In my diploma thesis, I studied whether water-soluble tracers are hydrophobic enough to be retained on a methacrylate support, which is weakly hydrophobic due to the presence of free epoxy groups. First, I determined the porosity and permeability of the columns, as these properties affect chromatographic performance. The porosity and permeability were determined by monitoring the passage of acetone, used as a tracer, through the system with and without a column at different flow rates.
I attempted to determine the hydrophobicity of the supports using various tracers, namely lysozyme, phenylalanine, tyrosine, tryptophan, butanol, hexanol, and plasmid DNA (pDNA). The measurements were performed at different concentrations of ammonium sulfate ((NH$_4$)$_2$SO$_4$) and different pH values, while the passage of the tracers was monitored using a UV–Vis spectrophotometer.
None of the water-soluble tracers used provided reliable evidence of complete retention on the methacrylate support that could be attributed to hydrophobic interactions. Therefore, under the applied experimental conditions and using the selected tracers, the hydrophobicity of the methacrylate support could not be determined.
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