INTERNAL SURFACE REVERSE PHASE LIQUID CHROMATOGRAPHY

INGA HELENE HAGESTAM, Purdue University

Abstract

Prior to this work, it was not possible to analyze serum drugs with reverse phase high performance liquid chromatography (HPLC) by injection of the serum sample directly onto the column. The serum proteins interact with and accumulate on the support, resulting in a rapid degeneration of column performance. In most laboratories, therefore, the proteins are removed before injection by time consuming sample preparation procedures. The goal of this research was to develop a reverse phase liquid chromatography support requiring no sample pretreatment. The novel support, termed the Internal Surface Reverse Phase (ISRP) packing is similar to ordinary liquid chromatography supports, except that the hydrophobic partitioning layer is confined to the internal surface of the chromatography particles, only. The external surface contains a hydrophilic, protein non-adsorptive layer. The pore sizes of the support are small, preventing the large serum proteins from reaching the internal hydrophobic surface. When a serum containing drugs is injected onto the ISRP support, the serum proteins, unretained by the external surface, elute in the interstitial void volume. The hydrophobic drugs partition with the internal phase and elute similarly to conventional reverse phase chromatography. The ISRP support was produced by first attaching hydrophilic glycerylpropyl silane to silica particles. Hydrophobic peptide moieties were then covalently bonded to the hydrophilic surface resulting in a hydrophobic layer attached to both the external and internal surfaces. The external hydrophobic groups were then cleaved off by carboxypeptidase A, leaving the outside of the particles hydrophilic again. Carboxypeptidase is too large to enter the pores, allowing the internal surface to remain hydrophobic. Additional synthetic methods utilizing chymotrypsin as the cleavage reagent are described within the thesis. Some application of the ISRP support are demonstrated. For example, the drug phenytoin was isolated from serum proteins with a large particle ISRP precolumn and quantitated by a conventional ODS HPLC column connected in-line with the precolumn. Furthermore, anticonvulsant drugs in human serum were resolved from each other and the serum proteins with a high performance ISRP analytical column.

Degree

Ph.D.

Subject Area

Analytical chemistry

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