The application of droplet counter-current chromatography (DCC) for the separation of acidic glycolipids

Droplet counter-current chromatography (DCC) a novel liquid-liquid partition technique, is a useful method for the separation of glycolipids. After a series of investigations on suitable experimental conditions for DDC, mouse brain chloroform-methanol-0.5% CaCl2-n-propanol (50:60:40:6) by the ascending method with 500 standard columns. GM1 and cerebroside sulfuric ester (CSE) from whale brain glycolipid fraction were separated by DCC into two peaks and three peaks, respectively, with chloroform-methanol-water-n-propanol (50:60:40:2) and 500 standard columns. The occurrence of two peaks of GM1 was due to the differences of sphingenine and fatty acid compositions. In the former peak of GM1, the majority of long chain base and fatty acid were 18:1 sphingenine and stearic acid, respectively, while the latter contained 18:1 (32%), 20:1 (68%) sphingenine and 16:0 (14.5%), 18:0 (61.4%), and 20:0 (21.0%) fatty acids. The three peaks of CSE were due to the difference of carbon chain length and to the presence or absence of a hydroxyl group in fatty acids. The first polar fraction (CSE-I) had hydroxy 23:0 (34%) and 24.1 (53.4%) fatty acids and the second fraction (CSE-II) contained hydroxy 24:0 (70.5%) and 25:1 (12.8%) fatty acids, while the third fraction (CSE-III) had normal 23:0 (11.9%), 24:1 (56.4%), hydroxy 25:0 (11.4%) and 26:1 (9.7%) fatty acids. Preparative DCC was also applied to the glycolipid fraction from Tay-Sachs' brain to give 242 mg of practically pure GM2 from 700 mg of crude brain extract without any preliminary purification.