Preparation Of Poly(2-hydroxyethyl Methacrylate) Cryogel Microbeads For Isolation Of Adenosine Triphosphate

Hydroxyethyl methacrylate(HEMA) copolymer has good hydrophilicity and biocompatibility.Porous HEMA microspheres have become hotspots in biochemical and pharmaceutica fields.The traditional preparation of poly(2-hydroxyethyl methacrylate)(pHEMA)microspheres includes suspension polymerization,emulsion polymerization,seed polymerization and so on.However,the preparation process had some problems about controling particle sizes and size distribution,complex preparation process and others.Researchers had been exploring new techniques for controlling the size distribution of pHEMA microspheres.In recent years,preparation of microspheres using micro-channels have become an effective method for obtaining controlled size and uniform microspheres.Cryogel prepared by cryo-polymerization methods under frozen conditions have been widely used in bio-separation areas.In this thesis,pHEMA cryogel microbeads were prepared by combining the microchannel with cryo-polymerization methods under frozen conditions and the preparation method was studyed.The pHEMA cryogel microbeads prepared by this process had narrow particle size distribution,high porosity and good connectivity.The cryogel microbeads were packed in a chromatography column and grafted with N,N-dimethylaminoethyl methacrylate monomer by initiator diperiodatocuprate(Cu(Ⅲ)solution).The chromatographic separations of triphosphate(ATP)from different fermentation broths were carried out by the grafted cryogel microbeads at the velocity of 2 cm/min.The results showed that the high purity ATP was recovered from the Saccharomyces cerevisiae fermentation broth containing different contents of ATP,adenosine diphosphate(ADP),adenosine monophosphate(AMP),other impurities and yeast cells.The maxium purities of the obtained ATP and the recoveries were about 93.4～96.9%and 47.7～83.0%,respectively.The chromatographic separations were also conducted at high velocities of 5 and 10 cm/min.The experimental results showed that AMP,ADP and other impurities have weak adsorption properties and were washed out from the bed easily,because there are large voids between the microspheres.However,by cryogel microbeads and gradient elution,high purity ATP could be obtained even at high flow rates.