Capillary electrophoresis of proteins in dextran-coated columns.

Abstract

A simple coating technique by using uncross-linked dextran has been developed for fused-silica capillaries to be used in capillary electrophoresis of basic proteins. The capillaries were first silanized with a heterobifunctional silane (gamma-aminopropyltriethoxylsilane), which served as a coupling agent between the capillary inner wall and the polysaccharide coating. Dextran of high molecular mass (about 70 kDa) was activated with 1,1'-carbonyldiimidazole. Then the activated dextran was coupled to the primary amino groups that were anchored onto the inner wall of the silanized capillaries. The residual reactive groups on the dextran were further substituted by neutral functions in a coupling reaction with excess ethanolamine. By using dimethyl sulfoxide (DMSO) rather than aqueous buffer as the reaction medium, the extent of substitution was improved by minimizing the residual reactive groups at the surface. Since they are ionogenic, the electrosmotic flow in the system is relatively low. The chemically bound dextran coating showed good reproducibility and stability. In electrophoretic experiments basic proteins were separated with high efficiency by use of the dextran-coated fused-silica capillary columns. The main advantage of the method described here is that both polysaccharide activation and amine-coupling reactions were carried out under mild conditions at room temperature without catalysts. For this reason, the method is recommended to coat the inner wall of microfluidic separation channels which would not tolerate a harsh treatment.