(631b) Hydrophobic Charge Induction Displacement Chromatography Visualized with Confocal Laser Scanning Microscopy

Displacement chromatography is a separation technique with high capacity and high resolving power. Mass transfer kinetics is an important factor that determines the separation efficiency of displacement chromatography, but few studies have been reported on the kinetics of the displacement process at a particle level. In this work, the kinetics of protein and displacer adsorptions, protein displacement and adsorbent regeneration in displacement chromatography were studied by both stirred-batch experiments and online visualization with confocal laser scanning microscopy (CLSM). Stirred-batch experiments showed that small-molecule displacers had higher effective diffusivities and mass transfer rates than proteins. For the displacement systems used in this work, the rate of displacement was dependent on the mass transfer and adsorption rate of the displacer. Direct visualization of the displacement process was achieved by labeling the protein with fluorescein isothiocyanate and by using a fluorescent displacer, rhodamine 6G. The concentration profiles obtained by CLSM clearly revealed the higher intraparticle mass transfer rate of the displacer and subsequent high rate of displacement. The observations by CLSM were mostly consistent with the results of stirred-batch experiments. However, although the direct visualizations by CLSM has provided valuable information of the microscopic processes in displacement chromatography, efforts are still needed to develop an improved displacement model system (proteins and displacers) that can provide more accurate and quantitative information of displacement processes.