High performance liquid chromatographic separation and direct ultraviolet detection of phospholipids

Abstract

A fast and efficient method for the separation of (phospho)lipids by high performance liquid chromatography using n-hexane, 2-propanol, water mixtures as the solvent system is described. The lipid separation occurs on a LiChrosorb Si-60 (10 micron) column and the individual components are monitored directly by ultraviolet absorption at 206 nm. Of a total lipid extract from erythrocytes a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are only partly separated under these circumstances. Furthermore, a mixture of synthetic phospholipids, i.e. 1,2-dilauroyl-sn-glycero-3-phosphatidic acid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-1'-sn-glycerol and 1,2-dioleoyl-sn-glycero-3-phosphocholine has been completely resolved. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some cases, as is shown for 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine and 1,2-didocos-13'-cis-enoyl-sn-glycero-3-phosphocholine.