Design and utilization of restricted-access media for high-performance liquid chromatography
Abstract
Development and performance of reversed-phase restricted-access high-performance liquid chromatographic packings are investigated for analysis by direct injection of small analytes in biological matrices. These supports, which prevent contact of proteins with the hydrophobic bonded phase, provide a solution to the problems of sample pretreatment. Cationic surfactants are used as pairing ions to extend application of Internal-Surface Reversed-Phase (ISRP) packings to include analysis of weakly acidic solutes. This study underscores the multifunctional nature of the tripeptide-based ISRP material. Nonionic polyoxyethylene-based surfactants are hydrophobically adsorbed on conventional reversed-phase columns, establishing a semipermeable hydrophilic layer over the alkylsilane surface, sterically prohibiting contact of proteins with the reversed phase, while permitting penetration and retention of small molecules. Adsorption behavior of nonionic surfactants on reversed-phase surfaces is described. Studies on the effects of surfactant adsorption on protein recovery and on retention behavior of small analytes show the viability of semipermeable-surface reversed-phase material for direct injection and provide the basis for the synthesis of a covalently bonded semipermeable-surface reversed-phase packing. A derivatizable reversed phase is modified by covalent attachment of polyethylene glycol to produce a semipermeable-surface reversed-phase packing. Preliminary evaluation includes results of elemental analysis after each synthetic step, as well as examination of protein recovery and small-molecule retention.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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