Separation of plant membrane lipids by multiple solid-phase extraction.
Plant membrane lipids were separated by multiple solid-phase extraction (SPE) in a single run. Elution was performed continuously through the modulated stationary phase employing only non-aqueous solvent systems. At the different stages of the glycerolipid separation the SPE manifold combined arninopropyl, arninopropyl/silica gel and silica gel/aminopropyl weak anion exchanger columns. The glycerolipid extract of pigment-containing plant tissues was cleared from the pigments onto the aminopropyl column. The aminopropyl column with the glycerolipid extract was then connected to a silica gel column from which monogalactosyldiacylglycerol, phosphatidylethanolamine, phosphatidylglycerol and digalactosyldiacylglycerol were eluted as individual fractions. The elution was performed under polarity, pH and temperature gradient conditions. To continue the separation, the aminopropyl column was discarded and the silica gel column containing the remaining glycerolipid extract was connected to an aminopropyl anion exchanger column. Individual fractions of sulfoquinovosyldiacylglycerol, phosphatidylcholine and phosphatidylinositol were now eluted. The separation process was supported by ammonium counter ions and by the polarity gradient of the elution systems used. The membrane lipids were isolated from pigment-containing (rice and maize leaves and rice leafy stems) and pigment-free (rice roots) tissues. The repeatability for a standard glycerolipid mixture was 2-6% (n=7), and for rice leaf lipid extracts, 3-7% (n=5). Glycerolipid recovery was 87-95%.