Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment

Abstract

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 pH hysteresis effect of fused silica to facilitate electrophoresis of the strongly adsorbing protein β(2) gpI in plain-fused silica capillaries at neutral pH. 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) gpI adsorbed on the surface was probed using all these independent techniques after electrophoresis at neutral pH on capillaries pretreated with HCl, background electrolyte (BGE), and NaOH. 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 NaOH, but after acidic pretreatment the loss was consistently less than 20%. NaOH most efficiently removes adsorbed protein between runs. A theoretical calculation of the pH change of the BGE showed that electrolysis affects the pH more than the deprotonation of silanols during electrophoresis. We conclude that acidic pretreatment of fused silica capillaries diminishes adsorption of β(2) gpI by decreasing charge-dependent wall adsorption.