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研究生:傅明棟
研究生(外文):Ming-Dong Fu
論文名稱:類黃酮素以液相層析與毛細管電泳之定量分析及取代基影響機制之研究
論文名稱(外文):Study on the effect of substituent group and quantitative analysis of flavonoids by high performance liquid chromatography and capillary electrophoresis
指導教授:王書蘋
指導教授(外文):Su-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:2005/02/
畢業學年度:93
語文別:中文
論文頁數:112
中文關鍵詞:液相層析分離機制毛細管電泳類黃酮素
外文關鍵詞:flavonoidscapillary electrophoresisseparation mechanism.liquid chromatography
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類黃酮化合物普遍存在於自然界植物中,由於此類化合物具有抗癌及預防心血管疾病等性質,因此近年來引起廣泛的研究。但到目前為止,卻較少有文獻對這一類化合物的分離機制做進一步的討論。本論文即選擇具有相同主結構之九種類黃酮化合物,探討其在層析及電泳分離時之機制。
液相層析使用C18的層析管柱,以5 mM phosphate buffer (pH 6.9) 及MeCN梯度沖提可得到良好的分離。至於毛細管電泳的研究,則採用最簡便的CZE分離模式,以 32 mM tetraborate buffer ( pH 9.2),加入5 mM SDS作為添加劑,施加15 kV之電壓,可同時分離多種結構異構物。
由分離機制的討論中得知,一般OH取代愈多者極性愈大,但具有兩個相鄰之OH取代者,其pKa卻比只具單取代者小。而具有C3-OH者會導致flavone與flavanone的流析順序相反。此外,C5-OH與C4-carbonyl group由於形成分子內氫鍵,而造成極性降低。具單取代化合物中,C6-OH的極性最小,C4’-OH與C7-OH取代之極性相近,而C7-OH最早解離其pKa最小。
在分離機制的討論中,除了瞭解取代基數目及位置對於分離機制之關係外,並且針對Wulf et al.及Wang et al.二者結果矛盾之處,找到了合理的解釋,是在於C3位置有無氫氧取代基之差異。
Much attention was paid on flavanone because of their potentials in the prevention against cardiovascular diseases and cancer. However, many previous papers had focused on the analysis of flavanones whereas few dealt with the migration and retention of flavanones. For investigation of separation mechanism, we selected nine compounds with identical skeleton of flavanone in this study.

The optimum separation of LC with C18 column was performed by gradient of acetonitrile and 5 mM phosphate buffer (pH 6.9). About capillary electrophoresis, we adopted CZE mode for its simple and convenient. The optimum electrolyte buffer of CZE was 32 mM sodium tetraborate buffer (pH 9.2), 4.5 mM SDS, and apply 15 kV. It can also be achieved for separating of isomers.

Generally, more hydroxyl groups will increase the polarity of compounds. For flavanones with dual adjacent hydroxyl groups, their pKa are less than singular substituent. Nevertheless, the compounds with hydroxy at C3 would cause adverse elution order of flavone and flavanone in LC. In addition, the polarity would be reduced because of forming intramolecular hydrogen bond within hydroxy at C5 and carbonyl group at C4. Moreover, in single substituted compounds, the polarity of flavanone with hydroxy at C6 is the smallest, and compounds with hydroxy at C4''and C7 are closely. But the pKa of flavanone with hydroxy at C7 is the smallest for its easier dissociation.

Through the discussion on mechanism, we not only demonstrated the effect of substituent, but also found a reasonable explanation for the contradictory results of Wulf et al. and Wang et al. was due to whether the hydroxy sustituent at C3 exist.
目錄………………………………………………………………..….…..Ⅰ
謝誌……………………………..………………….………………...…...Ⅳ
中文摘要……………………………...…………………………………..Ⅴ
英文摘要……………………………………………………….…………Ⅵ
表目錄………………………...…………………………………………..Ⅶ
圖目錄…………………………………………………………………….Ⅸ
第一章 緒論……………………………………………………………...1
第一節 前言…………………………………………………………..1
第二節 化合物結構…………………………………………………..5
第三節 相關文獻……………………………………………………..8
第四節 研究目的……………………………………………………19
第二章 液相層析法……………………………………………………...21
第一節 原理…………………………………………………………21
第二節 實驗部分……………………………………………………23
一、 儀器設備……………………………………………….….23
二、 藥品…………………………………………….………….24
三、 藥品配製………………………………………..…………25
四、 定性之判斷依據…………………………………………26
五、 定量依據…………………………………………………27
第三節 結果與討論…………………………………………………28
一、 移動相組成之討論…..……..……………………………29
二、 定量結果…………………………………………………42
第四節 分離機制之討論…………………………………………...46
第三章 毛細管電泳法…………………………………………………...50
第一節 原理…………………………………………………………50
一、 發展簡介………………………………………………….50
二、 儀器裝置………………………………………………….51
三、 影響分離效率的因子…………………………………….56
四、 操作模式…………………………………………………60
第二節 實驗部分……………………………………………………67
一、 儀器部分…………………………………………………67
二、 藥品………………………………………………………67
三、 藥品配製………………………………………………….68
四、 毛細管的清洗步驟……………………………………….69
五、實驗過程………………………………………….………69
第三節 結果與討論…………………………………………………72
一、儀器幫浦穩定性探討……………………………………..73
二、建立毛細管電泳最佳化條件………….………..…………74
三、定量結果………………….………………………….….…90
第四節 分離機制之討論…………………………………………….94
第四章 液相層析法與毛細管區帶電泳法之比較……………………...95
第五章 真實樣品之檢測……………………………………….………..98
第一節 實驗部分……………………………………………………99
一、 設備……………………………………………………….99
二、 SPE Cartridge活化步驟…………………………………99
三、 前處理步驟……………………………………………….99
四、前處理後flavanone之回收率..………………………….100
五、測定方式…………………………………….…………….100
第二節 結果與討論………………………………………………..101
一、回收率之測定…………………………………………….101
二、真實樣品分析……………………………………………101
第六章 結論…………………………………………………………….106
第七章 參考文獻……………………………………………………….108
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