2.5.1. Determination of fatty acids
Fatty acids were determined by gas chromatography (GC) after
transesterification of glycerides to methyl esters of fatty acids. Specifically,
in a 200 ml spherical flask 1 g of the fat that was extracted
by the Soxhlet method and then 15 ml of a mixture containing
methanol and benzene (3:1) and 0.2 g p-toluenesulfonic acid were
added. The flask was connected with a vertical condenser and
heated in a water bath at 100 C for 2 h. After cooling the content
was transferred to a 250 ml separation funnel and 100 ml of distilled
water were added. Extraction was carried out twice with
50 ml petroleum ether. Sodium sulphate was added (0.7 g) to dry
the solution and the solvent was removed using a rotary evaporator.
The residual liquid was diluted with 5 ml toluene and 2 lL
were injected in the injection port of a Shimadzu Gas Chromatograph
GC-8A (Kyoto-Japan) with a FFAP type stainless steel column
(3 m). The carrier gas was N2 at 20 mL/min. The injection port and