Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis

Abstract

A butyl methacrylate- co-ethylene dimethacrylate (BuMA- co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 μm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1 cm monolith was reached after loading 48 ng of protein. Finally, the BuMA- co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.