ANALYTICAL

BIOCHEMISTRY

78, 244-251 (1977)

Gas Chromatographic Retentions Identification Criteria1 K. YABUMOTO,~ University

W.G.

JENNINGS,~

of California,

Davis,

as

AND M. YAMAGUCHI~ California

95616

Received July 20, 1976; accepted November 5, 1976 On high-resolution wall-coated open tubular glass capillary columns, retention data are realistic criteria for identifying compounds. Two-dimensional plots of Kovats indices measured on two columns of different polarity are particularly useful in this regard. Temperature usually exercises only minor effects on Kovats indices, but because the magnitude and direction vary with the nature of the compound and the liquid phase, temperature shifts may affect the elution order of components of complex mixtures,

Since the very early days of gas chromatography, retentions have been proposed as criteria for identification of compounds. The reliability of these identifications was remarkably low, largely because of the limited separation efficiency of the gas chromatographic column. Interlaboratory comparisons were further complicated by differences between liquid phases, reactivities of solid supports, and limited control of parameters such as temperature and flow rates. These discrepancies have been well reviewed by Rijks (I), who found that temperature variations exceeding 10°C were not uncommon in older-model commercial gas chromatographic ovens. Open tubular columns overcome difficulties experienced with the reactivity of the solid support, and open tubular glass capillary columns offer even greater freedom from reactivity, plus the advantage of much higher resolution. Several commercial chromatographs available today control column oven temperatures with greater precision and uniformity. Tourres (2) and Walraven (3) demonstrated that retention indices (4) on liquid phases of two different polarities, or at two different temperatures, produced plots in which isomers differing only in the degree of branching produced lines of parallel slopes. ’ Portions of this work are from a thesis submitted by the senior author in partial satisfaction of the requirements for the Ph.D. in Agricultural Chemistry. z Present address: Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065. 3 Department of Food Science and Technology. 4 Department of Vegetable Crops. 244 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN OGQ3-2697

GAS CHROMATOGRAPHIC

APPARATUS

RETENTIONS

245

AND METHODS

Gas chromatography. A Hewlett-Packard 57llA gas chromatograph was adapted to wall-coated open tubular (WCOT) glass capillary columns coated (5,6) with Carbowax 20M or methyl silicone SE-30, admixed with benzyltriphenylphosphonium chloride (7) by a modified Golay technique. Complex flavor essences recovered from muskmelon, Cucumis melo (8), were chromatographed at three different isothermal temperatures (50,70, and 90°C for the SE-30 column; 80, 100, and 120°C for the Carbowax 20M column). Identification of the constituents had been established by gas chromatography-mass spectrometry, precise retention comparisons, and infrared spectroscopy (8). RESULTS AND DISCUSSION

Although retention data, including Kovats retention indices, have long been used as identification criteria, it is difficult to eliminate the possibility of two compounds exhibiting identical retention behavior in a given gas chromatographic column. This possibility is especially troublesome in low-resolution packed columns. The major value of retention data lies in the fact that one can rule out (by exclusion) many otherwise possible compounds. The level of confidence can be increased by utilizing columns of higher separation capability and establishing retentions on columns of two different polarities. Figure 1 shows an isothermal chromatogram of a

0 TIME

3b

I

-

IN

IO MINUTES

20

40

FIG. 1. Typical gas chromatogram of Porapak essence of cantaloupe headspace volatiles. Kovats index determination; at 100°C isothermal. 0.3 ~1 on 0.25 mm x 80 m wall-coated open tubular glass capillary column coated with Carbowax 20-M. admixed with benzyltriphenylphosphonium chloride (5%) and Ionox 330 (1%). Split ratio on injection, 1:lOO; inlet temperature, 250°C.

246

YABUMOTO,

JENNINGS,

AND YAMAGUCHI

muskmelon essence on a WCOT glass capillary Carbowax-20M column at 100°C and Fig. 2 shows a chromatogram of the essence on an SE-30 WCOT glass capillary column at 70°C. Hydrocarbon standards were cochromatographed in the same injection. The columns achieved baseline separation for peaks differing by two index units (i.e., separation number, ca. 50). Isothermal runs at + 20”from the above temperatures (i.e., Carbowax-20M at 120 and 80°C; SE-30 at 90 and 50°C) were also made, Kovats indices are expressed by the following equation at each selected temperature: Ig = 1% Tdsubstancd - 1% T,(n) b log T&z + 1) - log T,(n)

+ N x 1oo

1

where1 = Kovats retention index; a = liquid phase; b = column temperature; Ta(substance) = corrected retention time of the substance; T,(n) = corrected retention time of the leading hydrocarbon; T,(n+ 1) = corrected retention time of the trailing hydrocarbon; and N = carbon number of the leading hydrocarbon. Corrected retention times were determined by subtracting the dead time from the overall time from injection through the peak maximum. According to Rijks (l), the leading edge of methane peak gives an accurate measurement of dead time, and this value was used in these determinations. The hydrocarbon peaks were selected so that the peak of interest came between the standard hydrocarbons (which differed by only one methylene group) to minimize errors. It has been pointed out by early workers (9,lO) that while Kovats indices exhibit a hyperbolic shift as the temperature is increased, within a

I! L J

TIME

IN

i0

MINUTES

FIG. 2. Typical gas chromatogram of Porapak essence of cantaloupe headspace volatiles. Kovats index determination at 70°C isothermal. 0.3 ~1 on 0.25 mm x 80 m wall-coated open tubular glass capillary column coated with SE-30, admixed with Igepal CO-880 (7%). Split ratio on injection, 1: 100; inlet temperature, 250°C.

GAS

CHROMATOGRAPHIC

RETENTIONS

247

relatively small range (e.g. SO’), this shift is approximately linear. Therefore, the temperature dependence of the Kovats indices is expressed in the following equation (9): AZ/lo”C

=

1, - 1, (H - L)/lO

where AZ/lo”C = the retention index increment per 10°C; H = the higher and L = the lower temperature of temperature of determination; determination. Therefore, for Carbowax-20M,

and for SE-30, AZ/lo”C

=

zg - zg 4

.

The AZllO”C values obtained by this calculation indicate the direction and magnitude of the shift when the operating temperature was raised by 10”. Table 1 shows the Kovats indices observed in the two liquid phases and values of AZ/lO”C. Most of the index values were in close agreement with reference values (11,12); alcohols exhibited the greatest deviation. This was probably because alcohols have relatively large AZ/lo”C values (Table I), and the reference values were determined at different temperatures than were the indices in this study. Agreement was much closer when a temperature correction was applied. To assist in assigning identities to unknown compounds, the Kovats indices for the two liquid phases for each compound were plotted in a two-dimensional plane, as described by Rijks (l), who reported that the isomers of hydrocarbons tend to show recognizable patterns depending on the molecular weight. Figure 3 shows such a plot and indicates that isomeric esters exhibit the same pattern. Since the mass spectra of esters whose molecular weight is between 130 and 200 give a barely detectable or undetectable molecular ion with conventional electron impact mass spectra (13), two-dimensional plots of this type are particularly useful in estimating molecular weights. The graph also enabled characterization of unknown compounds by their position relative to the other group of compounds. For example, butanol and isobutanol have small retention times on SE-30, but relatively large retention times on the more polar Carbowax-20M, and their plots lie far removed from the group of esters in the graph. Moreover, the homologous series of compounds show straight line relationships in the plot. This is, by definition, equivalent to the fact

248

YABUMOTO,

JENNINGS, TABLE

KOVATS

INDICES

AND YAMAGUCHI 1

FOR COMPOUNDS

OF MUSKMELON”

Carbowax-20Mb Compound

I IllDo

Methyl acetate Ethyl acetate Propyl acetate Isopropyl acetate Butyl acetate Isobutyl acetate Pentyl acetate 2-Methylbutyl acetate 3-Methylbutyl acetate Hexyl acetate Ck-3-hexenyl acetate Methyl propionate Ethyl propionate Butyl propionate Isobutyl propionate 2-Methylbutyl propionate Methyl butyrate Ethyl butyrate Propyl butyrate Butyl butyrate Isobutyl butyrate 2-Methylbutyl butyrate Methyl isobutyrate Ethyl isobutyrate Butyl isobutyrate Isobutyl isobutyrate Methyl pentanoate Ethyl pentanoate Methyl 2-methylbutanoate Ethyl 2-methylbutanoate Propyl 2-methylbutanoate Butyl 2-methylbutanoate Isobutyl 2-methylbutanoate 2-Methylbutyl 2-methylbutanoate Methyl hexanoate Ethyl hexanoate Ethyl 3-hexenoate Ethanol Butanol Isobutanol Pentanol 2-Methylbutanol Hexanol

818 878 969 890 1068 1008 1169 1119 1119 1269 1311 901 952 1139 1079 1189 983 1033 1122 1218 1158 1265 919 963 1147 1091 1086 1133 1008 1053 1141 1234 1178 1282 1185 1232 1295 917 1122 1066 1237 1187 1337

AIllOT +0.2 0.0 0.0 0.0 -0.5 -0.5 -0.2 -0.2 -0.2 +0.2 +os +0.2 0.0 0.0 0.0 +0.5 -to.2 -0.5 +0.2 0.0 +0.2 +0.7 +0.7 -0.2 0.0 0.0 0.0 0.0 0.0 +0.2 +os 0.0 +0.7 +1.5 +os 0.0 0.0 -3.5 -3.5 -2.8 -2.3

SE-30” I 700 524 608 706 653 805 766 905 872 870 1004 1000 626 702 899 859 962 716 792 891 985 946 1048 679 750 944 905 821 893 772 843 937 1031 994 1096 916 990 997 692 655 801 765 897

AIllOT -d

-1.5 -0.7 -1.0 -0.5 -0.2 -1.0 -0.5 -0.5 -0.2 -1.3 - 1.0 -0.7 -0.2 0.0 -0.2 -0.2 -0.5 -0.2 -0.2 -1.0 -0.2 -0.2 -0.2 0.0 0.0 +0.5 0.0 0.0 -0.2 +0.2 0.0 -0.5 -0.5 -4.0 -3.7 -3.2 -3.2

GAS CHROMATOGRAPHIC TABLE

249

RETENTIONS

1 (Confinued) Carbowax-20M*

Compound Cis-3-hexenol Acetaldehyde Dimethyl disulfide Ethylene

SE-30C

I 100~

AlilWC

I 700

AlllO°C

1369 695 1076 -

-1.0

894 742 -

-3.0

-

+1.8

(1All determinations utilized wall-coated open tubular glass capillary columns. * Carbowax-20M admixed with 5% BTPPC and 1% Ionox 330. e SE-30 admixed with 7% Igepal CO-880. d Not measured.

that an homologous series of compounds shows a linear relationship between their molecular weights and the logarithm of the retention times. It is notable that homologous series of compounds show not only the linear relationship of the logarithm of their retention times to their molecular

8-B

600 i

FIG. 3. Kovats indices on Carbowax 20M vs Kovats indices on SE-30. Charts of this type permit prediction of retention dam for known compounds, or estimation of the molecular weight and degree of branching of an unknown compound from its retentions on the two liquid phases.

250

YABUMOTO,

TIME

JENNINGS,

AND YAMAGUCHI

--F

FIG. 4. Effect of separation temperature on gas chromatographic relative retentions and elution orders. All determinations measured isothermally on WCOT glass capillary columns coated with Carbowax 20M. The abscissas on the loo” and 120” chromatograms have been expanded to facilitate comparison. Components shown: a, pentyl acetate; b, isobutyl 2-methyl butyrate; c, methyl hexanoate; d, 2-methyl butyl propionate; e, 2-methyl butanol; f, butyl butyrate.

weight as do the straight-chain hydrocarbons but also the increment of the logarithms by a methylene unit is almost equal to that of the straight-chain hydrocarbon series (by definition, 100 index units) in both the Carbowax20M and SE-30 liquid phases; i.e., the Kovats indices of those members of the acetate series higher than propyl acetate are about 100 index units apart in both liquid phases. Figure 4 illustrates a phenomenon occasionally encountered in gas chromatographic analysis, a change of elution order. This can be experienced when the sample contains compounds of different functional groups, and the temperature of analysis is changed. REFERENCES 1. 2. 3. 4.

Rijks, J. A. (1973) Thesis, Tech. Univ. Eindhoven, The Netherlands. Tourres, D. A. (1967) J. Chromatog. 30, 357-377. Walraven, J. J. (1968) Thesis, Tech. Univ. Eindhoven, The Netherlands. Kovats, E. (1965) in Advances in Chromatography (Giddings, J. C., and Keller, R. A., eds.), Vol 1, pp. 229-247, Dekker, New York. 5. Jennings, W. G., Yabumoto, K., and Wohleb, R. H. (1974) J. Chromarogr. Sci. 12, 344-348.

GAS CHROMATOGRAPHIC 6. 7. 8. 9. 10.

RETENTIONS

251

Jennings, W. G. (1975) Chrumafographia 8, 690-692. Malec, E. J. (197l)J. Chromatogr. Sci. 9, 318-320. Yabumoto, K., Jennings, W. G., and Yamaguchi, M. submitted for publication. Ettre, L. S. (1964)Anal. C/rem. Xi,8 3lA-41A. Harris, W. E., and Habgood, H. W. (1966) Programmed Temperature Gas Chromatography, Wiley, New York. 11. McReynolds. W. 0. (1966) Gas Chromatographic Retention Data, Preston Tech. Abst. Co., Evanston, Ill. 12. Jennings, W. G. Unpublished data. 13. Silverstein, R. M., and Bassler, G. C. (1967) Spectrometric Identification of Organic Compounds. 2nd ed., Wiley, New York.