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研究生:李晉綱
論文名稱:使用無孔性吸附劑器之親和層析及其模式解析
論文名稱(外文):Experimemtal Study and Model Analysis on Affinity Chromatography using Non-porous Adsorbents
指導教授:李文乾
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:1995
畢業學年度:83
語文別:中文
論文頁數:119
中文關鍵詞:無孔性層析吸附蛋白質
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無孔性層析吸附的最大優點在於適合蛋白質之快速分析及微量的製備分離,因為使用無孔性吸附劑沒有像多孔性吸附劑一樣有溶質進出細孔的質傳擴散作用,因此溶質可被快速分離,且層析之解析度也較高。再者,快速分離對某些易失活性之溶質(如蛋白質)也有提高回收率之功能。
本文主要是針對無孔性顆粒為基材進行親和層析之模式解析與實驗操作。於模式解析部份,是以質量守恆配合反應動力學剖析得其數學模式,採用Galerkin有限元素法求得數值解,探討數值系統之穩定性、溶液參數等對層析之溶出波峰與未吸附分率之影響,深入瞭解以無孔性吸附劑為基材時之親和層析行為。當此親和性管柱層析應用於微量純化分離,或定量分析用途時,希望能藉著本文模式解析的結果,發掘層析時前、後拖尾,波峰變寬,溶出時間太長等不良現象產生之原因,進而謀求改善之道。本文提出之模式,除了可預測溶出時間、溶出峰及未吸附峰之波形變化等之外,也可以找出以波峰高度作為檢量線之範圍。
至於實驗方面,由於聚苯乙烯機械強度佳,將它製成親和性吸附劑可於較高的壓力與流速下操作,且在強酸、強鹼環境下,也不至於變形。因此本研究合成微米無孔性聚苯乙烯顆粒,經表面修飾後固定配位體作為親和層析之吸附劑。聚苯乙烯表面具疏水性苯環基,須先經硝化反應,再經氫化把顆粒表面還原成具有胺基(-NH2)之親水性擔體,最後再經重氮化反應,產生反應性極強之重氮鹽與蛋白質(ConA)結合,形成親和性配位體,做為親和性吸附劑。所製備之吸附劑經充填成高效液相層析管柱後用來層析ConA專一性醣類、纖維素分解酵素之層析結果並不理想,究其原因可能是硝化反應時所導入硝基太少。纖維素分解酵素的吸附成分雖經由添加競爭性的醣類衍生物或將pH值調為2.8(甘氨酸/HCl)而溶出,但這些溶離方式數次操作之後吸附能力會喪失,因此溶出的條件尚需更進一步探討。


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.

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