Preparation And Performance Evaluation Of Macroporous Polymer Cation Exchange Chromatography Media By Surface Grafting

The rapid development of upstream technology of biopharmaceuticals puts forward higher requirements for downstream technology of separation and purification.The development of fast and efficient purification methods has attracted much attention,especially the development of chromatographic media.Among them,cation exchange chromatography is a widely used type of ion exchange chromatography.The selection of its stationary phase is crucial to the purification efficiency of cation exchange chromatography.The development and preparation of new cation exchange chromatography media has always been the focus of chromatography researchers.In this paper,the macroporous polymer cation exchange chromatography media was prepared by surface grafting method.The chromatographic performance of the two kinds of media were evaluated.The design ideas and research contents are shown as follows:Idea 1:Macroporous cation exchange chromatography media（PCMS-EDMA-SP）based on a copolymer of vinyl benzyl chloride-ethylene glycol dimethacrylate（PCMS-EDMA）was prepared through grafting copolymerization by surface-initiated atom transfer radical polymerization（ATRP）.The main works are as follows:（1）PCMS-EDMA microspheres were prepared by suspension polymerization initiated by common free radical polymerization.By investigating the types and dosages of dispersants,monomer content,the reaction temperature,the types and ratios of porogens,the microspheres with good dispersion,controllable pore size and uniform distribution of benzyl chloride were finally obtained.（2）Potassium 3-sulfopropyl methacrylate（SPM）was grafted onto the surface of PCMS-EDMA microspheres by atom transfer radical polymerization.On the other hand,we investigated the impact of grafting reaction conditions on ligand density and static binding capacity of the media.Based on the results of the experiment,ligand density first increased and then decreased as monomer（0.5-1.5 mmol/m L）and catalyst（0.0125-0.2 mmol/m L）concentration increased.The reaction temperature（25-45℃）showed a similar result.Meanwhile,the rule of static binding capacity was consistent with the ligand density.With the extension of reaction time（6-12 h）,the ligand density and static binding capacity of the media increased first and then tended to be gentle.The maximum ligand density was 0.1683 mmol/m L and the maximum static binding capacity was 186.5 mg/m L.Idea 2:The redox graft polymerization initiated by cerium ion(Ce⁴⁺)was used to graft polymer chain on the surface of gigaporous poly（glycidyl methacrylate-ethylene glycol dimethacrylate）microspheres（PGMA-EDMA）to prepare strong cation exchange chromatography media（PGMA-EDMA-SP）.The detailed works are as follows:（1）Ce⁴⁺-initiated redox polymerization was used to graft SPM polymer chain on the surface of PGMA-EDMA microspheres.The graft polymerization reaction conditions were optimized with the media ligand density as the parameter.The best graft polymerization conditions were obtained as follows:the monomer concentration was 0.300 mmol/m L,the initiator concentration was 0.018 mmol/m L,the H⁺concentration was0.600 mmol/m L,the reaction temperature was 45°C,and the reaction time was 12 h.The highest ligand density could reach 0.326 mmol/m L.（2）The relationship between static binding capacity and ligand density of media was investigated.It was found that with the ligand density（0-0.16 mmol/m L）of media increased,the static binding capacity increased.The maximum static binding capacity in the test range was up to 86.2 mg/m L.The performance of the two kinds of media were evaluated,including the dynamic binding capacity,mass transfer,and the separation and purification of lysozyme.The obtained regulations are as follows:（1）The dynamic binding capacity of PGMA-EDMA-SP media increased with the increase of its ligand density,While the dynamic binding capacity of PCMS-EDMA-SP media increased first and then decreased with the increase of its ligand density.The dynamic binding capacity of PCMS-EDMA-SP media（55.98-124.56 mg/m L）was higher than that of PGMA-EDMA-SP media（15.19-73.89 mg/m L）with similar ligand density.（2）The mass transfer performance of the media with different pore sizes was studied.The elution peaks of the two kinds of media at different flow rates were evaluated.In addition,we tested the difference between the dynamic binding capacity and the static binding capacity of the two kinds of media.It was found that the increase of pore size was beneficial to improving the mass transfer performance of the media.（3）Using egg white as the separation model,the lysozyme in egg white was separated by two kinds of media and commercial media,respectively.High purity lysozyme was obtained by one-step purification.It indicated that the three kinds of media have good separation efficiency.