Sample adsorption to the inner surface of fused silica capillaries is a common problem in
CE when analyzingmacromolecules and is harmful to the analysis. We previously utilized
the pH hysteresis effect of fused silica to facilitate electrophoresis of the strongly adsorbing
protein β2gpI 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 β2-glycoprotein
I has been investigated using electroosmosis markers, SDS mobilization, and imaging
based on indirect immunofluorescence microscopy for direct visualization. The amount
of β2gpI 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 β2gpI by decreasing charge-dependent wall adsorption.
Weinheim, Germany: John Wiley & Sons, 2012. Vol. 33, no 12, p. 1695-1702
Acidic pretreatment / Capillary electrophoresis / pH-hysteresis effect / Protein adsorption