Methylatioln of Fatty Acids by Pyrolysis of Their Tetramethylammonium Salts in the Gas Chromatograph Donald T. Downing Departments of Dermatology and Biochemistry, Boston University School of Medicine, Boston, Mass. 02118 Richard S. Greene Department of Dermatology and Evans Memorial Department of Clinical Research, University Hospital, Boston, Mass. 02118 QUANTITATIVE CONVERSION of saturated fatty acids into their methyl esters occurs when the dry tetramethylammonium salts of these acids are pyrolyzed in the vaporizer unit of the gas chromatograph (1). This reaction has proved useful in the analysis of aqueous solutions of mono- and dicarboxylic acids (2, 3). The principal advantages of the technique are avoidance of the loss of volatile fatty acids during manipulations and the capability of working with aqueous solutions. In practice, the convenience of the pyrolysis methylation and the use of the tetramethylammonium hydroxide reagent in any preliminary extractions have proved to be added benefits. It has therefore seemed desirable to extend the use of the pyrolysis methylation to mixtures of fatty acids containing unsaturated components, as in most naturally-occurring lipids. It was expected that polyunsaturated fatty acids might present special difficulty, but conditions have been found under which these compounds may conveniently be examined. Pyrolysis methylation and the standard boron trifluoridemethanol esterification were compared by application to free fatty acids prepared from commercial reference mixtures of methyl esters. EXPERIMENTAL
Apparatus and Materials. The gas chromatograph (F and
M Scientific Corp. Model 400) was fitted with a flame ionization detector and modified for probe injection as described previously ( 1 ) . The 5-ft by 0.125-in 0.d. stainless-steel column was packed with 1 4 z diethylene glycol succinate on Diatoport S (Applied Science Labs. Inc.). The column was maintained at 175' C and the injection port at 280' C. Test mixtures of methyl esters of fatty acids were obtained from Applied Science Laboratories, Inc. Tetramethylammonium hydroxide was obtained as a 2 5 z aqueous solution (Mallinckrodt Chemical Works). Procedure. Direct gas chromatographic analysis. A hexane solution (10 mg/ml) of each of the test mixtures of methyl esters was first subjected to gas chromatographic analysis. Percentage compositions of the mixtures were then calculated from the peak areas, with allowance being made for the relationship between detector response and proportion of carbon in the respective molecules (4). Preparation of Free Fatty Acids from the Reference Mixtures. A 10-mg sample of each of the test mixtures was saponified by heating at 50' C in 0.5N ethanolic KOH for 30 min, after which the mixture was acidified and extracted with hexane. The hexane extracts containing the free fatty acids were washed free from mineral acid by shaking with water, and the hexane layer was dried over Na2S04. (1) (2) (3) (4)
D. T.Downing, ANAL.CHEM., 39,218 (1967). D. T. Downing and R. S. Greene, LIPIDS, 3, 96 (1968). D. T. Downing and R. S. Greene, J. Inoesr. Derm., in press. R. G . Ackrnan, J. Gas Chromatog.,2, 173 (1964).
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Figure 1. Chromatograms of a mixture of (1) methyl stearate, (2) methyl oleate, (3) methyl linoleate, and (4) methyl linolenate obtained: (A) by direct injection of the esters. (B) by pyrolysis methylation of the alkaline
solution of the tetramethylammonium salts of the liberated acids. (C) by pyrolysis of the tetramethylammonium salts after bringing their solution to pH 7.5-8.0. Boron Trifluoride Esterification. One half of each hexane solution of free fatty acids was evaporated under a stream of nitrogen and the residues were treated with 1-ml of 14x boron trifluoride-methanol reagent (Applied Science Laboratories). After heating at 50" C for 15 min in a screw-cap test tube, the mixtures were diluted with 1 ml of water and extracted with 0.5 ml of hexane. The mixtures of methyl esters in the hexane solutions were then analyzed by gas chromatography. Pyrolysis Esterification. The remaining half of each hexane solution of free fatty acids was shaken with 0.5 ml of a 1 solution of tetramethylammonium hydroxide in 50 aqueous ethanol. The lower, aqueous layers were transferred to VOL 40, NO. 4, APRIL 1968
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Evaluation of Pyrolysis Methylation for the Analysis of Saturated and Monounsaturated Fatty Acids Composition of test mixture K102 determined by:a Known composition Direct injection BF methylation Pyrolysis methylation Weight % Weight % Weight % Errorb Weight Err0l.b 14:O 12.29 12.5 f 0.33 12.3 f 0.63 -1.6 12.6 f 0.26 +O. 8 16:O 23.57 23.8 f 0.30 23.8 f 0.41 0.0 24.0 i 0.39 +0.8 16:l 6.43 6.3 f 0.20 6.4 f 0.10 +1.6 6.3 f 0.20 0.0 18:O 23.66 25.2 f 0.40 24.7 f 0.56 -2.0 25.0 i 0.54 -0.8 18:l 34.05 32.4 f 0.47 32.9 f 0.58 +1.5 32.0 4Z 0.68 -1.2 Each analysis is the average from six chromatograms. 6 Error refers to the difference between the values for direct analysis and each re-esterification procedure. Table I.
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Table 11. Evaluation of Pyrolysis Methylation for the Analysis of Polyunsaturated Fatty Acids Composition of test mixture H105 determined by:" Known composition Direct injection BF3 methylation Pyrolysis methylation Weight Weight Weight Errorb Weight Errorb 18:O 26.9 27.1 f 0.80 26.5 i.0.32 -2.2 26.9 f 1.02 -0.7 18:l 24.6 24.8 f 0.35 25.0 =t0.23 +1.2 25.5 f 0.57 +2.8 18:2 28.6 28.8 4Z 0.30 28.9 f 0.51 +O. 3 28.9 f 0.76 +O. 3 18:3 19.9 19.6 f 0.22 19.5 f 0.31 -0.5 18.7 f 0.75 -4.6 a Each analysis is the average from six chromatograms. % Error refers to the difference between the values for direct analysis and each re-esterification procedure.
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clean tubes and 3-pl aliquots of these solutions of the tetramethylammonium salts of the fatty acids were taken up in a capillary probe, dried at 100" C for 5 min and then injected into the gas chromatograph. The methyl esters produced by pyrolysis in the vaporizer were fractionated in the instrument in the usual way. When polyunsaturated fatty acids were present, it was necessary to modify the pyrolysis esterification procedure in one respect. The solution of the tetramethylammonium salts, which is normally highly alkaline, was brought to pH 7.5-8.0 by careful addition of 5% acetic acid. The resulting solution was then taken in the probe, dried, and injected into the gas chromatograph. RESULTS AND DISCUSSION
The pyrolysis methylation procedure as previously applied to saturated fatty acids gave excellent results with a mixture of saturated and monounsaturated acids. Close agreement was obtained with the results of standard procedures (Table I). However, when the same pyrolysis conditions were used with a mixture containing polyunsaturated acids there were major differences between the chromatograms obtained with the original esters (Figure 1A) and those from the pyrolysis methylation of the liberated acids (Figure 1B). This difference appeared to be caused by the alkalinity of the solution of tetramethylammonium salts. The effect was overcome when the pH of the solution was reduced to 7.5-8.0 by addition of acetic acid. With this modification of the original procedure there were no detectable extraneous peaks in the chromatogram (Figure 1C) and acceptable quantitative analyses were obtained (Table 11). The demonstration that the pyrolysis methylation can be used with the acids present in naturally-occurring lipids makes available a technique which is more rapid and convenient than existing procedures. This advantage exists even when the material to be analyzed is an anhydrous mixture containing only free fatty acids. Under other circumstances the pyrolysis methylation may have additional advantages. If the fatty acids are present in a mixture containing neutral compounds, from which they must first be extracted, the use 828
ANALYTICAL CHEMISTRY
of tetramethylammonium hydroxide as the extracting agent provides a solution requiring no further manipulation before being taken into the injection probe. When fatty acids are present as esters, in a lipid mixture containing unsaponifiable material, existing analysis procedures will involve a number of time-consuming steps for recovery of the acids in a form suitable for esterification. If the pyrolysis procedure is used, the usual saponification, removal of unsaponifiable material, and extraction of liberated acids into an organic solvent may immediately be followed by extraction of the fatty acids into tetramethylammonium hydroxide. The most significant limitation of the pyrolysis methylation is the special injection procedure required. Although a probe injection device is easily constructed and connected to most commercial gas chromatographs ( I ) , few instruments are at present modified in this way. A recent report by Bailey (5) suggests that in some circumstances at least the usual syringe injection may be employed for pyrolysis methylation, but such conversions are probably only quantitative at the very high concentrations employed by Bailey. With syringe injection, as established by Robb and Westbrook (@, the yield of methyl esters decreases with the concentration of the injected solution, particularly in the presence of water. However, the pyrolysis methylation procedure seems ideally suited to certain systems designed for automatic, unattended operation of gas chromatographs, when the solutions for analysis are evaporated onto metal gauze pellets for loading into a magazine. RECEIVED for review December 1, 1967. Accepted February 5 , 1968. This investigation was supported in part by Research Grant AM 07388 and Graduate Training Grant AM 05295, National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.
(5) J. J. Bailey, ANAL.CHEM., 39, 1485(1967). (6) E. W. Robb and J. J. Westbrook, Ibid., 35, 1644 (1963).
