High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane--isopropanol--water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks. A second system was developed utilizing sulfuric acid for ion suppression. The behavior of phospholipids in this system was found to be dependent on the presence of quaternary ammonium, amino, or hydroxyl groups. Except for plasmalogen, phospholipids were recovered intact. This system was optimized to provide baseline resolution of essentially all phospholipid species commonly found in mammalian tissues.