**5. Extraction and purification of individual molecular species of soybean phospholipids**

Extraction and purification of phosphatidic acid of C18 fatty acids

Powdery soybean phospholipids containing 20% PA are extracted with five folds (by weight) of 95% ethanol at 45 degrees Celsius for 2h with stirring at the speed of 100rpm. After centrifugation at 700×g for 10min, the supernatant is discarded. The above extraction procedure is repeated four more times until the ethanol fraction is colorless. The solid fraction is extracted with 5 folds (by weight) of methanol with a stirring speed of 100rpm for 12h at room temperature. The methanol fraction is obtained after centrifugation at 700×g for 10min. The methanol extraction procedure is repeated three times in total. The methanol fractions are combined. If the methanol is removed by evaporation, the solid residue will contain about 50% PA.

The pH of the methanol extract is adjusted to 8-9 by 1mol/L sodium hydroxide and obvious white precipitate is observed. The supernatant is obtained by centrifugation at 700×g for 5min. If the methanol is removed, the solid residue will contain about 70% PA. The pH of the supernatant is adjusted to 5-6 by 1mol/L hydrochloric acid before n-hexane of four times the volume of the methanol solution is added and mixed. The n-hexane phase is obtained after extracting for 15min and being left standing still for 2h. The n-hexane is evaporated at 45 degrees Celsius and the residue is dissolved in methanol of ten times the volume of the n-hexane phase. 60% zinc chloride solution is added into the methanol solution until no more white precipitate is formed. The precipitate is obtained by centrifugation at 700×g for 10min, washed three times with acetone which is removed by filtration and dried under nitrogen gas steam. The solid obtained is PA of C18 fatty acids of about 98% pure. The yield is 1/60 of the raw materials (Liu et al., 2008).
