Study On Chromatographic Properties Of Allyl Ester-based Monolithic Column In The Separation Of Proteins

Monolithic column,as an important stationary phase of high-performance liquid chromatography,has specific advantages in the separation and analysis of biological macromolecules such as proteins.However,most of the monolithic columns present the disadvantages of small specific surface area,poor selectivity and easy swelling or shrinkage.In order to give full play of its advantages in protein separation,two allyl ester monomers were introduced into the preparations of monolithic columns to increase the specific surface and enhance the recognition sites between protein and stationary phase.By enriching the chemical groups on the surface of stationary phase,the selectivity of chromatographic stationary phase was improved;by strengthening the mechanical stability of the monolithic column,swelling or shrinkage of the resulting monoliths was avoided.Firstly,a diallyl maleate-based monolithic column was developed and the effects of the monomer/crosslinker ratio and porogenic solvents on the properties of monolithic columns were explored in detail.The morphology and pore properties of the prepared monoliths were measured by scanning electron microscopy,nitrogen adsorption-desorption method and mercury intrusion porosimetry,respectively,thus exhibiting a relatively uniform porous structure with macro-pores.The median pore diameter,porosity and specific surface area were 3.02?m,74.49%and 26.06 m² g^-1,respectively.The monolithic material was used as the stationary phase of high-performance liquid chromatography to achieve rapid separation of five standard proteins mixture in 3 min.The results showed that the observed retention time increased with the relative hydrophobicity of the proteins.Furthermore,the fast separation of human plasma proteins performed on the diallyl maleate-based monolithic column was achieved in 15 min.The chromatographic fractions were identified by liquid chromatography/mass spectrometry,and the results indicated that different fractions contained different kinds of proteins.The present method is an outstanding method for the fast and efficient fractionation separation of human plasma.Secondly,allyl phenoxyacetate containing aromatic group was used as the monomer to prepare monolith,aiming to enhance the specific surface area.The effects of the porogen composition,the volume of the crosslinker and the type of the monomer on the resulting monoliths were investigated.The morphologies of the monoliths were characterized using scanning electron microscopy and nitrogen adsorption-desorption method,and the pore structure was characterized using mercury intrusion porosimetry.The results indicated that the optimized monolith had a micro-,meso-and macro-multi-sized pore structure with a high specific surface area of 260.66 m2g^-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 effect of the three highest abundant proteins in the detection of middle-or low-abundance proteins in human plasma.Some of the chromatography fractions of human plasma protein were identified by liquid chromatography/tandem mass spectrometry technique and the results indicated that different fractions contained different types of proteins.The present method is an outstanding method for efficient fractionation separetion of human plasma,which is of great significance for plasma proteomics research,especially for exploring new disease marker and drug target.