Preparation And Application Of Diol Group Modified Magnetic Chitosan Microspheres

In the natural product,there are some low abundance of small molecules,which play an important role in maintaining the normal operation of the living body.Immobilized metal affinity chromatography has a specific adsorption function for adsorbed proteins.However,the confined macromolecules compete with the target small molecule for the adsorption site,which reduces the selectivity of the affinity separation.In order to excrete large proteins and enhance the adsorption of small active molecules,the immobilized metal affinity magnetic media were modified by diol groups in this study.In order to avoid the detrimental application of carrier uniformity,this study used membrane emulsification and in-situ method to prepare uniform affinity carrier.The results were as follows:(1)The chitosan microspheres were prepared by membrane emulsification method.The optimum conditions were as follows:acetic acid content of 3%,Span-80 volume fraction of 4%,emulsification pressure of 200 kPa,stirring rate of 400 r·min-1,cross-linking agent of 2 mL and pH 7.5.Under these conditions,the average particle size of chitosan microspheres was 5.95 ?m and the particle size distribution coefficient was 0.37.When the mass ratio of chitosan to FeCl2·4H2O was 1:1,the homogeneous magnetic chitosan microspheres(M-CS)with magnetic saturation intensity of 25.69 emu·g-1 could be obtained by in-situ method.(2)The carrier was activated by epichlorohydrin(ECH).The optimum conditions were as follows:activation temperature for 45?,ECH volume fraction of 50%,NaOH concentration of 0.6 mol·L-1 and reaction time of 6h.Under these conditions,the epoxy group density was 189.72 ?mol·g-1.The magnetic immobilized Cu2+ affinity media(M-CS@Cu2+)were prepared with IDA as the chelating agent.And using the media to adsorb bovine serum albumin(BSA),the optimum process conditions were as follows:BSA concentration of 1.0 mg·mL-1,PBS buffer of 0.01 mol·L-1,pH 6.8,35?,adsorption time of 1.5h,the adsorption capacity of M-CS@Cu2+ to BSA was 123.27 mg·g-1.It was found that the adsorption process of BSA could be described by Freundlich equation,and the adsorption was in accordance with pseudo-second order kinetic model.(3)The diol group modified M-CS@Cu2+@OH media were prepared on the basis of M-CS@Cu2+ under the condition that the amount of IDA was changed to control the reaction path of the epoxy group.The VSLPEW was the active small molecule model,and BSA was the macro molecular protein model.Comparing the adsorption of M-CS,M-CS@Cu2+,M-CS@Cu2+ @OH in the mixed system of BSA and VW-6,it was found that M-CS@Cu2+@OH had the best specific adsorption effect.And the content of VW-6 could be increased from 0.99%to 4.66%.The optimal elution conditions were as follows:pH 5.0,NaCl elution of 2.0 mol·L-1,25min of elution time and eluted three times.After repeated use of three times,the effect of media adsorption became worse,and the adsorption capacity of the media after treatment was basically stable.The casein hydrolyzate was used to simulate the actual system.By comparing the adsorption and activity of the hydrolyzate adsorbed by M-CS,M-CS@Cu2+ and M-CS @Cu2+@OH,it was found that the protein molecules adsorbed by M-CS@Cu2+@ OH had the highest activity.