Abstract: Short packed silica gel column chromatography has been performed to optimize the production of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) from hen egg yolk with very low or no toxic solvents. The effects of silica type, sample loading amount, dimension of the glass chromatotube, and mobile phase compositions were investigated and high separation efficiency was achieved: gradient elution as 200 mL ethanol followed by 300 mL 95% ethanol to fractionate PE and PC after neutral lipids (NL) removed by 120 mL ethyl acetate, 40 mm silica gel (54 to 74 μm) bed height of the chromatotube with 22 mm inner dia (ID), and 0.25 g sample loading amount. By this procedure, 3.69 g PE and 2.88 g PC per 100 g egg yolk lipids were obtained, respectively. The refined PE and PC were identified by high-performance liquid chromatography/ultraviolet detector (HPLC-UV) with purity over 96%. The fatty acids in egg yolk revealed that PE and PC characterized higher ratios of n− 6/n− 3 (PE, 7.41; PC, 8.99). 18:2 n− 6 of PC (15.21%) predominated over PE (10.29%), whereas the level of 20:4 n− 6 of PC (8.78%) was lower than PE (15.67%).