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研究生:楊濡碩
研究生(外文):Ru-Shuo Yang
論文名稱:香豆素類化合物以毛細管電泳測定與分離機制探討之研究
論文名稱(外文):Study on the Determination and Separation Mechanism of Coumarins by Capillary Electrophoresis
指導教授:王書蘋
指導教授(外文):Shu-Ping Wang
學位類別:碩士
校院名稱:靜宜大學
系所名稱:應用化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:102
中文關鍵詞:解離常數測定香豆素毛細管電泳分離機制討論
外文關鍵詞:coumarinscapillary electrophoresisseparation mechanismdissociation constant
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摘要

香豆素 (coumarins) 廣泛存在於許多植物中,已被證實具有多種生物活性,其用途廣泛且多元,在藥理學上具有抗癌及治療水腫之功效,在過去文獻中已有相當數量的研究報告發表,但較少研究針對其分離機制進行討論。因此,本研究針對十種用途較廣且結構相近之香豆素類化合物,探討其分離機制並測定之。
由酸鹼值、界面活性劑、有機溶劑分離機制的討論中得知,4-hydroxycoumarin 可與 lactone ring 形成共軛系統而較易解離,其 pKa 值較同樣是醇基取代者小。酸性香豆素的遷移行為,直接受到本身 pKa 值及緩衝液 pH 值大小影響,而中性物種則和 sodium dodecy sulfate (SDS) 微胞有不同分配作用,分離選擇性高。再者,取代基種類直接影響分析物的極性大小,是支配分離機制的重要因素。適量有機修飾劑可調節微胞和中性香豆素的作用,提供良好的波峰形狀。
利用毛細管電泳測定酸性香豆素的解離常數,以代數法計算可成功測得四種酸性香豆素 (6,7-dihydroxycoumarin、7-hydroxycoumarin、coumarin-3-carboxylic acid、4-hydroxycoumarin) 之 pKa 值,分別為 7.53 (pKa1)、6.52、4.64 及 4.32。建立 pKa 值有助於了解並預測該類化合物在生物系統及環境中的吸收、散佈、代謝及排泄的狀況,且也利於藥物製備及相關產品開發。
本研究所建立之方法具有方便及快速分析之優點,可同時分離多種香豆素類化合物,並且可應用於真實樣品 (如食品、尿液) 之分析。線性相關係數皆在 0.9955 以上,遷移時間及積分面積的RSD值分別低於 1.6% 及 5.4% 以下,顯示此分析方法具良好的再現性。
Abstract

Coumarins are widely spread in plants and essential oils, and have been used to treatment of cancer and oedemas. In past years, lots of attention was paid on analysis of coumarins, but few of them were dealt with the effect of substituents on migration behavior in capillary electrophoresis (CE). Thus, for investigating the separation mechanisms, we selected ten compounds with identical skeleton of coumarin in this study.

Through the discussion on the mechanisms, 4-hydroxycoumarin possesses the smaller pKa than others with various position of hydroxy group. We supposed resonance may be formed within lactone ring and hydroxy group that caused the proton was dissociated facilely. The value of pKa of acidic coumarins and the used pH of running buffer both would affect the migration behaviror directly. Furthermore, micelle of SDS may have various partition interaction with neutral species and enhance the selectivity. Moreover, the substituents of coumarin would affect the polarity and be the key factor of the separation mechanism. Finally, the adequate methanol (20%) would modify the interaction between coumarin and micelles and provide a suitable peak shape.

There are two methods, algebraic method and differential method, to determinate the value of pKa by the CE. Through the algebraic method, the pKa values of 6,7-dihydroxycoumarin, 7-hydroxycoumarin, coumarin-3-carboxylic acid and 4-hydroxycoumarin were obtained successfully (7.53, 6.52, 4.64, 4.32). Determination of pKa of acidic coumarins would be helpful for recognizing and estimating the absorption, distribution, metabolism, and excretion of the compounds in biological system and in environment. This would be valuable to develop medicine and relative products as well.

The proposed method of CE were simple, fast and simultaneous for separation, and it also could be applied to analysis of food and urine with good precision. The correlation coefficient were above 0.9955, and the RSD of migration time and peak area were under 1.6% and 5.4%, respectively.
目錄

目錄………………………………………………………………..….……I
謝誌……………………………..………………….………………...…...Ⅳ
中文摘要……………………………...…………………………………..Ⅴ
英文摘要……………………………………………………….…………VI
表目錄………………………...…………………………………………VII
圖目錄…………………………………………………………………..VIII
第一章 緒論……………………………………………………………...1
第一節 前言…………………………………………………………..1
第二節 分析物結構與性質…………………………………………..5
第三節 相關研究情況及文獻整理………..…..……………………..8
第四節 研究目的……………………………………………………14
第二章 酸性香豆素解離常數 pKa 值之測定………………………...15
第一節 前言…………………………………………………………15
第二節 實驗部分……………………………………………………17
一、 儀器設備………………………………………………….17
二、 藥品……………………………………………………….18
三、 藥品配製…………………………………………………19
四、 毛細管清洗方式…………………………………………22
第三節 結果與討論…………………………………………………22
一、 理論計算……………..……………………………………22
二、 取代基與所得 pKa 值之關係……………………………30
第三章 毛細管電泳……………………………………………………...33
第一節 原理…………………………………………………………33
一、 發展簡介………………………………………………….33
二、 儀器裝置………………………………………………….35
三、 影響分離效率之因素…………………………………….41
四、 操作模式……………………………………………..……46
第二節 實驗部分……………………………………………………52
一、 儀器設備…………………………………………………52
二、 藥品………………………………………………………52
三、 藥品配製………………………………………………….53
四、 毛細管的清洗方式……………………………………….56
第三節 結果與討論…………………………………………………57
一、 香豆素遷移行為與分離機制之探討……………………58
二、 最佳分離條件之建立與定量結果評估…………………71
第四章 真實樣品之檢測………………………………………………...82
第一節 實驗部分……………………………………………………83
一、 前處理步驟……………………………………………….83
二、 測定方式………………………….…………...…………..84
第二節 結果與討論…………………………………………………87
第五章 結論……………………………………………………………...91
第六章 參考文獻………………………………………………………...93
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