Adjusting the electroosmotic flow for CE separation of proteins by using mixed polycations/polyzwitterions poly(α-L-lysine)-based 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. Beside these adsortion phenomena, the electroosmotic flow (EOF) has also 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 setpwise the ε-amino functions of this polycation into carboxamides, the total number of positive charges decreases, leading to reduced EOF. To keep simultaneously the analyte-surface interactions as small as possible, carboxylic acids armed with a zwitterionic functionality based structurally on a sulfobetaine motiv 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 invsetigated as outermost layers of the SMIL coatings, clearly showing that the EOF decreases depending on the degree of functionalization while keeping the high efficiency.