Fast preparation of monolithic immobilized pH gradient column by photopolymerization and photografting techniques for isoelectric focusing separation of proteins

A new method was developed to prepare monolithic immobilized pH gradient (M-IPG) columns in UV-transparent fused-silica capillaries by the 5-min photopolymerization of acrylamide and N,N'-methylenebisacrylamide, followed by the 20-min photografting of the focused ampholine-derived glycidylmethacrylate monomer on the monolithic matrix, by which the preparation time was reduced, and the stability of the formed pH gradient was improved, compared with our previous methods. Using the prepared M-IPG column, the baseline separation of proteins was achieved according to their pIs. Without carrier ampholytes added in the running buffer, the separated components could be detected with high sensitivity by UV at low wavelength.