0.5 hour after dissolution of the sample. Twenty samples can be analyzed by an analyst in a single work day. ACKNOWLEDGMENT
(2) Burke, K . E., Davis, C. M., ANAL. CHEM.36,172 (1964). (3) Dagnall, R. hf., West, T. S., Young, P., Analysl90, 13 (1965). (4) Gentry, C. H. R., Sherrington, L. G., Ibzd., 71,432 (1946). (5) Hill, U. T., ANAL. CHEM.31, 429
The author t'hanks Thomas Ruppert for his assistance.
(10) Ibid., p. 329; Ibid., 2124. ( 1 1 ) Pakalns, P., Anal. Chim. Acta 32, 57 (1965). (12) Rooney, H. R. C., Analyst 83, 54.6 (1958). (13) Strafford, N., Wyatt, P. F., Ibid., 68,319 (1943). KEITHE. BURKE
The International Nickel Co., Inc. Paul D. Merica Research Laboratory Sterling Forest, Suffern, N. Y. 10901
LITERATURE CITED
( 1 ) Burke, K . E., Anal. Chim. Acta 34,
Presented, 152nd hleeting, ACS, New York City, September 1966.
485 (1966).
Analysis of Methyl Octadecenoate and Octadecadienoate Isomers by Com bined Liquid-Solid and Capillary Gas-Liquid Chromatography SIR: Liquid-solid chromatography on silver nitrate-impregnated adsorbents is frequently used for the separation of unsaturated fatty acid methyl esters according to their geometry (cis and trans) or degree of unsaturation (monoene, diene, triene, etc.) (1,6,16). Capillary gas-liquid chromatograply is used extensively for the study of complex mixtures of isomeric esters, particularly the methyl octadecenoates (11, IS) and octadecadienoates (10, 12, 16). However, almost all reported analyses of fatty acid methyl esters on capillary columns have been confined to the separation of postional isomers of a given geometry or geometrical isomers at a given bond position. We have applied a combination of the above chromatographic techniques to the analysis of partially hydrogenated vegetable oils. The successful analysis of samples containing such a multiplicity of isomeric fatty acids has not been reported previously. I n the present work liquid-solid chromatography on silver nitrate-silica gel was used to separate monoenoic fatty
acid methyl esters into trans and cis isomer fractions and dienoic methyl esters into several distinct fractions depending upon geometry and bond position. These fractions mere then analyzed by infrared spectrometry, capillary gas chromatography, and oxidative cleavage to give a fairly complete picture of the fatty acid composition of partially hydrogenated fats and oils. A similar combination of nicthods was recently used by the authors to analyze a much less complex mixture of internal olefins (8). EXPERIMENTAL
Preliminary Treatment of Sample. Methyl esters of the fatty acids were prepared from the fat or oil by basecatalyzed alcoholysis according to procedures previously described ( 7 , 8). For oils containing appreciable dienoic fatty acids, the monoenoic and dienoic methyl esters were first separated by liquid-solid chromatography of their mercury derivatives (8). The unsaturated ester fractions thus isolated were then further separated by chromatography on silver nitrate-silica gel as described below. For samples of 21.5%
38.1%
13.3%
6.1%
-
-I
Sample 0.6309. Recovery- 100 %
ro
Eluant 60 140 PE1 Benzene
50150
"
'I
40160 'I " 30170 " '' 901 I O Benrene/Ether
O/lOO
"
'I
OJ-
IO
20
30
40
FRACTION NUMBER
50
60
I 70
Figure 1. Liquid-solid chromatography of Cis dienoates from hydrogenated corn oil
low or negligible diene content the methyl esters can be chromatographed directly on silver nitrate-silica gel. Liquid-Solid Chromatography. The technique used here was a modification of that reported by DeVries (5, 6). Silica gel impregnated with about 30'% silver nitrate was used in 39- x 2-cm. columns cont.aining 75 to 80 grams of adsorbent. The adsorbent was sometimes mixed with Celite 545 filter aid in 20: 1 or 30: 1 ratio to facilitate solvent flow. Sample sizes averaged about 1.0 gram for saturated-monoenoic methyl ester mixtures and 0.65 gram for dienoic methyl esters. The chromatography required 20 to 48 hours depending upon the complexity of the sample. Petroleum ether-benzene mixtures were used to elute the various components: 80:20 for the saturated esters, 70:30 for the trans monoenoic esters, and 55: 45 for the cis monoenoic esters. For dienoic ester mixtures, the elution was started with 60: 40 petroleum ether-benzene, followed by 50:50, 40:60, and 30:70 mixtures. The final dienoic ester fraction was eluted with 9O:lO benzeneethyl ether. The composition of these fractions will be discussed later. Ethyl et,her was used to strip the column of the non-ester components. The "valleys" 1
I
125
I20
TIME, MINUTES
Figure 2. Gas chromatographic separation of trans Cls monoenoate isomers VOL 38, NO. 1 1 , OCTOBER 1966
1611
3
I
205
200
I95
TIME, MINUTES
Figure 3. Gas chromatographic separation of cis CIE monoenoate isomers
between the major fractions were determined by taking numerous small individual fractions. Prior knowledge of the composition of the sample, either by a preliminary chromatographic run or other independent data, is of help in determining which fractions should be composited to keep coelution to a minimum. Solvent was added to all individual fractions immediately after obtaining the initial weight to minimize oxidation before making the composites. The adsorbent was prepared in a manner similar to that described for silver nitrate-alumina (3). Davison Grade 923 100-200 mesh silica gel was used as received. The silver nitratesilica gel was dried overnight a t 120' C. rather than a t the 180' C. used for the alumina. The final adsorbent usually contained 19 to 21y0 silver and 1 to 2y0 moisture (weight loss after three hours a t 180' C.). Best results were obtained with adsorbents containing 1.6% moisture. Gas-Liquid Chromatography. All gas chromatographic analyses were performed on 150-foot by 0.01-inch capillary columns (pancake configuration) in a Perkin-Elmer Model 226 chromatograph. The trans-octadecenoate isomers were analyzed on an XE-60 coated capillary under programmed temperature conditions: 10G170' C. a t 0.5' C. per minute. The cis-octadecenoate and all the octadecadienoate isomers were analyzed on a polyphenyl ether coated column (OS-138, Perkin-Elmer Corp.) : the
former at 100-180' C. a t 0.5' C. per minute and the latter a t 140-180' C. a t 0.5' C. per minute. The inlet pressure was 20-30 p.s.i.g. and all other conditions were as noted previously (3). Peak areas were determined by the peak height times width at half height method. Samples of the cis and trans isomers of methyl 6-,9-, and 11-octadecenoates and the geometric isomers of methyl 9,120ctadecadienoate were run to aid in peak identification. Oxidative Cleavage. Details of the periodate-permanganate oxidation and subsequent programmed temperature gas chromatography of the products as well as typical chromatograms have been reported (9). Monoand dicarboxylic acid cleavage fragments (C4 and up) are recovered and determined quantitatively (as their methyl esters) by this method. Infrared trans Determinations. Analytical and instrument conditions employed in the infrared method for trans unsaturation have been described (2). A calibration curve prepared from binary mixtures of methyl elaidate and methyl oleate was used. Because of the limited sample available some of the infrared trans values were obtained on weights as low as 20 mg. (in a 1-ml. volume). RESULTS A N D DISCUSSION
Methyl esters were prepared from a mixed cottonseed-soybean oil which had been hydrogenated to an iodine
-
Table 1.
Major Methyl Octadecadienoate Classes Separated (Figure 1 ) by Liquid-Solid Chromatography NO.of CH,
Fraction
% IR trans
A
136
B C
91
D E
86 10
1612
58
ANALYTICAL CHEMISTRY
groups between double bonds 0 1 1 1 2 2 2
Major components 20% conjugated dienes
7570 trans,trans dienes cis,trans dienes 70% cis,cis dienes 30% trans,trans dienes czs,trans dienes cis,cis dienes
I 115
110 TIME, MINUTES
Figure 4. ration of tion A
Gas chromatographicsepadienoate isomers-frac-
CIS
value of 48. The methyl esters were chromatographed directly on silver nitrate-silica gel to give a clear separation of the methyl esters into saturated (46.6790), trans monoenoic (37.970), cfs monoenoic (13.3%) esters and dienoic esters plus non-ester components (2.2y0). The trans monoenoic ester fraction analyzed 96% trans, the cis monoenoic ester fraction
