Shielded phases for protein separations
Abstract
Shielded phases for protein separations in both high performance liquid chromatography (HPLC) and high performance capillary electrophoresis (HPCE) have been described. Applications of these phases in biotechnological and pharmaceutical areas have also been discussed. The retention mechanism of both small analytes and proteins was examined on two types of semipermeable surface sorbents (SPS): (i) polyoxyethylene (POE)-based non-ionic surfactant coated packings and (ii) alkyl silane/POE silane bonded phases. Low molecular weight analytes were divided into three groups according to their retention behavior on SPS columns. Proteins and peptides are generally unretained under physiological conditions. In contrast, changes in the structure of either the polypeptide or sorbent surface under denaturing conditions cause substantial retention of proteins and peptides. By covalently linking bovine serum albumin (BSA) to porous silica, a protein coated shielded anion exchanger was created. This packing showed good recovery of proteins and was also useful in the separation of oligonucleotides. Scanning tunneling microscopy (STM) was used to image proteins packed on a surface to further study the mechanism of this packing. It was shown that there are gaps in between protein molecules through which small molecules could gain access to the surface below. A gradient displacement model for RPC has been described and applied to predict the retention behavior of proteins and small molecules in RPC and ion exchange chromatography. Several polymer coatings for HPCE have been explored among which the Trisacryl coating gave the best separation and recovery of proteins. A procedure for using internal standards to quantitate protein recovery was developed and applied to the evaluation of surface coatings in HPCE.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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