Optimization of capillary coating by hydroxyethyl methacrylate for capillary zone electrophoresis of proteins

This work describes further improvements of coating fused silica capillaries with 2-hydroxyethyl methacrylate (HEMA) by atom transfer radical polymerization (ATRP). First, endcapping with a sterically less bulky silanyl reagent reduces the electrosmotic flow (EOF) by 25% in addition to the 40% EOF reduction caused by HEMA coating compared to a bare fused silica capillary. An additional hydrolysis step was introduced into the preparation of HEMA coated capillaries and leads to better reproducible migration times. The influence of the solvent during ATRP and the resulting polymer coating was investigated by replacement of DMF with water or water–methanol mixtures. The quality of the optimized coating was characterized by protein separations at pH 3. HEMA coated capillaries reveal up to 746 000 plates. The polyvinyl alcohol (PVA) coated capillary provides only half of this efficiency. A long-term test at pH 9 shows good stability of the HEMA coated capillaries in basic medium. Also the numbers of plates in this medium was about 30% higher than for separations with the PVA capillary. In addition, the phosphate buffer was replaced by a volatile ammonium acetate buffer for later use with mass spectrometry (MS).