Preparation, Characterization And Applications Of Large-Scale Cryogels

Cryogels have interconnected supermacropores with size of several to several hundreds of microns,which can be used to separate target biomolecules directly from unclarified feedstocks or crude cell homogenates at high flow rates.They have been applied to capture biological products such as cell,plasmid,virus,enzyme and nuclear guanylic acid.The purpose of this thesis is to prepare large-scale cryogels,and to explore the feasibility of their industrial applications.The frozen conditions of the large-scale cryogel matrices produced with 7% acrylamide（AAm） by radical co-polymerized in a specialized stainless steel mold were measured.The results showed that the pore structures of large-scale cryogels are influenced by freezing rate and terminal freezing temperature greatly.Lower terminal freezing temperature ensured the heat generated during the formation of matrices be removed timely;and the appropriate freezing rate ensured the connectivity of large-scale pores and the good elasticity of the cryogels.Therefore,the cryogels with well interconnected supermacropores of diameters from 10～100 microns, porosity of 80%and swelling degree of 14 were obtained under the the freezing rate of 0.21℃/min and the terminal freezing temperature of-25℃in a stainless steel mold with a height of 20 mm and a diameter of 143 mm. N,N’-Diethylaminoethyl methacrylate（DEAEMA） with diethyl aminoethyl functional group（DEAE） can be successfully grafted onto the pore wall surface of the matrices to prepare weak anion-exchange cryogels.The graft polymerization was initiated by potassium diperiodatocuprate（K₅[Cu（HIO₆）₂]） in an in-situ manner.The properties of the small cryogel column（10 mm of diameter） maintained stable with graft polymerization of DEAEMA.The adsorption capacity of BSA did not vary significantly with the flow rate.The BSA adsorption of the small size cryogel grafted with 1 M DEAEMA reached 2.88 mg/（mL cryogel）, which was higher than that grafted with higher concentration of monomer.The size of column reached 2300 mL by stacking large-scale cryogel matrices in a glass column with a diameter of 140 mm and a height of 150 mm.0.056 M K₅[Cu（HIO₆）₂]solution and 1 M NaCl solution were mixed with volume ratio of 2:1 as catalyst.1 M monomer was grafted to large-size cryogel column under temperature of 48℃for 2 hours.The large-scale cryogel columns grafted with DEAEMA abtained high separation efficiency,and maintained good permeability with large and interconnected supermacropores in it.The method using large-scale anion-exchange cryogels was developed to isolate adenosine triphosphate（ATP） directly from crude fermentation broth of Saccharomyces cerevisiae.Deionised water was used as running buffer and the elution was carried out using 0.03,0.4 and 1 M NaCl at flow velocity of 2 cm·min^-1 during the chromatographic separation.The majority of impurities can be eluted by 0.03 M NaCl solution and higher purity ATP can be harvested by the elution of 0.4 M NaC1 solution.The purities of the obtained ATP were analyzed quantitatively by high performance liquid chromatography（HPLC）.The results showed that the purity of ATP by the one-step separation method reached 95%～97%,and the recovery rate reached 49.1%.Compared with previous work,this method has the advantages of simpler process and less time-consuming to reduce the risk of ATP degeneration.In the chromatography process,deionised water was employed and proved to be effective as an alternative running buffer beyond hydrochloric acid,which can decrease the cost of bioseparation.