Preparation Of Continuous Supermacroporous Cryogels Via Cryo-Copolymerization

Continuous supermacroporous cryogel monolithic chromatography is a new method for separation and purification of bioproducts. Cryogel has interconnected supermacropores with pore size of several microns to several hundreds of microns, which permit the solid particulates in the culture fluids to pass through freely without blockage. It can be applied to capture and separate target biomolecules directly from unclarified feedstocks or crude cell homogenates at high flow rates.Solvent freezing crystallization is a crucial step in the preparation of cryogels and the formed ice crystals act as porogen. In this work, the measurements of liquid-solid phase transition of solvent were carried out to reveal the freezing characteristics of aqueous solution. Freezing curves of this solution system under various concentrations were obtained to determine the corresponding values of freezing point temperature Tmc and the actual initial solvent crystallization temperature T_c at various cooling conditions. The results showed that a supercooling state occurred at each freezing curves. The effect of freezing rate on T_mc can be neglected when the freezing rate was low enough. However, T_c changed randomly in a certain range with monomer concentration and freezing rate, while T_mc decreased with the increase of monomer concentration under freezing condition at a constant freezing rate and a linear correlation of T_mc and monomer concentration existed.Polyacrylamide-based supermacroporous monolithic cryogel beds have been prepared under the variation freezing condition. The formation of cryogels under the present situations has been considered as a coinstantaneous process of solvent crystallization and monomer copolymerization. The results showed that the pore microstructures, liquid dispersion characteristics, water permeability and porosity of the cryogel depended strongly on the cooling temperature and cooling rate,which control the solvent crystallization process, and the redox initiator concentration, which controls the monomer polymerization. Good porous cryogel with well-interconnected pores and with smooth gel walls can be obtained by appropriate combining control of these conditions.The Cu²⁺-IDA, Zn²⁺-IDA and Ni²⁺-IDA metal chelated cryogels have also been successfully prepared. In order to reveal the protein adsorption characteristics in these cryogels, BSA was used as model protein to study the adsorption and desorption behaviors of protein in these metal chelated cryogels. The results showed that the maximum adsorption capacity of Cu²⁺-IDA bed was 3.29 g BSA/L cryogel at pH 5.0, and the maximum adsorption capacity of Zn²⁺-IDA and Ni²⁺-IDA beds were 4.45 and 3.92 g BSA/L cryogel at pH 7.2, respectively. The presence of NaCl affected the adsorption capacity of BSA on the Zn²⁺-IDA and Ni²⁺-IDA beds, while had weak effect on Cu²⁺-IDA bed.The liquid flow rates had no significant effect on the BSA breakthrough and the protein adsorption capacities at the given flow rates （1～6cm/min）. 0.2 M imidazole solution was satisfied to elute the adsorbed BSA and a low elution liquid velocity was beneficial to protein desorption from the metal chelated cryogels.A novel continuous supermacroporous cryogel embedded with Fe304 nanoparticles with pore size of 10 to 50μm has been prepared under the conditions of freezing-temperature variation. The axial liquid dispersion coefficients of the obtained cryogel were weak. The chromatographic performance of BSA in the cryogel column embedded with Fe₃O₄ nanoparticles was shown that the protein adsorption capacity of the present cryogel was improved due to the embedded nanoparticles in the gel matrix and the obtained protein adsorption capacity is 1.93 g BSA/L cryogel.