This proposal is a continuation of our research in the development of high performance liquid chromatography (HPLC) to problems of biochemical importance. Our focus in the HPLC of biopolymers, especially proteins, but also including oligonucleotides. The strategy involves a two pronged attack on the problem -- synthesis of bonded phases and examination of the physiochemical phenomena involved. Three separate programs are proposed. (1) First, we shall examine in detail separation based on high performance hydrophobic interaction chromatography, using newly developed stable and reproducible polyether phases. We shall systematically vary the hydrophobic character of the surface, both in terms of extent of hydrophobicity of individual ligands and the spacing between ligands. Of particular interest is the use of phases derived from cholic acid, in which a broad interaction to a relatively rigid group can be envisioned. Adsorption isotherms to explore hysteresis and the kinetics of desorption will also be studied. (2) Secondly, we shall examine metal chelate chromatography using new approaches to ligand binding. This work will involve the role of metal chelate structure, metal and spacing between bonded ligands. Attempts will be made to map the surface topography of complexing amino acids on proteins. (3) A third area will involve a continuation of the study of conformational and other macromolecular behavior in HPLC. Biopolymers which reversibly, e.g., ribonuclease, and irreversibly, e.g., papain, convert from native to denatured states will be studied by various HPLC methods including reversed phase, hydrophobic interaction and size exclusion chromatography. Adsorption on various structurally controlled surfaces will be examined, particularly with a view toward large scale operation.
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