Estimation of the amount of β 2 ‐glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment

Sample adsorption to the inner surface of fused silica capillaries is a common problem in CE when analyzing macromolecules and is harmful to the analysis. We previously utilized the p H hysteresis effect of fused silica to facilitate electrophoresis of the strongly adsorbing protein β 2 gp I in plain‐fused silica capillaries at neutral p H . In the present paper, the effect of different pretreatments of the capillary on the adsorption of the β 2 ‐glycoprotein I has been investigated using electroosmosis markers, SDS mobilization, and imaging based on indirect immunofluorescence microscopy for direct visualization. The amount of β 2 gp I adsorbed on the surface was probed using all these independent techniques after electrophoresis at neutral p H on capillaries pretreated with HCl , background electrolyte ( BGE ), and N a OH . BGE pretreatment was included as a positive control. We found that 80% or more of the starting material was adsorbed to the inner surface of the silica capillaries during electrophoresis after pretreatment with only BGE or with N a OH , but after acidic pretreatment the loss was consistently less than 20%. N a OH most efficiently removes adsorbed protein between runs. A theoretical calculation of the p H change of the BGE showed that electrolysis affects the p H more than the deprotonation of silanols during electrophoresis. We conclude that acidic pretreatment of fused silica capillaries diminishes adsorption of β 2 gp I by decreasing charge‐dependent wall adsorption.