The Rapid Preparation of Fatty Acid Esters
for Gas Chromatographic Analysis L. D. METCALFE
and A. A. SCHMITZ
Armour Industrial Chemical Co., McCook, III. The routine application of gas chromatography to the determination of the composition of fatty acid samples made it essential to prepare
esters rapidly and simply. trifluoride-methanol converts fatty acids to their methyl esters in about 2 minutes. The esters are comparable to those obtained by other
methyl Boron
procedures, most important applications of gas-liquid chromatography (GLC) is the determination of the fatty acid composition of lipides. When this technique is used, the fatty acids are usually converted to their methyl esters. To apply GLC to the routine plant control of large numbers of fatty acid samples, it is essential that the methyl esters be prepared rapidly and simply. A number of authors (1-5, 8, 9) have described procedures for preparing methyl esters of fatty acids prior to GLC analysis. These procedures usually use anhydrous methanol containing an acidic or basic catalyst and involve reflux for 0.5 to 2 hours. Diazomethane in ether methylates fatty acids rapidly, but it must be freshly prepared each time it is used and it is toxic and explosive. Mitchell (6, 7) has described the use of a boron trifluoride-methanol reagent for esterifying carboxylic acids in an analytical procedure employing Karl Fischer reagent. The present authors found that fatty acids boiled in an excess of this reagent were essentially completely esterified in about 2 minutes. The esters were easily recovered and analyzed by GLC. of the
One
EXPERIMENTAL
BF3-Methanol Reagent. One liter of reagent grade methanol, in a 2-liter flask, is weighed and cooled in an ice bath. With the flask still in the bath, BF3 is bubbled through a glass tube into the methanol until 125 grams is taken up. This operation should be performed in a good fume hood, and the gas should not flow so fast that white fumes emerge from the flask (The BF3 must be flowing through the glass tube before it is placed in and until it is removed from the methanol, or the liquid may be drawn into the gas cylinder valve
system.) This reagent has an excellent shelf life and has been used up to 4 months after preparation. Esterification Procedure. One hundred to 200 mg. of fatty acid is placed in a 20 X 150 mm. test tube, and 3 ml. of BF3-methanol reagent is added. The mixture is boiled on a steam bath for 2 minutes, and the esters are recovered by the appropriate one of the following two procedures.
Only Acids of More Than 10 Carbons Present. The boiled mixture is washed into a 125-ml. separatory funnel with 30 ml. of petroleum ether (b. p. 40-60° C., reagent grade, redis-
tilled) and 20 ml. of distilled water is added. The funnel is shaken vigor-
ously, the layers are allowed to separate, the aqueous-methanol layer is drained off and discarded, the petroleum ether layer is drained through filter paper into a 50-ml. beaker, and the solvent is evaporated on a 60° C. w7ater bath. The last traces of solvent may be removed under reduced pressure, although this is usually unnecessary. Acids of Less Than 10 Carbons Present. The boiled mixture is transferred to the separatory funnel with 20 ml. of water, the contents are mixed and allowed to separate, the ester layer on top is then physically separated from the aqueous layer, and the sample is filtered or centrifuged to remove small amounts of entrapped w7ater. By either procedure, the total elapsed time is about 10 minutes, and the esters are ready for GLC analysis. RESULTS
AND DISCUSSION
A series of commercial fatty acids, containing 6 to 18 carbons, and two dibasic acids w7ere esterified by the BF3methanol procedure. The per cent recovery (determined by weighing) and the free fatty acid values (determined by titration with base) are shown in Table I. The free fatty acid remaining in these esters is on the order of 1%. This small amount appears to have no effect on the GLC results. Table I als,o shows the effect of petroleum ether on the recovery of esters, particularly of the lower acids. Petroleum ether is used to prevent crystallization of the higher molecular weight esters, but large fractions of the lower esters are lost in the evaporation of petroleum ether at 60° C. Even wrhen petroleum ether is removed at room temperature by a stream of air, some
In this series of experiments, no effort was made to recover esters quantitatively. The procedure w7as specifically designed for plant control work where large amounts of sample are available. When all possible care is taken to make recovery quantitative (as with costly biological samples), the recovery is well above 90%. A number of commercial fatty acids (C12 to Cis) were mixed to form a synthetic mixture. These acids wrere not pure, so the exact composition of the mixture is not known. Portions of the mixture were esterified by the dry methanolic-HCl method (
