Fatty acid methyl esters are prepared in a two-stage reaction (to avoid trans-isomerization) using CH3O−Na+ and CH3OH/HCl according to the AFNOR method (AFNOR 1984). Methyl esters are more volatile and less polar than fatty acids.
In 100 mg of microbial lipids we add 10 ml of CH3O−Na+, which contain phenolofthaleine as a pH indicator and the mixture is boiled at low temperature. 20 min later, CH3OH/HCl is added until the mixture’s pH become acidic, and then the mixture is boiled again for 20 min. The reaction is stopped by adding water and the fatty acid methyl esters are collected with a triple hexane extraction.
The fatty acid methyl esters are analysed in a Gas Chromatographer using a 60 m×0.25 mm HP-88 J8W Scientific column, Chrompack; helium was the carrier gas (2 ml/min). The analysis was run at 200 °C with the injector and detector at 250 °C. The fatty acid analysis lasts approximately 20 min. Fatty acid methyl esters were identified by reference to authentic standards
(1). 1 g of sodium is diluted in 100 ml of methanol:benzol (70:30) to prepare sodium methoxide solution.
(2). 10 ml of acetyl-chloride are added slowly in 125 ml of methanol to prepare the hydrochloric methanol solution. The reaction is very exothermic.