Preparation of a hydroxyethyl-based monolithic column and its application in the isolation of intact proteins from complex bio-samples

In this work, a monolithic hydroxyethyl-based column was fabricated in a stainless-steel column (50 mm × 4.6 mm i.d.) via radical polymerization technique using hydroxyethyl methylacrylate as the monomer. The morphology and pore size distribution indicate that the optimized monolith has a relatively uniform structure with macro-pores. The homemade monolith was used as the stationary phase of high performance liquid chromatography for the separation of intact proteins from complex bio-samples, including human plasma, egg white and snailase. The resulting monolith shows excellent selectivity for intact proteins mainly depending on the different relative hydrophobicity of the objective proteins with reversed-phase liquid chromatographic mechanism. Besides, the hydrogen-bond interaction and electrostatic interaction were the additional interactions in the chromatographic separation owing to hydroxyl groups present in the surface of monolithic material.