Purification of gangliosides by liquid-liquid partition chromatography

Tomohisa Kato,Kenichi Hatanaka

doi:10.1194/jlr.d800033-jlr200

Abstract

A liquid-liquid partition chromatographic technique was applied to separate amphiphilic glycolipids. A two-phase solvent system composed of n-butanol-t-butyl methyl ether-acetonitrile-water at a volume ratio of 3:1:1:5 was found to be suitable for separating the gangliosides from total lipids extracted from rat brain by liquid-liquid partition chromatographic systems, namely centrifugal partition chromatography (CPC) and high-speed counter-current chromatography. GM1 could be separated rapidly by using the upper phase as stationary phase for both systems. Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) could be separated individually by using the lower phase as stationary phase by CPC. The sample can be recovered without loss by these systems.

Highlights

A liquid-liquid partition chromatographic technique was applied to separate amphiphilic glycolipids
centrifugal partition chromatography (CPC) and high-speed counter-current chromatography (HSCCC) are both liquid-liquid partition chromatographic techniques that have been widely used for separation and purification of natural products [9,10,11,12,13]
We describe the successful separation and purification of gangliosides by two liquid-liquid partition chromatographic systems, CPC and HSCCC

Summary

MATERIALS AND METHODS

Materials All organic solvents were of analytical grade and were purchased from Nacalai Tesque, Kyoto, Japan. Rat brain tissue (Rockland) was purchased from Funakoshi, Tokyo, Japan. The strong anion exchanger cartridge (InertSep SAX) was purchased from GL Science, Tokyo, Japan. Apparatus CPC was performed using the CPC240 (Sensyu Scientific, Tokyo, Japan). Total rotor volume of this model is 240 ml, and num-. Total column volume of this model is approximately 320 ml. Centrifugal concentrator used was a Tomy CC-105 system with TU-105 low temperature trap, and GCD-0513 (Tomy Seiko Co., Ltd., Tokyo, Japan)

Lipid extraction from tissue

Column procedures

RESULTS AND DISCUSSION

Separation by HSCCC