Modified Rapid Preparation of Fatty Acid Esters from Lipids for Gas Chromatographic Analysis SIR: In this letter we describe a modification of the rapid technique for preparing fatty acid methyl esters from triglycerides according to Metcalfe, Schmitz, and Pelka (I). Their method consists of a saponification with a methanolic alkali solution ( 5 minutes) followed by boiling the soap with B F r methanol in the same vessel for 2 minutes (Procedure B). The methyl esters are then floated out of the mixture using a saturated salt solution. We used this technique for the analysis of a sample of cocoabutter. As a reference we used the fatty acid composition of the same cocoa-butter as determined via the methyl esters obtained by a trans-esterification method using methanolH2SO4(Procedure A). The latter method, which is basically in conformity with the procedure given by the American Oil Chemists’ Society (2), is used routinely in our laboratory and is thoroughly checked for quantitative results. The reported data are the averages of at least two analyses that did not differ more than 0.4x (absolute) for the major fatty acids. As can be seen from Table 1, the fatty acid composition as obtained through the Procedures A and B do not agree. With the BF3-methanol procedure the most important differences are a too small amount of palmitic acid and a too high stearic acid content. These differences are not caused by an incomplete reaction as was shown when the methyl esters were extracted from the mixture with heptane directly after the BF3 treatment (Procedure C). The fatty acid composition was in good agreement with our reference (see Table I). This result suggests that a nonrepresentative part of the methyl esters remains in the methanol-water phase. This effect diminished when the total amount of methanol was reduced to half the original quantity (Procedure D in Table I). Although the deviations from the reference composition are reduced, the result is still not correct. To optimize the circumstances, we added a few milliliters of h e p tane to the reaction mixture. To promote a good extraction of the methyl esters, the mixture was boiled for another minute. Then the saturated salt solution was added and the fatty acid composition of the heptane solution determined (Procedure E). From Table I it is seen that the result is now in good agreement with the reference composition. Modified Procedure. Approximately 150 mg of fat is added to a 50-ml roundbottom flask equipped with a ground glass joint. Two milliliters of 0.5N methanoiic sodium hydroxide is added and the roundbottom flask is connected to a helicoil condenser. The mixture is boiled under total reflux on a sand bath until the fat globules go into solution. Usually this step will take 2 to 5 minutes. Two milliliters of BF3-methanol is added via the condenser. (The BF3-
(1) L. D. Metcalfe, A. A. Schmitz, and J. R. Pelka, ANAL. CHEM., 38, 514 (1966). (2) J . Am. Oil Chemists’ Soc., 43, 12A (1966).
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ANALYTICAL CHEMISTRY
Table I. Fatty Acid Composition (Weight %) of a CocoaButter Sample by Gas Chromatography of Methyl Esters Prepared by Several Modifications of a BFS-Methanol Procedure Fatty Proced ureb A B C D E acid. 14:O 16:O 16:l 17:O 18:O 18:l 18:2 18:3 20:o
0.1 25.4 0.3 0.2 34.8 34.8 3.3 0.1 1.0
0.1 25.7 0.3 0.2 34.5 34.7 3.3 0.2 1.o
0.1 23.1 0.3 0.2 37.3 34.1 2.9 0.2 1.2
0.1 24.3 0.3 0.2 36.4 34.1 3.1 0.3 1.2
0.1 25.1 0.4 0.3 34.6 34.8 3.4 0.2 1.1
Fatty acid notation: first figure indicates the number of carbon atoms, second figure the number of double bonds in the molecule. A. Reference composition; HzS04-methanol procedure (2). B. BFa-methanol; Metcalfe, Schmitz, and Pelka (I). C. As B, methyl esters extracted with heptane. D. As B, half the amount of methanol used. E. As D, 2 ml of heptane added before NaCl solution. 5
Table 11. Fatty Acid Composition (Weight %) of Palmkernel Oil Sample by Gas Chromatography of Methyl Esters Prepared by Some Modifications of the BF3-Methanol Procedure Procedureb Fatty acids A B E F 8 :O 3.3 2.2 3.5 3.4 10:o 12:o 14:O
16:O 18:O 18: 1 18:2 a
3.5 48.7 16.2 8.2 2.4 15.4 2.2
2.8 46.3 17.0 9.1 3.0 17.2 2.5
3.5 48.4 16.1 8.3 2.6 15.3 2.4
3.6 48.6 16.2 8.3 2.5 15.3 2.1
See Table I, footnote a. A. Reference composition. B.
BF,-methanol technique, Metcalfe, Schmitz, and Pelka (I). E. Modified BF8-methanol procedure. F. As E, saturated salt solution replaced by water.
methanol reagent was obtained as a boron trifluoride methanol BF, from the British Drug complex, containing about 14 Houses, Ltd.) The boiling is continued for 2 minutes. One and a half to 4 ml of heptane are added via the condenser and the mixture is boiled for another minute. Enough of a saturated sodium chloride solution is added to bring the liquid level into the neck of the roundbottom flask. About 1 ml of the upper (heptane) layer is pipetted into a glass-stoppered tube. Some anhydrous sodium sulfate is added to bind traces of water. This solution is suited to be injected directly on a gas chromatographic column.
DISCUSSION We applied the modi iied esterification procedure to different types of natural fats. These included beef tallow (rich in saturated fatty acids), lish oil (rich in highly unsaturated fatty acids), and palmkernel oil (rich in lower molecular weight fatty acids). In all cases the modified technique gave results that were in very good agreement with our routinely determined reference compositions. Metcalfe, Schmitz, and Pelka (1) used a saturated salt solution to prevent thr: loss of lower molecular weight fatty acids in the water-methanol phase. But, as was shown in Table I, losses of fatty acids with a relatively small number of carbon atoms in the molecule still occurred when cocoa-butter was analyzed with this technique. The same effect was observed for the methyl esters prepared from the lower fatty acid containing palmkernel oil (Table 11). However, when the modified procedure was used it made no difference whether water was used instead of a saturated salt solution (Table 11). The amount of heptane that is used as the extraction solvent is not critical. This means that the quantity can be chosen in such a way that the concentration of the methyl esters in the heptane is just suited ior a direct injection on the gas chromatographic column. Although we did no1 analyze a butterfat completely, we did determine the ratio of the butyric acid and the decanoic acid content. For this ratio we found 2.2, a considerable higher value than 1.6 as was l’ound by Metcalfe, Schmitz, and Pelka (I). This result suggmts that with the modified procedure relatively more butyric acid will be found. The modified BF8--methanol procedure was evaluated relative to our routine procedure. This technique leads to quantitative results when the fats analyzed do not contain fatty acids with less than 12 carbon atoms. If lower fatty acids are present, however, the results are no longer correct as can be seen in Table 111. In this table the results are given of the analysis of a text mixture of triglycerides containing lower as well as medium molecular weight fatty acids. The two methods are again in good agreement with each other but the absolute values of the percentages of octanoic acid and decanoic acid are lower than the values of the quantities weighed into the test mixture. This effect is not due to the response ol’ the flame ionization detector as was
z)
Table 111. Fatty Acid Composition (Weight of Triglyceride Test Mixture Containing Lower Fatty Acids Methyl esters prepared by different procedures Weighed H~SOIBF3Fatty acid0 composition methanol methanol 8:O 1o:o
12:o 14:O 16:O 18:l
4.0 7.0 9.9 16.3 20.2 42.6
3.2 6.6 9.8 16.4 20.5 43.5
3.4 6.6 9.8 16.3 20.3 43.4
See Table I, footnote a.
shown by analyzing the National Heart Institute Fatty Acid Standard E (3). The quantitative results agreed with the stated composition data with a relative error less than 3 for major components ( > l O z of the total mixture) and less than 5 % for minor components (
