High-performance liquid chromatography of proteins on functional polysaccharide-coated silica supports

Abstract

Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combined the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. The passivation of the inorganic phase was obtained by a coating with dextran or agarose substituted with a calculated amount of positively charged diethylaminoethyl (DEAE) functions. The passivation of the silica beads can be improved by a second coating with native polysaccharide. The supports presented minimal non-specific adsorption as tested by high-performance size-exclusion chromatography. Because of their polysaccharidic overlayer, these phases were easily grafted with active ligands like anticoagulant compounds (heparin or functional dextrans) or amidine derivatives and used in high-performance affinity chromatography. The affinity of these active supports for thrombin in solution was evaluated and their performances in affinity chromatography were determined. On these supports, the non-specific interactions were minimized and the importance of the specific binding between the active ligand and thrombin in the overall interaction mechanism was enhanced.