Separation of proteins in capillary electrophoresis (CE) is often hindered by their interactions with the silanols groups on the inner surface of the fused silica capillary. In particular, the positive charges of alkaline proteins establish electrostatic interactions with the negative charges of the surface, leading to peak broadening and loss of separation efficiency. Moreover, uncontrolled electroosmotic flow (EOF), severely impacts on analyte mobility causing either loss of resolution or loss of efficiency. Among several strategies, coatings are widely employed to mask capillary surface silanols and so to reduce protein adsorption and EOF. Here we report on the synthesis and characterization of a novel family of adsorbed polymeric coatings, which provide improved performance in terms of prevention of protein adsorption and EOF regulation. In particular, we have added to the polymer backbone, made of N, N-dimethylacrylamide, different ionizable monomers (weak acrylamido acids and bases commercially available with the trade name of Immobilines) to confer a net positive or negative charge to the polymer chain depending on the buffer pH. As a consequence, the separation of alkaline protein is drastically improved in different pH conditions, because the interactions with the inner capillary wall were completely prevented by electrostatic repulsion. The content of these ionogenic monomers can be adjusted, permitting a perfect control of the surface charge density, so EOF is finely and precisely regulated. We also investigated the conformational variation of the polymer on the surface by changing buffer pH using Dual Polarization Interferometry (DPI). The coating procedure is very simple and fast as it consists in the adsorption of a diluted aqueous solution of the polymer on the capillary walls. In addition, the coating is very stable under harsh conditions, can be used for several runs without any re-conditioning or re-coating steps and it is compatible with MS volatile buffers.
Sola, Laura, Marcella Chiari
Journal of Chromatography A