Preparative separation of algal polar lipids and of individual molecular species by high-performance liquid chromatography and their identification by gas chromatography—mass spectrometry

Abstract

A crude polar lipidic extract of the green fresh-water alga Chlorella kessleri cultivated under heterotrophic conditions was separated by high-performance liquid chromatography (HPLC) on a preparative silica gel column into a total of twelve lipid classes, in which the content of fatty acids was determined by means of gas chromatography—mass spectrometry (GC—MS). The separation was by gradient elution from hexane—isopropanol (6:8) to hexane—isopropanol—water (60:80:14) lasting 20 min and then isocratically for 30 min. A total of 17.5 mg of lipids were injected. Individual types of lipid classes were further separated into eighteen molecular species by isocratic HPLC on a reversed-phase C 18 column using a mixture 20 m M choline hydrochloride in methanol—water—acetonitrile (90.5:7:2.5) in the preparative mode (5 mg injected). Phosphatidylcholine and phosphatidylethanolamine were hydrolized by phospholipase C and corresponding diglycerides were identified by GC—MS on a polar capillary column. In mono- and digalactosyldiglycerols, fatty acids in position 1 were identified after a specific hydrolysis by lipase. The recovery obtained with an UV detector in HPLC and a mass spectrometer in GC—MS is discussed. It was shown that the relative response of the UV detector decreases with increasing saturation of acids, whereas the relative response of the mass spectrometer increases.