Preparation And Chromatographic Characterization Of Poly Hemin Monolithic Column

As the active macromolecules in cell, proteins are the main disease-related molecules. The change of their expression level is directly related to diseases. Therefore, it is a meaningful work to study the separation and identification of the proteins.Monolithic column has been used as the solid phase in high performance liquid chromatography(HPLC) to separate and analyze proteins, because it has the advantages of simple preparation and excellent permeability. In this work, two kinds of poly hemin monolithic columns were prepared via two polymerization methods, respectively, and which were successfully used as the stationary phases of HPLC to separate several standard proteins and real bio-samples.Firstly, a novel poly(Hemin-co-GMA-co-EDMA) monolithic column was synthesized by in situ radical polymerization with hemin and glycidyl methacrylate(GMA) as co-monomers, ethylene glycol dimethacrylate(EDMA) as crosslinking agent, 1,4-butanediol, propanol and N, N-dimethylformamide as dual porogens, azobisisobutyronitrile as initiator. The obtained monolithic column was assayed by scanning electron microscopy, infrared spectrometry, nitrogen adsorption-desorption method, and mercury intrusion method, respectively. The monolith was successfully used as the stationary phase of HPLC to separate the mixture of three standard proteins in 8 minutes. Furthermore, it was used to separate egg white and enzymatic hydrolysates of cytochrome C. The results showed that the addition of hemin improved the selectivity for protein of the monolith that had ability to separate real protein samples.Secondly, in order to improve the uniform of pore structure and enhance selectivity for protein, atom transfer radical polymerization(ATRP) was utilized to prepare poly(Hemin-co-GMA-EDMA) monolith. The obtained monolith was successfully used to separate the mixture of three standard proteins, human plasma, and snailase, respectively. Compare to the in-situ monolith, the present poly(Hemin-co-GMA-EDMA) monolith prepared with ATRP had more uniform structure and better selectivity for proteins.