Two procedures to remove polar contaminants from a crude brain lipid extract by using prepacked reversed-phase columns

Abstract

Two procedures were developed using prepacked, reversed-phase columns (Bond Elut) for the separation of lipids from water-soluble contaminants. A crude lipid extract from brain tissue, was diluted stepwise with a methanol/water (method 1) or a methanol/saline (method 2) mixture and, with each step, was passed through the column. As the polarity of the solvent was increased, all lipids became bound to the column, while the water-soluble compounds remained in the eluate. After three subsequent dilutions and column elutions, the eluate containing the more polar contaminants was discarded. The bound lipids were then eluted with a small volume of chloroform/methanol (1∶2, v/v). Alternatively two fractions were eluted, the first fraction eluted with methanol/water (12∶1, v/v), contained gangliosides, phosphatidyl-serine, phosphatidylinositol, phosphatidic acid and sulfatides. The second fraction, eluted with chloroform/methanol (1∶2, v/v), contained all remaining phospholipids, cerebrosides and cholesterol. For both methods a quantitative recovery of cholesterol and phospholipids was obtained. In method 2, when water was replaced by saline in the dilution solvent mixture, gangliosides were also bound and quantitatively recovered.