Preparation of Macroporous Strong Anion Exchange Chromatography Media by Surface Grafting
Objective:A surface grafting method was explored for the preparation of macroporous anion-exchange chromatography media.The chromatographic performance of the medium obtained was evaluated in the present study.Methods:A macroporous anionic chromatography medium was prepared by redox-initiated graft polymerization of 3-(methacryloylamino)propyl trimethylammonium chloride using polyacrylate-based macroporous microspheres as the substrate.The optimal preparation process was determined by single-factor experiments,and the effects of the reaction factors on the chromatographic performance of the medium were investigated in detail.The esterase-like activity of proteins was measured before and after adsorption to determine the change in activity due to the interaction of the medium with the protein molecule.The pressure resistance of the medium was tested under different flow conditions.The stability properties of the medium were tested using 1 mol/L NaOH solution.Th non-specific adsorption of proteins on the medium was tested under non-adsorptive conditions.Plate heights for media packed columns were determined at different flow rates to evaluate column efficiency.High-purity protein was purified from egg white using anion-exchange chromatography.Results:The static binding capacity of the protein on the prepared medium could reach up to 138.94 mg/mL under the optimal preparation conditions.Recovery of protein activity from the medium was up to 98％.The dynamic binding capacity of the medium decreased from 99.234 mg/mL to 93.981 mg/mL in the linear velocity range of 143 to 574 cm/h.The column pressure increased linearly in the range of 143 to 3 871 cm/h linear velocity.The surface and internal morphology of the medium was examined using a scanning electron microscope and it was found that grafting polymers had no effect on the pore diameter of the medium.The minimum plate height was 0.059 9 mm at an eluent flow rate of 535 cm/h.The anion-exchange chromatography medium adsorbed 0.136 mg/mL of bovine serum albumin under non-adsorption conditions.The static binding capacity of the protein in the medium decreased by only 2.4％after 96 h of immersion in 1 mol/L NaOH solution.The one-step purification of egg white proteins with the medium resulted in high purity of lysozyme,ovalbumin and ovotransferrin.Conclusion:The media have better mass transfer performance,higher protein activity recovery and lower non-specific adsorption values,with good stability and rigidity compared to conventional media.This type of medium has great potential for high-throughput protein separation.
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