The Synthesis And Protein Absorbance Of Crosslinked Modified Chitosan Micropheres

Objective:The aim of this paper was to investigate new polymer microsphere materials as the potential chromatography stationary phases for protein separation and purification.Methods:CM-CTS and chloride trimethyl chitosan quaternary ammonium (HACC),were separately prepared by reacting CTS with chloroacetic acid and epoxypropyl trimethyl ammonium chloride(GTA),and then were cross-linked into microspheres by emulsion-glutaraldehyde.The reaction conditions were investigated and optimized and the performances of synthetic microspheres were studied.The following are the main contents and conclusions:1 By using CM-CTS as the original material to synthesize CM-CTS microspheres,we set up the best reaction system in the light of the microspheric morphology:2g CM-CTS,50mL H₂O,50mL paraffin(contains 2.5%Span80),10 mL glutaraldehyde,mainly from the form of the microspheres.We used the candidating BSA and Cyto-C for the study on protein adsorption of synthesized CM-CTS microspheres.The maximum adsorption of BSA reached 69.10mg/g.In addition,in order to validate whether the adsorption is reversible or not,we also tested the microspheres for the protein desorption.Besides,the porogens,PEG and CaCO₃, is not able to work.2 We used CTS to synthesize the CM-CTS,and tried to calculate the DS of CM-CTS to optimize reaction parameters.Parameters including the reaction temperature,reaction time,the amount of chloroacetic acid and sodium hydroxide, and alkalization temperature,were fixed on to set up best reaction system.Studies have shown that chloroacetic acid,sodium hydroxide and the amount of original materials are the most important factors affecting the synthesis of CM-CTS.The best reaction system is:3g CTS,20g NaOH,200mL isopropanol,55℃for 12h,4g Chloroacetate,60℃for 5h,and the maximum of DS is 0.7295.3 Through measuring the DS,the set up reaction system is not very accurate.So we adopted a two-step reaction process to synthesize CM-CTS microspheres,and by detecting the protein adsorption capacity of CM-CTS microspheres,we finalize the best parameters for synthesis.Synthesized CTS is on one hand and the second is cross-linked CM-CTS into CM-CTS microspheres.We used BSA to figure out the protein adsorption capacity of CM-CTS microspheres.Experimental results showed the optimum synthesis parameters for CM-CTS:2g CTS,25mL 50%NaOH solution,5g Chloroacetate,25mL isopropylalcohol,60℃.The highest adsorption amount of BSA was 47.03mg/g.Dissatisfactorily,we still could not obtain the desired microspheres with a good protein adsorption capacity.4 Because of the complexity of the carboxymethylation reaction and existing by-products,it was difficult to achieve a good result as we initially devised.So we added quaternary ammonium groups to CTS and then cross-linked the products into HACC microspheres for functional BSA adsorption and infrared spectroscopy.The optimum reaction system for HACC synthesis was:5g CTS,100mL solvent (isopropanol:water=9:1),20g GTA,reaction for 8h.For the cross-linking,5mL glutaraldehyde was further added into.The highest BSA adsorption was 132.2mg/g. Meanwhile,we tested to build the BSA adsorption isotherm curve for the HACC microspheres and its infrared characterization.