Study On The Preparation And Evaluation Of Immobilized Metal Chelating Chromatography Medium Based On Polymer Macroporous Microspheres

In this study,a polymer chromatographic medium with high nickel content was developed by a simple redox graft method.Graft copolymerization of glycidyl methacrylate modified by iminodiacetic acid was carried out on the surface and pore of macroporous polyacrylate microspheres.The influencing factors of grafting copolymerization initiated by Ce⁴⁺were systematically investigated.It was found that with the increase of monomer concentration（0.075–0.4 mol/L）,initiator concentration（0.0075–0.025 mol/L）,hydrogen ion concentration（0.2–0.4 mol/L）and reaction temperature（30–60℃）,the grafting amount of monomer increased first and then decreased.When the monomer concentration exceeds 0.32 mol/L,the probability of homopolymerization competing with graft copolymerization increases,which reduces the amount of monomer grafting.When the initiator concentration exceeds0.32 mol/L,the termination reaction rate of Ce⁴⁺with free radicals increases and resulted in the decrease of monomer grafting amount.When the concentration of hydrogen ion exceeds 0.3 mol/L,the generation of free radical active sites induced by Ce⁴⁺is inhibited.When the reaction temperature exceeds 45℃,the free radical termination reaction rate is greater than the initiation rate,which is not conducive to the graft copolymerization of monomer.When the reaction time exceeds4.5 h,the monomer consumption is completed,and the grafting amount remains unchanged.The optimum preparation conditions were determined as follows:2 g polyacrylate microspheres,0.32 mol/L modified monomer,0.0225 mol/L initiator,0.3 mol/L H⁺,reaction temperature 45℃,reaction time 5 h.Based on this preparation method,the chelating ligand of Ni chelating chromatography medium can reach0.20 mmol/m L,and the nickel content can reach 180μmol/m L.The functional groups and surface morphology of microspheres were characterized by infrared spectroscopy and scanning electron microscopy.The static binding capacity of proteins on PGMA-IDA-Ni medium was122.5 mg/m L using bovine hemoglobin as the model protein.Then,the chromatographic performance of PGMA-IDA-Ni medium was characterized.When the flow rate was 1 m L/min,the dynamic binding capacity（10%flow through）of bovine hemoglobin（1 mg/m L）was 17mg/m L,which was better than that of commercial polymer chromatographic medium（12 mg/m L）.The binding capacity of proteins on PGMA-IDA microspheres chelated with different metal ions(Cu²⁺,Zn²⁺,Co²⁺)was also investigated.The results showed that the binding capacity of protein on PGMA-IDA microspheres chelated with copper ions was the highest.The static binding capacity and the dynamic binding capacity were 174.4mg/m L and 24.5 mg/ml,respectively.PGMA-IDA-Ni medium was eluted with phosphoric acid buffer that was a common mobile phase of metal chelating chromatography medium.After 100 column volumes were eluted,the nickel content of PGMA-IDA-Ni medium only decreased by2.3%.After ten cycles of regeneration test,the nickel content was basically unchanged.This indicated that the medium can be used repeatedly.In addition,tetradentate ethylenediamine diacetic acid and pentadentate aminosuccinic acid were modified by glycidyl methacrylate,tetradentate and pentadentate modified monomers were grafted on the surface of macroporous polyacrylate microspheres by Ce⁴⁺.The chemical functional groups and surface morphology of the grafted tetradentate and pentadentate modified monomers microspheres were characterized by infrared spectroscopy and scanning electron microscopy.PGMA-IDA-Ni medium with different grafting amount of GMA-IDA monomer was used to purify the recombinant maltose binding protein and SL11 protein expressed in Escherichia coli.The results showed that both the protein recoveries and the purity of the target proteins were better than those of the similar commercial medium.