Weak cation‐exchange monolithic column for capillary liquid chromatography of peptides and proteins

Abstract A stable poly(2‐carboxyethyl acrylate‐ co ‐poly(ethylene glycol) diacrylate) monolith was synthesized inside a 75‐μm id capillary by direct in situ photo‐initiated polymerization in a binary porogenic solvent consisting of methanol and ethyl ether. The resulting monolith was evaluated for weak cation‐exchange capillary liquid chromatography of peptides and proteins. A high dynamic binding capacity of 72.7 mg lysozyme per cm 3 column volume was measured. Fast mass transfer was demonstrated by steep breakthrough curves. The resulting monolith exhibited negligible hydrophobicity, leading to good separation of peptides and proteins. Peak capacities of 11 for peptides with a 10‐min salt gradient and 39 for proteins with a 20‐min salt gradient were measured. An efficiency of 37 000 plates/m for proteins was obtained under isocratic conditions. The effects of functional group concentration, porogenic solvent composition, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run‐to‐run relative standard deviation (RSD) <1.93% and column‐to‐column RSD <4.63% were achieved.