In situ synthesis of a monolithic material with multi-sized pores and its chromatographic properties for the separation of intact proteins from human plasma

Abstract

The fabrication of a monolithic allyl phenoxyacetate-based material was proposed via the in situ radical polymerization using ethylene dimethacrylate as the crosslinker and 2,2′-azobisisobutyronitrile as the initiator within a stainless steel column (50 mm × 4.6 mm i.d.). The effects of the porogen composition, the crosslinker amount and the monomer type on the resulting monoliths were investigated. The morphology of the monoliths was characterized using scanning electron microscopy and a nitrogen adsorption-desorption instrument, and the pore structure was characterized using mercury intrusion porosimetry. The results indicate that the optimized monolith has a micro-, meso- and macro- multi-sized pore structure with a high specific surface area of 260.66 m2 g−1. The resulting monoliths were used as stationary phases for the separation of proteins from bio-samples, including a mixture of six standard proteins, chicken egg whites, snailase and human plasma, using high-performance liquid chromatography. Compared to optimized glycidyl methacrylate-based and styrene-based monolithic columns, the allyl phenoxyacetate-based monolithic column exhibited improved selectivity in the separation of proteins. Furthermore, the present method avoids the masking of highly abundant proteins, such as human serum albumin, immunoglobulin G and human fibrinogen, in the detection of middle- or low-abundance proteins in human plasma. The protein identification results obtained from liquid chromatography/mass spectrometry indicate that the present method is an outstanding method for efficient fractionation of human plasma, which will be especially useful in future plasma proteomics research.