Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

ABSTRACT

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.