Abstract: The effects of initial water content in alumina and water content of mobile phase on the simultaneous purification of phosphatidyl choline(PC) and phosphatidyl ethanolamine(PE) by alumina column chromatography were studied systematically.The results showed that the initial water content in alumina had great influence on the purification process, but only little changes could be seen for the water content of mobile phase within the experiment range.Baseline separation and higher recovery of PC could be reached at 8%(mass) initial water content in alumina and 0.1%—1.1%(vol) water content of mobile phase.The recovery of PC was more than 90% and the recovery of PE over 40%.Explanation and discussion of the results were given.In addition, reproducibility of the experiments was tested. Not only could the work give some guides in the preparation of high purity PC and PE, but also it would be very helpful in R&D of other natural products.