Preparation And Characterization Of Ion-exchange Cryogels By Grafting Polymerization

Ion-exchang supermacroporous cryogel is a novel chromatographic media for bioseparation, which attracts the increasing interests for direct isolation of target biomolecules from fermentation broths or unclarified feedstocks with cells or cell debris in downstream processes. The mass transfer resistance of the target molecules from the feedstock bulk to the binding sites on the pore surface is low and the adsorption equilibrium is established quickly. Therefore, high throughput of crude feedstocks with effective purification can be performed in cryogel beds. This technique has potential applications in bioseparation areas.In this paper, we prepared novel cation- and anion-exchange cryogels with functional binding groups by in-situ graft polymerization. The effects of the grafting conditions on the properties of the cation-exchange cryogels with sulfo binding groups were explored. The influences of salt type and concentration on the chromatography properties of the anion-exchange cryogel were also investigated. Moreover, isolation of ATP from fermentation broth of Saccharomyces cerevisiae was performed by anion-exchange chromatography.The results showed that graft polymerization of monomer chains with expected functional groups onto the matrix pore surfaces by initiator is an effective approach for introducing ion-exchange groups to cryogel matrix to get anion- or cation-exchange supermacroporous cryogels. The grafting polymerization was achieved in an in-situ manner which was performed by pumping the initiator and the reactive solution of graft monomer with sulfo or amido binding groups directly through a cryogel bed pre-produced in a glass column under frozen condition.The effects of grafting conditions on the properties of a novel cation-exchange cryogel by grafting of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) onto polyacrylamide-based cryogel (pAAm cryogel) using potassium diperiodatocuprate (Cu(III) solution) as initiator were investigated experimentally. Grafting conditions, such as concentrations of monomers and initiator, graft reaction time, graft temperature and the volume ratio of Cu(III) to NaOH influenced the liquid dispersion behaviors and the flow resistance as well as the protein binding capacity of the grafted cryogels. The protein binding capacity increased with the increase of concentration of AMPSA and Cu(III), the graft temperature and the graft reaction time in a wide range under the present conditions. Compared with these factors, the volume ratio of Cu(III) to NaOH in the initiator solution in the considered situations had a relatively weaker influence on the cryogel microstructures and protein binding capacity. The maximum binding capacity of lysozyme reached 2.6 mg/mL cryogel in these grafted cryogels.The salt type and concentration influenced the chromatography property of the anion-exchange cryogel. The protein binding capacity decreased as the salt concentration increased; and the more electric charges, the more obvious the salt type induced changes of the protein binding capacity.We also developed a novel one-step method to directly separate ATP from fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using superaiacroporous cryogel. The maximal purity of ATP by the one-step separation method was 95.5% using 0.01 M HCl as running buffer in this work.