Separation of Phospholipids from Soybean by Step-Gradient NP-HPLC

초록

Normal-phase HPLC was used to separate the useful phospholipids, phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC) from soybean lecithin. The mobile phase used in this experiment were hexane, isopropanol and methanol. Step-gradient mode was applied as the three components could not be separated by isocratic mode. To find the optimum separation condition, the concentration profiles of effluents were simulated by the retention factor and the plate theory in step-gradient mode. The retention factor was correlated by the equation of , where A, B, C, D, E were the experimentally determined constants and F, G were the volume fraction of isopropanol and methanol, respectively. From the calculated results, PE was separated with hexane/isopropanol/methanol (90/5/5 vol.%) in isocratic mode, while PI and PC were resolved in the operating conditions of 10 min. gradient time and the second mobile phase of hexane/isopropanol/methanol (50/20/30 vol.%) in step-gradient mode. The agreement between the calculated concentration profile and the experimental data was fairly good, so the methodology developed in this work can be used to obtain the optimum separation condition in step-gradient mode.