Preparation and characterization of lauryl methacrylate-based monolithic microbore column for reversed-phase liquid chromatography

Abstract

Poly(lauryl methacrylate-co-ethylene dimethacrylate) monoliths were in situ synthesized within the confines of a silicosteel tubing of 1.02 mm i.d. and 1/16″ o.d. for microbore reversed-phase HPLC. In order to obtain practically useful monoliths with adequate column efficiency, low flow resistance, and good mechanical strength, some parameters such as total monomer concentration (% T), cross-linking degree (% C) and polymerization temperature were optimized. High-efficiency monoliths were successfully obtained by thermal polymerization of a monomer mixture (40% T, 10% C) with a binary porogenic solvent consisting of 1-propanol and 1,4-butandiol (7:4, v/v) at a high temperature of 90 °C. The morphology and porous structure of the resulting monoliths were assessed by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC), while the column performance was evaluated through the separations of a series of alkylbenzenes in acetonitrile–water (50:50, v/v) eluent. At a normal flow rate of 50 μL/min (corresponding to 1.66 mm/s), the optimized monolithic columns typically exhibited theoretical plate numbers of 6000 plates/10 cm-long column for amylbenzene ( k > 40), and the pressure drop was always less than 1 MPa/10 cm. The monoliths, which were chemically anchored to the tube inner wall surface using a bifunctional silylation agent, exhibited adequate mechanical strength of up to 12–13 MPa, and were properly operated at 10 times higher flow rate than normal, reducing the separation time to one tenth. The lauryl methacrylate-based monolithic column was applied to a rapid and efficient separation of ten common proteins such as aprotinin, ribonuclease A, insulin, cytochrome c, trypsin, transferrin, conalbumin, myoglobin, β-amylase, and ovalbumin in the precipitation-redissolution mode. Using a linear CH 3CN gradient elution at a flow rate of 500 μL/min (10-times higher flow rate), 10 proteins were baseline separated within 2 min.