Studies Of Protein Adsorption And Mass Transfer Kinetics In Ion-exchange Chromatography

The mechanism research on chromatography is of great significance for the rational design of chromatographic supports and the optimization of such an operation. Concentrating on presently the most widely used liquid chromatographic technology, the ion- exchange chromatography, the author carried out the following research work:(1) A parallel diffusion model was derived from the Maxwell-Stefan theory. The parallel diffusion model based on the MS approach (MS-ParD model) and its simplified form, the surface diffusion model (MS-SD model), were analyzed using the experimentally obtained uptake data, and compared with those based on the Fick's law (F-ParD and F-SD models). The validity and superiority of the mass transfer models based on Maxwell-Stefan theory was thus confirmed. Then,the MS-ParD model was extended to the situation of two-component protein adsorption kinetics (BiMS-ParD model). Combining with the multicomponent Langmuir isotherm, the BiMS-ParD model was used to analyze the experimental result for the adsorption of BSA/BHb mixture in the cation exchanger SP Sepharose FF. A reasonably good result was obtained.(2) A structured parallel diffusion model (SParD model) was developed to describe the intraparticle mass transport of proteins in porous chromatographic matrices. This model takes into account surface diffusion, hindered pore diffusion, pore size distribution of porous supports, and the shrinking of effective pore radius by protein adsorption. The dynamic adsorption data previously obtained were analyzed by the SParD model. The analysis confirmed the validity of this newly developed model and provided better understanding of the mechanisms of intraparticle mass transport.(3) In virtue of a homemade micro-column, the in situ observation of protein adsorption in transparent chromatographic matrix was realized on the confocal laser scanning microscopy (CLSM). The ubiquitous light attenuation effect impeded quantitative analysis carried out with CLSM. A general attenuation equation was derived from the imaging mechanism of CLSM, which realized the quantitative analysis based on CLSM. Thereafter, the experimental setups of micro-column were mended. The micro-column was evolved into a micro-flow cell. The attenuation equation was also developed. Thus, a self-contained experimental and theoretical system based on the technique of CLSM was set up. In virtue of this system, the proteins'chromatographic adsorption behavior can be quantitatively analyzed on a single particle level.