Ioannidis, Nicolas (2009)
Ph.D. thesis, University of Birmingham.
Chromatography remains the most commonly employed method for achieving high resolution separation of large-sized biomolecules, such as plasmid DNA, typically around 150-250 nm in diameter. Currently, fractionation of such entities is performed using stationary phases designed for protein purification, typically employing pore sizes of about 40 nm. This results into a severe underexploitation of the porous structure of the adsorbent as adsorption of plasmid DNA occurs almost exclusively on the outer surface of the adsorbent. In this study, the effect of two processing parameters, the ionic strength of agarose solution and quenching temperature, on the structure of the resulting particles was investigated. Three characterization methods, Atomic Force and cryo-Scanning Electron microscopy, as well as mechanical testing of single particles where used to quantify the effect of these parameters on the pore size/size distribution and mechanical properties of the adsorbent. In the presence of salt, it was found that agarose fibres tend to aggregate, leading to a gel with large pore size and wide pore size distribution. In fact, for the narrow range of ionic strength used (0-0.1m), a five-fold increase in pore size of the gel was observed. The same type of enlarged agarose structures was observed when slow cooling was applied during the gelation of agarose. The increase in pore size of the gel was also accompanied by an increase in the compression strength and the elastic modulus of the particles, i.e. particles with 200 nm pore size were found to have higher compression strength (1.5-fold difference) than those with 40 nm pore size.
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