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Evaluation of entropic interaction chromatography media

University of British Columbia

Entropic interaction chromatography (EIC) media were synthesized by aqueous atom transfer radical polymerization (ATRP) of N,N -dimethylacrylamide (DMA) from the surfaces of non-porous and porous matrices. This novel chromatographic medium demonstrated efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into the layer of polymer attached to the support. Reaction conditions influencing the physical properties of the grafted layer were investigated. The use of "grafting from" initiators on a hydrolyzable ester linkage enabled exhaustive characterization of the grafted layer when cleaved from the surface. Negatively charged sulfate groups on the surface of the support were required for growth of dense polymer brushes and a combination of high surface charge and initiator concentration were necessary for the growth of dense brushes. The molecular weight of the grafts could be controlled by modifying the concentration of DMA used for the polymerization. Low polydispersities for the grafted layer demonstrated that the polymerization was well controlled. Scale up permitted the targeted synthesis of sufficient material grafted with 55.7 kDa grafts of high density (0.164±0.005 chains/nm ²) to be packed into a 30 x 1 cm chromatography column. Evaluation of a wide variety of columns synthesized with varying graft molecular weights or densities revealed that by exercising synthetic control over physical properties of the grafted layer, the selectivity curve could be tuned. Reducing the graft density allowed greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing the graft molecular weight also altered selectivity, but more directly affected column capacity by increasing the volume of the grafted layer. Through the use of modeling studies, it was predicted that the shear force could act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning predicted by this situation was observed experimentally. The large scale EIC column has potential application towards rapid desalting applications of bimolecular feeds and the ease by which graft properties can be tuned suggests that the synthesis of custom chromatography media may be possible.

Text

2010-01-20

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http://hdl.handle.net/2429/18756

Chemistry

University of British Columbia

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