Trans-esterification with BF3-methanol and gas chromatography

Fatty acids in the extracted lipids were derivatized into their methyl esters by trans-esterification with boron trifluoride-methanol. The derivatized methyl esters were redissolved in a small volume of hexane and purified by thin-layer chromatography using n-hexane-diethyl ether-acetic acid (50:50:2, v/v/v) as the developing solvents. After development, plates were dried and sprayed with Premulin. Fatty acid methyl ester bands were identified under ultraviolet light by comparing the rf of methyl heptadecanoate (C17:0) standard (rf, 0.67) applied side by side on the same plate. Methyl esters were extracted from thin-layer chromatography powder with diethyl ether concentrated under nitrogen and redissolved in 100 μl hexane. The lipids’ fatty acid compositions were analyzed by gas chromatography (GC) as follows. FAMEs were analyzed with 1 μl sample injection on an Agilent GC machine, model 6890N equipped with a flame ionization detector, an autosampler model 7693 and a ChemStation software for data analysis. An Agilent HP 88 with a 30-m GC column with a 0.25-mm inner diameter and 0.20-mm film thickness was used. Hydrogen was used as a carrier gas, and nitrogen was used as a makeup gas. The analyses were conducted with a temperature programming of 125 to 220°C. The fatty acid components within unknown samples were identified with respect to the retention times of authentic standard methyl ester mixtures run side by side. The fatty acid components were quantified with respect to the known amount of internal standard added, and the calibration ratio was derived from each fatty acid of a standard methyl ester mixture and methyl heptadecanoate internal standard. The coefficient of variation for GC analyses was within 2.3% to 3.7%.