| Concentrating is one of the important processes of protein industry. It has affected the quality and cost of the product. Nowadays the methods of concentrating protein solution include heating-evaporation, freeze-drying, ultrafiltration, precipitation, etc. There are many disadvantages of these methods like wasting energy and time, and may damage protein activity due to a high or low temperature or toxicity of the solvent. Since the adsorb-separate method using hydrogels means low-energy consumption, easy-operation and few-damage process for protein solution concentration. The superabsorbent hydrogels were synthesized and modified based on classic polyacrylamide, polyacrylic acid superabsorbent hydrogels in this paper. And a series of smart temperature-sensitive hydrogels were made by adding another special monomer (N-isopropyl acrylamide). The factors and conditions were discussed to gain a kind of hydrogel with excellent efficiency using for easy-harmless protein activity concentrating process. The results as below:(1) The Polyacrylamide, Poly (acrylamide-acrylic acid) and Poly (acrylamide-acrylic acid -N- isopropyl acrylamide) temperature-sensitive hydrogels were synthesized by radical polymerization. The structure and morphology characterization were analyzed by infrared spectroscopy (IR) and scanning electron microscopy (SEM).The hydrogel with super water absorption capacity was obtained by adjusting parameters of the polymerization. The product with max equilibrium swelling degree was the recipe of AM: AA=1:1 with 786 g/g water absorption. The swelling kinetics of hydrogels were studied. The results shows the swelling process of all products can satisfy the first order kinetics equation in the initial stage and the second order kinetics equation in the expand stage. The fitting calculating results can match the experiment water capacity. And the half swelling time order was:terpolymer hydrogels< PAM hydrogels< copolymer hydrogels. The product with fastest selling equilibrium time was recipe of AM:AA: NIPAM=2.2:2.2:0.6, its half swelling time was 1.4h(2) The water state in swelled hydrogels was analyzed by DSC. The results shows, when the hydrogel started to adsorb water, the water molecules was fixed in the polymer chain by hydrogen bond and formed the "non-freezing water", which was first hydration layer. And then, the non-freezing water was saturated, the next water molecules (intermediate state) was forming the second hydration layer. Finally, the outside layer was filled with lot of free water. The saturated quantity of non-freezing water in PAM, copolymer and terpolymer hydrogels was 0.9 g/g,1.4 g/g,1.1 g/g, respectively.(3) The temperature-sensitive properties of terpolymer hydrogels were proved by changing temperature. The higher NIPAM content led to a more obvious temperature response. Changed the temperature above or below its LCST, the hydrogels showed well repeatability and a bigger swelling degree was obtained in the second swelling process.(4) Chose BSA, pepsin and trypsin as objective protein to test the concentrating efficiency of hydrogels. Used the directly concentrating method, the concentrating efficiency to same hydrogel in different solution was:BSA>pepsin>trypsin; to the same protein solution, the concentrating efficiency was:copolymer>terpolymer>PAM. The concentration of BSA solution can be improved to 5.16×10-5mol/L from 10-5mol/L by using a 0.01 g copolymer hydrogels with the recipe of AM:AA=1:1 in 100 mL protein solution. To the terpolymer hydrogels, used a special "adsorb-release" method to concentrate protein solution based on its temperature-sensitive properties. The results shows that using very few hydrogels as concentrating agent (0.01g hydrogels/100mL protein solution), the concentration of BSA, pepsin and trypsin can be improved to 2.66×10-5、3.72×10-5、4.36×10-5mol/L respectively from 10-5mol/L. The concentrating efficiency was highest in the entire tests, and far higher than the similar literatures (0.24g hydrogels concentrating 100 mL protein solution, the concentration ratio was only 4.2).(5) The difference of swelling in the deionized water and the protein solution was studied by SEM, swelling kinetics analyze and DSC. The competitive phenomenon of protein molecule and water molecule to gather in the hydrogels surface and penetrate into the hydrogel’s internal structure was described by data analyzing. The lower protein molecular weight and higher concentration meant competitive phenomenon was stronger and its effect on the process of water molecule diffusing into hydrogels was greater. |
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