Separation of Unsaturated Chondroitin Sulfate Disaccharides in Thin-Layer Chromatography on Silica Gel and Their Quantitative Determination by Densitometry

Abstract

A simple and rapid method has been developed to determine the relative amounts of 4-and 6-sulfate in chondroitin sulfate or proteoglycan preparations. Unsaturated disaccharide products derived from the samples by chondroitinase ABC digestion in 0.01 M Tri-HCl buffer (pH 8.0) were applied to a silica gel-coated thin-layer chromatographic plate and separated in a solvent system of n-propanol-isopropanol-n-butanol-water (55 : 20 : 5 : 20, v/v) containing 0.04M NaCl and 0.01 M ammonia without a desalting step, which was necessary in paper chromatography or thin-layer chromatography on cellulose. The developing time was as short as 2-2.5 h at room temperature. After staining of the separated products with a carbazole reagent followed by immersing the plate in liquid paraffin, the ratio of monosulfated disaccharides, generally found as major components in chondroitin sulfate chains, was estimated by densitometry.