Study On Preparation Of Cellulose Acetate Membrane Chromatography Functionalized With Short Peptides And Its Separation Performance

With the widely application of active macromolecules used in the society, many fields, such as medicine, biology, etc., focus on the growing demands for the performance of separation technology. Membrane chromatography has been recognized as an efficient and environmental separation method. Additionally, it is easy to pack and industrialize. This study aims to prepare an affinity membrane chromatography via grafting appropriate ligands to the membrane, and then use for the purification of corresponding bioactive macromolecules.Firstly, the feasibility study of cellulose acetate membrane chromatography preparation via phase-inversion method was performed in this study. After the optimization of concentration, solvents ratio, additive, humidity of steam bath, temperature, etc., the water flux of the cellulose acetate membrane was increased from 283 Lm-2h-1 to 1598 Lm-2h-1. After grafting β-cyclodextrin to this membrane, the water flux was 639 Lm-2h-1 .However, when used in the preparation of membrane chromatography, the maximum number of packing layers was only 3, and the pore structures in the membrane were destroyed with increasing pressure. Consequently, the cellulose acetate membrane prepared by phase-inversion method is not suitable for the application in affinity membrane chromatography.Secondly, electrospinning method was investigated in this work to prepare cellulose acetate membrane. This membrane possessed high porosity and mechanical property. After the optimization of concentration, solvents ratio, receive distance, flow rate, voltage, etc., its pore size were between 264 nm and 5.12μm, in good shape. Subsequently, the membrane was treated by deacetylation and grafting of β-cyclodextrin. The results showed that it has good stability with packing 25 layers and continuously testing for 24 h. The crude puerarin sample was purified by this membrane chromatography, and its purity was increased from 42.52% to 84.1%, indicating the cellulose acetate membrane prepared by electrospinning method can be applied in affinity membrane chromatography.Finally, the small polypeptide LPPLPLPPLP, can specifically recognize the SH3 domains of protein, was synthesized in this study. With treating by deacetylation, oxidation, and grafting LPPLPLPPLP, the grafting ratio of the cellulose acetate membrane prepared by electrospinning method could reach 15.1μg/mg. In addition, BSA (as impurity) and phospholipase A2 were mixed as crude sample, and then be used to evaluate the performance of the membrane chromatography. The result showed when this crude product was treated by the membrane chromatography equipped with 20 layers of the modified cellulose acetate membrane, the phospholipase A2 could be efficiently immobilized on the membrane, and the saturated adsorption was 7.2μg/mg. And then after elution and concentration, the phospholipase A2 could be purified.