Rigid porous polyacrylamide-based monolithic columns containing butyl methacrylate as a separation medium for the rapid hydrophobic interaction chromatography of proteins

Macroporous poly(acrylamide-co-butyl methacrylate-co-N,N'-methylenebisacrylamide) monoliths containing up to 15% butyl methacrylate units have been prepared by direct polymerization within the confines of HPLC columns. The hydrodynamic and chromatographic properties of these 50 mm x 8 mm I.D. columns-such as back pressure at different flow-rates, effect of percentage of hydrophobic component in the polymerization mixture, effect of salt concentration on the retention of proteins, dynamic loading capacity, and recovery-were determined under conditions typical of hydrophobic interaction chromatography. Using the monolithic column, five proteins were easily separated within only 3 min.