LIQUID CHROMATOGRAPHY OF BIOLOGICAL SUBSTANCES

Yale University, New Haven CT

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Abstract

DESCRIPTION: (applicant's abstract) This proposal is aimed at further expanding the scope of modern liquid chromatography in a major way to meet the needs in medicine and life sciences for analytical separation methods of high speed and resolution. The main focus is on the development and use of novel packed capillary columns to explore the full potential of capillary electrochromatography. This liquid chromatographic technique employs a packed capillary column and high electric field to drive the mobile phase and promises the realization of high peak capacities attainable so far only in high resolution gas chromatography and capillary zone electrophoresis in the analysis of volatile and charged sample components respectively. A major research effort will be made to shed light on the physico-chemical underpinning of the nature, generation and control of eluent flow by electrosmosis and to develop a family of novel columns especially tailored to meet the particular requirements in capillary electrochromatography. Furthermore, the application of this new technique to the separation and analysis of complex mixtures of biological significance varying from carotenoids through peptides and proteins to glycoconjugates and polysaccharides will be explored. The proposed research will be carried along several closely related lines. Following studies on the energetics of the chromatographic process in the last project period, a method will be developed to estimate preferential interaction parameters which are useful in the characterization of biological substances including therapeutical proteins, from data readily obtained in hydrophobic interaction chromatography. High throughput screening in combinatorial chemistry requires rapid analytical methods and in continuation of earlier "studies, the research" on high temperature HPLC with packed capillary columns will be augmented. The advantages of capillary electrophoresis will be further examined at subzero temperatures with readily interconverting conformers of peptides, proteins and carbohydrates, and in the separation of various agglomerated forms of such substances. Novel applications for the promising use of cyclodextrins in capillary electrochromatography and capillary electrophoresis of peptides, proteins and other biopolymers will be investigated. All studies proposed will greatly benefit from fused silica based novel capillary columns that are under development. They include in situ formed fluid impervious polymeric innertube or porous monolithic packing, each having surface properties appropriate for the particular application.