Electrophoretic separations of proteins in capillaries with hydrolytically-stable surface structures

Abstract

A procedure for obtaining highly stable coated capillaries for use in capillary electrophoresis (CE) is described. Reaction of surface-chlorinated fused silica capillaries with the Grignard reagent, vinyl magnesium bromide, followed by reaction of the vinyl group with acrylamide, results in an immobilized layer of polyacrylamide attached through hydrolytically stable Si-C bonds. This method is an extension of the capillary coating procedure described previously by Hjerten, differing in the means by which the polyacrylamide layer is bonded to the capillary walls. Capillaries treated in the manner described here can be used over a pH range of 2-10.5, without noticeable decomposition of the coating. In comparison to uncoated capillaries, separations of proteins using such coated capillaries are improved due to a reduction in protein adsorption to the capillary walls, although interaction is still present to some degree as evidenced by an inability to obtain plate counts as high as those predicted by theory. Electroosmotic flow is virtually eliminated in the coated capillaries, resulting in improved reproducibilities of protein migration times in comparison to uncoated capillaries. Additionally, peak skew is evaluated for model proteins and improvements are noted for the coated capillaries. Results are presented for separations of model protein mixtures, comparing the performance of the vinyl-bound polyacrylamide coated capillaries and uncoated capillaries at both high and low pH extremes.

Selectivity change in the separation of proteins and peptides by capillary electrophoresis using high-molecular-mass polyethyleneimine

“…The use of polymeric coatings has led to high efficiency and reproducible protein separations preventing the surface adsorptions. Polymers ranging from highly hydrophobic to highly hydrophilic were used for this purpose to obtain efficient separations of proteins in CE [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16].…”

The influence of surface treatments on the electroosmotic flow in micellar electrokinetic capillary chromatography

“…Also, silanization by trimethylchlorosilane (TMCS) was used to silylate the column surface and reduce the EOF [13,21]. Again, polymer and a number of other coatings were applied by different authors [22][23][24][25][26][27][28] and a decreased EOF was generally observed. Terabe et al [29] investigated the effects on the EOF of hydrophobic and hydrophillic coatings.…”

Capillary electrophoresis of proteins 2003–2005

“…There are several chemistries applied to polymer attachment on the wall: silane bifunctional reagent [71], Grignard chemistry [72], thermal immobilization [73], and vapor deposition.…”