Preparation and characterization of methacrylate-based semi-micro monoliths for high-throughput bioanalysis

Hexyl methacrylate (HMA)-based monolithic semi-micro columns were prepared by in situ polymerization within the confines of 1.02-mm-i.d. silicosteel tubing for reversed-phase and/or precipitation-redissolution liquid chromatography. Practically useful monolithic columns with adequate separation efficiency, high permeability, and good mechanical strength were successfully obtained using a polymerization mixture comprising 24% hexyl methacrylate (HMA), 6% ethylene dimethacrylate (EDMA), 44.5% 1-propanol, and 25.5% 1,4-butanediol. The column performance was evaluated through the separations of a series of alkylbenzenes. At a normal flow rate of 50 mu L min (-1), the produced HMA-based monolithic columns typically exhibited 3,000 theoretical plates for a 20-cm-long column, and the pressure drop was generally less than 1 MPa per 20 cm. The monolithic columns were resistant to at least 15 MPa, and could be properly operated at 15-20 times higher flow rate than normal, reducing the separation time to 1/15-1/20. The HMA-based monolithic columns were applied to rapid and efficient separations of proteins such as ribonuclease A, cytochrome c, transferrin, and ovalbumin in the precipitation-redissolution mode. Using a CH3CN gradient elution at a flow rate of 1,000 mu L min(-1), four proteins were baseline separated within 20 s.