Sodium hydrogen sulphate

The fatty acid composition was determined following the ISO standard ISO 5509:2000 (ISO 2000). In brief, one drop of the oil was dissolved in 1 mL of n-heptane, 50 μg of sodium methylate was added, and the closed tube was agitated vigorously for 1 min at room temperature. After addition of 100 μL of water, the tube was centrifuged at 4500 g for 10 min and the lower aqueous phase was removed. Then 50 μL of HCl (1 mol with methyl orange) was added, the solution was shortly mixed, and the lower aqueous phase was rejected. About 20 mg of sodium hydrogen sulphate (monohydrate, extra pure; Merck, Darmstadt, Germany) was added, and after centrifugation at 4500 g for 10 min, the top n-heptane phase was transferred to a vial and injected in a Varian 5890 gas chromotograph with a capillary column, CP-Sil 88 (100 m long, 0.25 mm ID, film thickness 0.2 μm). The temperature program was as follows: from 155°C; heated to 220°C (1.5°C/min), 10 min isotherm; injector 250°C, detector 250°C; carrier gas 36 cm/s hydrogen; split ratio 1:50; detector gas 30 mL/min hydrogen; 300 mL/min air and 30 mL/min nitrogen; manual injection volume less than 1 μL. The peak areas were computed by the integration software, and percentages of fatty acid methyl esters (FAME) were obtained as weight percent by direct internal normalization.