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Abstract
A high‐performance liquid chromatographic method coupled with evaporative light‐scattering detector was used for the accurate quantification of soybean phospholipids. This method is based on normal‐phase chromatography with silica gel as stationary phase and a ternary gradient with n‐hexane, isopropanol, and water as mobile phase. Major soybean phospholipid classes were separated by optimizing the solvent systems. The ternary solvent of n‐hexane:isopropanol:water (53:42:5, by volume) was suitable for the separation of neutral lipids (NL), glycolipids (GL), and phosphatidylethanolamine (PE). By using hexane:isopropanol:water (17:66:17, by volume), phosphatidylcholine (PC), sphingomyelin (SM), and lysophosphatidylcholine (LPC) were separated. PE and phosphatidylinositol (PI), phosphatidic acid (PA), and PC were base line resolved by carefully adjusting the flow rate and switch time of intermediate gradients. The analysis was completed in 32 min, and repeated injections of the samples were possible. The method has good repeatability and accuracy from the view point of high recovery, and low coefficients of variance in retention time and peak area for each phospholipid. Comparison was made between three detectors, i.e., ELSD, UV, and RI. The results show that ELSD is the best in phospholipids analysis.
