Monophosphates such as the phospholipid lecithin poorly adsorb to hydroxyapatite because of anionic repulsion with hydroxyapatite phosphate groups. Here, we attempted to optimize the adsorption and elution of lecithin from hydroxyapatite chromatographic columns. The addition of Ca2+ ions to the running buffer masked the hydroxyapatite phosphate groups and increased the adsorption of lecithin from 38.9% to 96.1%. The stably adsorbed lecithin was eluted with isopropanol at 99.1% efficiency. By establishing an isopropanol gradient using 2-morpholinoethanesulfonic acid buffer supplemented with Ca2+ ions, the chromatographic method developed here may be suitable for the purification of various phospholipids, including monophosphates.