl esters. The final standard mixture consists of 2pentanol combined with the 2-alkanones (C4-Cs). Gas Chromatography of Standards The nine standard mixtures (alcohols, acetates, isobutyrates.. butvrates. isovalerates. valerates. hexanoates..2 -.~ e n t v l . esters, 2-alkanbnes plus 2-pkntanol) and the banana extract must be run on a t least two different GC columns in order to obtain reliable identification. These columns should be chosen so that they have widely differing characteristics. If two columns are used, the most logical choice is a column with a non-polar liquid phase and one with a polar liquid phase or one which has a special affinity for esters. Apiezon, sqnalane, and silicone are examples of the former, and Carhowax, FFAP, and DEGS are examples of the latter (8).A listing of the McReynold constants for liquid phases is useful in choosing columns (9). The actual choice will probably depend mainly on availability in the individual laboratory, and it is not critical. The two columns chosen here are Apiezon L and Carbowax 1540. both 3 mm X 2.5 m stainless steel with 10%loadine on ~hromosorhW. The GC is dual column with flame ionization detectors and is oven-temperature-programmed from 50°C a t 4°/min. This gives excellent separation of the major constituents of the banana extract, hut the analysis can be carried out isothermally with slightly less good separations. In fact, iwthermal ourration may give better rewroducibilitv of retention times, "nless the te*perature pr&amming is carried out carefully and is reproducible. It is possible to do qualitatke aiialysis hy using retention times only, hut it is more mtsininnful to use one of the ralcu. lated retention indices which appear in the literature since a retention time is characteristic of an individual instrument and set of operating conditions while a retention index is independent of instrument variables and essentially characteristic only of the compound being measured. Any retention index may be used (7) but the commonest, and the one used here, is the Kovats Index (lo), which is the retention time relative to the retention times of the n-alkanes. For a GC peak
(X) which falls between the peaks for the n-alkane with N carbons and the n-alkane with M carhons ( M > N), the Kovats Index (Kx) is given by (7): log Vx - log VN K x = lOON + 100(M - N) log VM - log VN for isothermal operation, where V is the retention volume (or corrected retention time) for the three peaks. For linear-temperature-programmed operation, the Kovats Index can be approximated by (11): Kx
= lOON = 100(M
- N)
(:: :3 -
(2)
where t is the uncorrected retention time. The usual method for calculatine Kovats Indices is to combine a mixture of n-alkanes witi the compounds to be measured, but this can be difficult with com~lexmixtures such as fruit extracts because ot ihr cxtmiivt~overlappingofpeaks (vide intra~.A faster and onlv slirhtlv less accurate method is to run the unknown com~oundsind mixture of alkanes separately, and keep conditions as nearly as possible the same during the two chromatograms. Thus, alkanes need not be mixed with any sample, and onlv 11samples need be run on each column (9 itan;lanl soluti;na, the aikane mixture, and the banana extract). The sraldity of isothermal conditions or the repr(8ducibilit) 11ftrmperiiture programming must be cht:cked hy wmparing tu80runs of the I I -alknnes. Isothermal operarim should prtsmr no problems, but prograniming may or may not be acceptable. For iostance, in our laboratory, a Hewlett-Packanl5710A GC with the 3702A wm ttmperature programmer is reproducible to within i 2 Kovats Units; a HP5XtOA-microurocessor-wntrolledGC gives unaccentable results (up to f20 Kovats Units), when they are programmed from 50-17O0C a t 4OImin. The data given here were obtained on the former instrument. When reproducible GC conditions cannot be attained, the alkane mixture must be combined with a portion of each of the samples to he run. Then. in anv chromatoeram where overlapping occurs hetween sample peaks and a&ane peaks, the clearly separated alkane peaks are used to calculate the indices for the clearly separated sample peaks, using eqns. 1 or 2. A chromatogram of the sample without alkanes is then obtained, and the sample peaks with already calculated indices are used as references to calculate the indices for the rest of the peaks, using slightly modified forms of eqns. 1or 2:
where S and L are the reference peaks (sample peaks with known indices) nearest to, and on either side of, the peak to be measured (KL > K X > KS) (11). Thus, whenever alkane peaks overlap with sample peaks, two chromatograms are necessary for complete assignment of indices. A mixture of alkanes with the banana extract will have overlapping peaks, but manv of the standard solutions mav not. For instance. a mixture of the valerates with alkanes will not have any overlauoine weaks on either the A~iezonor the Carhowax lianid phasesu(iee Fig. 1). For the banana extract and the standards used here. a mixture of Cs-Cl4 n-alkanes is sufficient for the ~ p i e z o nL column. This is shown in Fieure l(b). For the wolar column (Carhowax 1540), C9-CI7 GkaneS will cove; the desired range. The Kovats Indices for the 52 standards (the seven n-propyl compounds were omitted) are measured on both GC columns (Apiezon and Carbowax), and are listed in Table 1.The table is arranged in order of increasing Apiezon index; this facilitates matching unknown indices with the series of known values. Volume 61
Number 1
January 1984
63
Table 1. NO.
Compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
ethanol 2-butanone isobutyl alcohol ethyl acetate 2-pentanone ~ b u t yalcohol l 2-pentanol ethyl isobutyrate isobutyl acetate isoamyl alcohol ethyl butyrate 2-hexanone butyl acetate amyl alcohol 2-pentyl acetate ethyl ,sovalerate isoamyl acetate ethyl valerate isobutyl isobutyrate amyl acetate Bhexyi alcohol butyl isobutyrate isabutyl butyrate 2-pentyl isobutyrate butyl butyrate ethyl hexanoate
Kovats Indices of Standard Comoounds
Kovats Index Apiezan Carbowax
