Hydrophobic Charge Induction Displacement Chromatography: Methodology Development And Displacer Screening

Displacement chromatography is a powerful separation technique in which the solutes are sequentially displaced from the column in high concentration and purity by a high-affinity substance called the displacer. Promising as it is, the technique did not have much applications in bioseparation processes due to some theoretical and practical difficulties. This dissertation is dedicated to solve a number of problems that hinder the applications of displacement chromatography so as to make full use of its potential in bioseparation processes.First, a new separation technique,"hydrophobic charge induction displacement chromatography (HCIDC)", was proposed to solve the problem of column regeneration in traditional displacement processes. With the assistance of ligands with proper hydrophobicity and disassociation properties, adsorption and displacement of proteins can be achieved by hydrophobic interactions. By an adjustment of pH, the ligand can take on charges of the same polarity as the displacer and column regeneration can be achieved by charge repulsion. Lysozyme andα-chymotrypsinogen A was successfully separated by HCIDC on MEP Hypercel at pH 5.0, with benzethonium chloride as the displacer. Column regeneration was achieved with high efficiency at low pH values.In order to address the difficulties in displacer development, a displacer– immobilized ligand docking scheme was proposed for the virtual screening of displacers. The efficacy data of various cation, anion and hydrophobic displacers were used to verify the effectiveness of the docking scheme, and the Spearman ranking coefficient of all cases were over 0.5. This indicates that it is feasible to use the docking scheme for the screening of displacers. The scheme was then used to screen displacers for HCIDC from over 1000 commercially available compounds, with several novel displacers identified. Column displacement experiments showed that the displacers identified were efficacious in protein displacement.The microscopic mechanisms of displacement chromatography are of great importance in its development and optimization. However, no studies have been reported on this issue till now. In this dissertation, confocal laser scanning microscopy was used for the studies of the microscopic processes in displacement chromatography. Direct observation of the process was achieved with a fluorescently labeled protein and a fluorescent displacer. Result showed that the mass transport of small molecule displacers are extremely fast, while the desorption of the protein is the rate-limiting step of the whole process. It is expected that these observations can facilitate the process development of displacement chromatography.