Liddy, Alison Mary (2010)
Ph.D. thesis, University of Birmingham.
The aim of this study was to create a bi-layered packed bed chromatography support for the purification of nano-sized bioproducts. The effect of three different chemistry approaches, different solvent conditions, and microwave heating were investigated on construction of a bi-layered support. Sepharose CL-6B was activated with allyl glycidyl ether (routes 1&2). The inert outer layer was created in route 1 by reacting bromine with the allyl groups at the surface, followed by the addition of sodium hydroxide. Creation of the outer layer in route 2 was achieved by oxidation of surface groups with potassium permanganate. In both synthesises the allyl groups remaining were reacted with bromine and a charged amine ligand was coupled to the inner core. The activation step of route 3 resulted in the introduction of three membered epoxide groups throughout the support. Surface groups were reacted with sodium hydroxide or hydrochloric acid. Finally a charged amine ligand was coupled to the support by reacting trimethylamine hydrochloride with the remaining epoxide groups. Supports created by route 1 eliminated 91% of plasmid DNA binding whilst maintaining a high protein binding capacity. This was achieved by using DMS0 as the solvent in the bromination step and employing microwave heating. Route 2 proved to be the least successful in creating a bi-layered support. The beads created under hydrochloric acid-methanol conditions (route 3) reduced 91% of the plasmid DNA binding whilst maintaining a high protein binding capacity. This study revealed that microwave heating was a useful tool in the synthesis of chromatography supports. Subsequently, a comprehensive study was untaken investigating the effects of microwaves on numerous chromatography matrices.
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