毛細管電泳管內反應之探討和應用

On In-capillary Processes of Capillary Electrophoresis and the Applications

陳忠智(Jong-Jyh chen)

指導教授 ： 李允中 副教授

國立臺灣大學/生物資源暨農學院/生物產業機電工程學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.01688

摘要

本研究旨將一般生化分析轉用成具動態且可微量分析之毛細管電泳技術，並進行以下幾種管內反應之應用。首先，為將幾丁質類物質之親合特性作具體化的描述，採親合性電泳技術作幾丁質類物質之結合常數的驗證，本研究係藉由馬尿酸與幾丁寡醣(5〜9mer.)間具親合特性，故分析其親合物之結合常數約為0.56〜1.0174 M-1。由於該分析物在毛細管中的分離與結合係同時發生，所以對於分子間的動態交互反應提供相當多有用的資訊。

另外利用在毛細管管內進行呈色反應，可提高毛細管電泳圖譜分析的靈敏度，本研究以鄰苯二甲醛(O-phthalaldehyde；OPA)及硼酸液與葡萄糖胺可形成複合物，並對該兩種方法作一比較，結果顯示該OPA法較為靈敏(0.1~50mM, A340)且專一，且為克服OPA試液之衰減快速及再現性差的缺點，本研究之OPA法配合毛細管可線上反應、偵測之特點，成功地對於市售含葡萄糖胺之保健產品作有效的量測。

又毛細管電泳管內微萃取技術是另種對於樣品前處理的有效手段，在富含各種蛋白質的市售醬油中，毛細管電泳技術是無法有效地偵測出防腐劑成份的，本研究則將醬油先經酸化、再以乙酸乙酯萃取後之有機層液，微量注入具鹼性緩衝液之毛細管中進行電泳程序，則可簡化對醬油中防腐劑之分析，並達到定量的目的。

關鍵字

毛細管電泳 ； 葡萄糖胺 ； 保健食品 ； 微萃取

並列摘要

Dynamic micro-scaled biochemical processes were designed and conducted within the separation capillary of capillary electrophoresis (CE) system, and several possible applications were demonstrated. To characterize the bio-affinity properties of chitosan, ACE (affinity capillary electrophoresis) techniques were applied to evaluate the binding constant. From the shift in migration times of hippurate / chitooligosacharide complex, the binding constant of hippurate with chitooligosacharides (around 5〜9 mer.) was calculated to be in the range of 0.56〜1.0174 M-1. Since the binding process occurred simultaneously with the separation process, the method is rapid and provides valuable information about the dynamic molecular interactions.
Secondly, in-capillary chromogenic reaction was used to enhance the separation and sensitivity of CE system. Two labeling procedures for glucosamine using o-phthalaldehyde (OPA) and borate were developed and compared. The OPA method is more sensitive (0.1~50mM, A340) and specific, but the reaction is too quick to reproducibly operate in an off-line manner. OPA-labeling reaction was successfully performed on-line within the capillary, and the glucosamine contents in nutraceuticals were accurately measured.
Thirdly, in-capillary micro-extraction process served as an efficient sample pretreatment procedure. Benzoate derivatives are common preservatives in soy sauce; however, the CEgrams of untreated samples were too complicated and can not be effectively resolved / analyzed. The complicated samples were acidified and then extracted with ethyl acetate. The organic extract was directly injected and analyzed in capillary zone electrophoresis mode. The organic sample plug (benzoates in ethyl acetate) was extracted and then separated in the alkaline running buffer, which simplified the electropherogram for quantification.

並列關鍵字

Capillary electrophoresis ； Glucosamine ； Nutraceutical ； Micro-extraction

參考文獻

Albert, R.A., and E.L. King. 1951. Moving boundary systems formed by weak electrolytes. Study of cadmium Iodide complexes. J. Am. Chem. Soc. 73(2):517-523.

Albin, M., R.Weinberger, E. Sapp, and S. Moring. 1991. Fluorescence detection in capillary electrophoresis: evaluation of derivatizing reagents and techniques. Anal. Chem. 63:417-422.

Altria, K.D. 1993. Essential peak area normalization in capillary electrophoresis. J. Chromatogr. 35:177-182.

Altria, K.D. 1996b. Determination of drug-related impurities by capillary electrophoresis. J. Chromatogr. A. 735(1-2):43-56.

Amini, A., and D. Westerlund. 1998. Evaluation of association constants between drug enantiomers and human a1-acid glycoprotein by applying a partial-filling technique in affinity capillary electrophoresis. Anal. Chem. 70(7):1425-1430.