Electrophoretic separation of proteins on capillary columns grafted with non-fouling, stable films prepared by controlled radical polymerization [recurso electrónico]

Abstract

Proteins are complex macromolecules that are inherently active towards a variety of surfaces. The active sites of the surface can enable structural alteration and destabilization of many proteins leading to their adsorption. Capillary electrophoresis (CE) is one analytical application where the consequences of adsorption can be especially catastrophic when the protein solute becomes stuck on the capillary before reaching the detector. Chemical modification of the fused-silica capillary has been aimed at eliminating unwanted interactions between the inner wall of the capillary tube and the sample undergoing separation. The goal of this project is to provide a hydrolytically stable organic film that coats the inner-wall of the fused-silica tubes used in CE. We used a surface-confined grafting procedure based on atom transfer radical polymerization (ATRP) which was also surface-initiated from ¿-bromoisobutyryl groups. Immobilization of the initiator was achieved by hydrosilylation of allyl alcohol on hydride silica followed by esterification of the resulting propanol-bonded surface with ¿-bromoisobutyryl bromide. The modified surfaces were characterized by IR and solid-state NMR spectroscopies, atomic force microscopy, contact-angle measurements, etc. The focus of this thesis work was to quantitatively assess the extent of surface deactivation (non-fouling) upon grafting by examining the CE migration profile of several chemical probes, including proteins. Additionally, a systematic assessment of the stability of all new bonded materials was also an essential part of this work. Our results showed that the coated capillary tubes exhibit (i) a complete (100%) recovery of lysozyme; i.e., there is no adsorption of this challenging solute on the coated surface, and (ii) a high long-term hydrolytic stability under the inherently harsh conditions of capillary isoelectric focusing. As a by-product of the present work, we have come up with an improved analytical methodology to determine solute recoveries in CE.