BY COLUMN CHROMATOGRAPHY
Solid-phase extraction on micro-columns filled with various phases is at present the most rapid and reliable technique to obtain a lipid fraction enriched with glycosphingolipids from a complex lipid mixture. This technique is well adapted to small lipid amounts (from some micrograms to 10-20 mg) but may be scaled up using large columns. Moreover, low solvent volumes are needed and automated device may be used.
It must be emphasized that better and clearer results are expected if lipid extracts are previously saponified (mild alkaline methanolysis) to remove the interfering glycerolipids (triglycerides, phospholipids, glycoglycerolipids). Thus, the free fatty acids and the water-soluble moieties generated by glycerolipids are easily separated from the remaining glycosphingolipids.
- First procedure: The easiest way to isolate all glycosphingolipids is to use silica column and acetone elution as described previously. The eluted fraction contains neutral and charged glycolipids without overlapping with ceramides, fatty acids or sphingomyelin. This fraction may be further analyzed by TLC or HPLC.
- Second procedure: A sequential separation of glycosphingolipids may be effected using aminopropyl column and differential elutions of neutral and acidic lipids. The selected procedure allows the isolation of sphingomyelin and ceramides free of fatty acids. Free spingoid bases are recovered with neutral glycosphingolipids but are easily separated by TLC or HPLC.
- Third procedure: Another sequential separation of neutral (cerebrosides) and acidic (phosphorylated, sulfated or with hexuronic acid) glycolipids may be efficiently effected by ion exchange chromatography on DEAE cellulose or ion exchange resin. These procedures give the purest fractions when applied on saponified extracts. Furthermore, it may be easily scaled up.
An application note with details on the detection of sphingolipids with a light scattering device (Corona Charged aerosol detector) may be found on the manufacturer web site.