Monolithic porous layer open tubular (monoPLOT) columns for low pressure liquid chromatography of proteins

Abstract

Glycidyl methacrylate–ethylene dimethacrylate (GMA-co-EDMA) based monolithic porous layer open tubular (monoPLOT) columns (0.05 mm I.D., monolithic layer thickness ≈ 5 µm) have been fabricated using an automated column scanning technique, providing UV polymerisation at 365 nm. Columns were chemically modified to obtain desired diol groups on the surface, and the longitudinal homogeneity of the stationary phase was profiled using scanning capacitively coupled contactless conductivity detector (sC⁴D), before and after such modification. Using the automated scanning polymerisation technique, column-to-column production reproducibility, including longitudinal phase thickness, was within ±5% RSD. The prepared columns were tested to evaluate their liquid chromatographic stationary phase selectivity, efficiency and reproducibility, with a series of test protein mixtures. Under optimised gradient conditions, the separation of up to 8 proteins was demonstrated on the open tubular column (510 × 0.05 mm I.D.), with a column pressure drop of <1.5 MPa.