Experimemtal Study and Model Analysis on Affinity Chromatography using Non-porous Adsorbents

• Main Author: 李晉綱
• Other Authors: 李文乾
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/97716095013566399290

碩士 === 國立中正大學 === 化學工程研究所 === 83 === The most advantage of non-porous chromatographic adsorbents is that they are suitable for very rapid analytical and micropreparative affinity chromatography of proteins. Mass transfer effects due to pore diffusion of protein into and out of porous matrix are eliminated when non-porous adsorbents are employed as the packing material. The solute such as protein thus can be separated very rapidly without being denatured and with high resolution on chromatographic column. The yield of protein recovery is still high with a low binding capacity due to low surface area. What we studied in this article are the model analysis and experimental works on affinity chromatography of proteins using non-porous adsorbents. A mathematical model for designing affinity chromatography was formulated by following the mass balance and chemical kinetics. Numerical solution to the model based on the Galerkin finite element method was obtained the problem of numerical stability and the influence of solution parameters on the shape of the elution peak and the fraction of the non-retained peak were discussed. The chromatographic behavior of the affinity system with non-porous adsorbents was studied deeply. On the application of affinity column to micropreparative chromatography or quantitative analysis, we hope to know in prior whether the peak is asymmetric and boarder or not and the elution time is longer or not according to the conclusion from model analysis. The model proposed in this article provided a prediction of the shape of the elution peak and the non-retained peak and a range of calibration curve for quantitative analysis based on the peak height. Experimental results indicated that an affinity adsorbents for system operation under conditions of high pressure and flow-rate. In addition, the adsorbents could remained resistance against a strong acid or base. In this study, mondipersed, micrometer, non-porous polystyrene particle were synthesized and modified to introduced active groups and then immobihzed with the affinity ligand. The hydrophobic, aromatic groups on surface polystyrene beads were firstly nitrated. After nitration the surface of particles was reducted to yield hydrophilic, anuno groups by hydrogenation. Finally the aminopolystyrene was converted to that possessed via diazotization and coupled with concanavalin A (Con A). Con A specific sugars and cellulase were then analyzed by the high performance liquid chromatographic columns packed with the resultant affinity adsorbent. Although the experimental data were not good enough as we expected due to the low amino density we introduced to the surface of polystyrene, the bounded component of cellulase could be successfully eluted out by adding competing sugar derivative to the buffer or adjusting the pH value to 2.8 by using glycine/HCI. However, the adsorption capacity of the columns gradually decreased to zero after several times of sample injection. Therefore, the elution conditions need to be further investigated.