A simple and quantitative purification of glycosphingolipids and phospholipids by thin-layer chromatography blotting.

A new and simple method for purifying glycosphingolipids and phospholipids by using "TLC blotting" was established. Glycosphingolipids separated by two-dimensional thin-layer chromatography (TLC) were made visible with primuline reagent, and then bands were marked with a drawing colored pencil. The glycosphingolipids that separated on the HPTLC plate were transferred by TLC blotting to a polyvinylidene difluoride membrane together with the color marks. The marked areas were excised after which their glycosphingolipids were extracted and monitored by TLC. By this method, 20 glycosphingolipids showing homogeneous bands on a HPTLC plate were isolated from the neutral glycosphingolipid fraction of human meconium. Moreover, 10 kinds of acidic glycosphingolipids were purified as homogeneous bands from the bovine acidic glycosphingolipid fraction. The yields of glycosphingolipids (13 different ones) ranged from 68 to 92%, the mean value being 82.3%. The glycosphingolipids were confirmed to be purified as intact forms by mass spectrometric analysis and chromatographic mobilities on a HPTLC plate. The same procedure could also be used to purify phospholipids.