Adjusting the electroosmotic flow for CE separation of proteins by using poly(α-l-lysine)-based mixed polycationic/polyzwitterionic multilayer coatings

Abstract

Capillary electrophoresis (CE) is a robust, selective and highly efficient technique for the analysis of peptides and (intact) proteins. The anionic and/or hydrophilic character of the fused silica surface generally leads to protein adsorption by electrostatic interaction or H-bonding. Successive multiple ionic-polymer layer (SMIL) coatings are often used to limit adsorption and to improve the repeatability of migration times. Besides these adsorption phenomena, the electroosmotic flow (EOF) also has a strong influence on the resolution. Here, the frequently used and efficient cationic SMIL coating agent poly(α-L-lysine) (α-PLL) is modified for the systematic modulation of the EOF. In particular, by converting stepwise the ε-amino functions of this polycation into carboxamides, the total number of positive charges decreases, leading to reduced EOF. To simultaneously keep the analyte–surface interactions as small as possible, carboxylic acids bearing a zwitterionic functionality based structurally on a sulfobetaine motif were developed. Using the water-soluble and highly hydrolysis-resistant condensation agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), the degree of functionalization could be adjusted between 8 and 99%, depending on the applied stoichiometry. The obtained set of mixed polycationic/polyzwitterionic polymers based on the α-PLL scaffold were investigated as the outermost layers of the SMIL coatings, clearly showing that the EOF decreases depending on the degree of functionalization while maintaining high efficiency.