| A series of protein-based chiral stationary phases was synthesized by immobilizing serum albumins, obtained from different animal sources, onto various silicas. All surfaces were characterized at each stage of the synthesis using microcombustion analysis to determine the extent of surface modification as well as a variety of other analytical techniques to determine physicochemical properties of the surfaces. Numerical methods were employed to derive parameters such as the BET specific surface areas, pore size distributions, and energy distribution functions. The results furnished a detailed description of the influence of the different steps of the modification chemistry on the surface characteristics, which provided a better understanding of the sorption properties.; It was demonstrated, that inherent differences in the amino acid residues forming the tryptophan binding pocket of various serum albumins directly influence the chromatographic properties of the respective chiral stationary phases.; Temperature studies were carried out to evaluate enantioselective binding affinities for L-tryptophan and related compounds. Using a two-site model to describe the solute-protein interaction, binding enthalpies and entropies were derived from van't Hoff plots. The results indicate an equilibrium between at least two conformational states of the tryptophan binding site with different affinities for the analyte.; Furthermore, a number of different organic eluent modifiers were studied. The results show differences between the properties of various classes of solvents that are consistent with data obtained from solution studies using circular-dichroic spectroscopy. Certain hydrophobic compounds induce changes within the stationary phase, (i.e., solvent memory effects) which influence the chromatographic properties. A possible explanation for this is diffusion of the modifier molecules into the hydrophobic core of the protein, which effects the conformation of the binding site. Long term changes in the retention of the test solutes studied were attributed to trace amounts of solvents used in the column packing procedure.; Similarly, different mono- and oligo-saccharides were tested as eluent additives. The use of sugars as modifiers in liquid chromatography offers some unique features compared to more conventional modifiers, such as alcohols. In combination with memory effects, the application of sugars is potentially useful for tailoring chromatographic properties of stationary phases or adsorbates. |
