Preparation Of High Performance Chromatographic Media And Its Application In Fast Biochemical Separation

There are excellent chemistry and physical properties of chromatographic media using polymeric substance as matrix.It has been widely applied to the separation and purification of bimolecular. In this study, the main aim is the preparation of high performance chromatography medium for rapid separation of biomolecular.PS microspheres have good mechanical strength, but it is strong hydrophobicity in nature, thus non-specific adsorption for protein on its surface uaually occur. In this study, PS microspheres were modified through ATRP and the "one-step" method using two kinds of hydrophilic molecules(HEMA and ethylenediamine).The Chemical composition and surface morphology of the modified microspheres were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The contact angle of hydrophilic microspheres changed from 128.2 degrees to 2.5 degrees and 2.1 degrees respectively; Meanwhile, the protein recovery of the microspheres were more than 90%.PGMA-EDMA microsphere was prepared by ATRP suspension polymerization with dichloromethane and n-octanol as porogen, bromo ethyl propionate as initiator., The optimized conditions for preparation of microspheres were obtained, in which the main factor was the ratio of porogen. Based on the ratio, adding F-127 into the aqueous phase and oil phase to prepare two kinds of microspheres were studied. Firstly, F-127 as the porogenic agent was added into the aqueous phase to prepare the PGMA microspheres. The microspheres porosity of the microspheres is 82.4% that is less than that of commercial medium POROS(90%),while its surface area(130.9 m2/g) is nearly 2.5times of a commercial POROS microspheres(53.7 m2/g) Because of the high specific surface area, high load can be obtained forchromatography medium. When putting the F-127 as reverse micelles into the oil phase, the microspheres porosity reached 88%, and the average pore diameter(275.7 nm) is 1.35 times of commercially available media POROS(203.6 nm). High permeability is favor for rapid separation of the biological macromolecules.The anion exchange medium based on the matrix prepared with F-127 as the porogenic agent and in the water phase was prepared through chemical reaction. SEM showed that the modified PGMA microspheres had the uniform coating, meanwhile, the throughput pores were maintained in the microspheres. The column based on PGMA-PEI-GTA was measured in term of its permeability and the column pressure was only 3.25 MPa at the flow rate of 3000 cm/h. Both the static adsorption and dynamic adsorption capacity reached the level of commercialization medium. The mixed proteins were separated on the medium at different flow velocity. It was found that the model proteins can be separated within 5 min, even at the velocity of 1806 cm/h, the good resolution can be still obtained. Therefore, PGMA microspheres show a great potential in the rapid separation of biomolecules.