Modifying last layer in polyelectrolyte multilayer coatings for capillary electrophoresis of proteins

Protein adsorption on the inner wall of the fused silica capillary wall is an important concern for capil-lary electrophoresis (CE) analysis since it is mainly responsible for separation efficiency reduction. Suc-cessive Multiple Ionic-polymer Layers (SMIL) are used as capillary coatings to limit protein adsorption, but even low residual adsorption strongly impacts the separation efficiency, especially at high separa-tion voltages. In this work, the influence of the chemical nature and the PEGylation of the polyelectrolyte deposited in the last layer of the SMIL coating was investigated on the separation performances of a mix-ture of four model intact proteins (myoglobin (Myo), trypsin inhibitor (TI), ribonuclease a (RNAse A) and lysozyme (Lyz)). Poly(allylamine hydrochloride) (PAH), polyethyleneimine (PEI), epsilon-poly(L-lysine) (epsilon PLL) and alpha-poly(L-lysine) (alpha PLL) were compared before and after chemical modification with polyethyleneg-lycol (PEG) of different chain lengths. The experimental results obtained by performing electrophoretic separations at different separation voltages allowed determining the residual retention factor of the pro-teins onto the capillary wall via the determination of the plate height at different solute velocities and demonstrated a strong impact of the polycationic last layer on the electroosmotic mobility, the separa-tion efficiency and the overall resolution. Properties of SMIL coatings were also characterized by quartz microbalance and atomic force microscopy, demonstrating a glassy structure of the films.(c) 2023 Elsevier B.V. All rights reserved.