When: Friday, April 26, 2013, 10:00 am
Where: TRC Room 206
Student: Hui Guo
Dissertation Title: Development of Chromatofocusing Techniques Employing Mixed-Mode Column Packings for Biomolecule Separations
Abstract:
A novel category of mixed-mode separation methods was developed which combines the technique of chromatofocusing with complementary chromatographic methods that utilize nonelectrostatic interaction, such as hydrophobic interactions or size exclusion effects. For studies of the hydrophobic interaction chromatofocusing method, two commercial column packings, one based on mercapto-ethylpyridine and the other based on hexylamine were investigated with regard to their ability to separate proteins when using internally generated, retained pH gradients. The effects of added salt and urea on the behavior of the retained pH gradient and the protein separation achieved were also investigated for this case. It was found that these additives were useful for "tuning" the separation of proteins achieved by altering the balance between the hydrophobic attractive force and the electrostatic repulsive force. Numerical simulations were shown to be a useful method to select the compositions of the starting and elution buffers in order to achieve desired shapes for the pH and ionic strength gradients. The use of the method to fractionate blood plasma was explored as a prototype example application. The case of size-exclusion chromatography combined with chromatofocusing was investigated using a bonded silica column packing where the underivatized silanol groups provided the electrostatic functionality. A multiple-charge-state model was developed to describe the adsorption processes in these cases. This model accounts for the salt effect on the hydrophobic interaction, the effect of charge regulation where adsorption changes the protein charge, the size-exclusion effect, and the effect of changing pH. Calculations using both the Donnan potential and the surface potential to fit adsorption data for lysozyme suggested that the electrostatic repulsive component of the overall adsorption affinity in the mixed mode chromatography systems considered likely results from both localized charged regions on the protein adjacent to the column packing surface and the net overall charge on the protein. In addition, a family of tetrazole-functionalized mixed-mode column packings based on both polystyrene-divinylbenzene and sepharose was synthesized which incorporates both hydrophobic and weak-acid cation-exchange functionalities. This material was studied with regard to its usefulness in chromatofocusing.
Light refreshments will be served at 9:45 am