CAPILLARY MODIFICATION AND EVALUATION USING STREAMING POTENTIAL AND FRONTAL CHROMATOGRAPHY FOR PROTEIN-ANALYSIS IN CAPILLARY ELECTROPHORESIS

The modification of capillary surfaces in capillary electrophoresis requires accurate chemical characterization of the synthesized materials. A three-step procedure was developed for the evaluation of capillaries used for protein separations in capillary electrophoresis. These measurements include zeta-potential determination via streaming potential measurements, adsorption characteristics using frontal chromatography measurements of test solutes, and overall performance under running conditions using selected test solutes. The zeta-potential determined from the streaming potential reflects the degree to which the capillary wall can undergo electrostatic interactions with proteins as well as the magnitude of the electroosmotic flow. Frontal chromatography measurements with selected probe proteins can indicate both the amount of adsorption as well as the probable types of interactions involved in the adsorption. Kinetic information can also be obtained in some instances. Electrophoresis with test solutes reflects the overall effect of adsorption. For this study, three types of capillaries were evaluated: (1) bare fused-silica capillaries, (2) capillaries coated with several thickness of cross-linked polyethylene glycol and (3) cross-linked polyethyleneimine. The polyethylene glycol column displayed much weaker electrostatic and similar hydrophobic and/or hydrogen bonding interactions as compared with underivatized fused-silica columns. The polyethyleneimine column exhibited poor performance for the test proteins used. Good electrophoretic performance seems to be possible only if adsorption measurements were below 0.2 ng/cm2 (corresponding to 0.1% of available surface area) for all of the test proteins.