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研究生: 陳怡君
論文名稱: 毛細管電泳技術對葡萄糖偵測法的開發與研究
指導教授: 林震煌
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2010
畢業學年度: 98
語文別: 中文
中文關鍵詞: 葡萄糖電泳拉曼
論文種類: 學術論文
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  • 毛細管電泳(capillary electrophoresis, CE)雖然具有高分離效率、能提供各種分離模式、溶劑消耗量低…等多項優點,但是此方法對於不具UV吸收性質或非螢光性物質的分析,仍有不足之處。本研究以葡萄糖(D-glucose)做為測試樣品,分別探討當毛細管電泳結合拉曼光譜法、螢光衍生法及間接吸收光譜法時,對葡萄糖分離與偵測的效果。
    實驗結果發現,以綠光半導體雷射(500 mW, 532 nm)為激發光源,藉由CCD偵測器可同時觀測到葡萄糖具有多處拉曼特徵峰。其中以1129 cm-1的譜峰最適合做為電泳時的觀測峰。 在pH=12.1的氫氧化鈉溶液中,添加0.5 mM的界面活性劑-CTAB做為管壁修飾劑,以負電的模式進行電泳,可直接偵測到葡萄糖的單一譜峰。
    為了提高偵測的靈敏度,本實驗以2-氨基吖啶酮(2-aminoacridone)為衍生試劑,對葡萄糖進行螢光化反應。衍生物以10 mM硼酸鹽溶液稀釋後,配製於10 mM硼酸鹽及100 mM界面活性劑-SDS所組成的緩衝溶液中。在此條件之下,以藍光半導體雷射(100 mW, 473 nm)為激發光源,進行毛細管電泳分析。在觀測CCD偵測器的即時光譜時,發現衍生試劑(2-氨基吖啶酮)的最大螢光波長為520 nm,但經螢光衍生後則紅光位移至580 nm。此螢光標識法可將葡萄糖的偵測極限改良至~ 1.1 × 10-5 M。上述方法可提供蜂蜜純度的鑑定或快速分析糖尿病患檢體等研究。
    由於間接的方式無法得知分析物的光譜或質譜,本實驗嘗試將基質輔助雷射脫附游離飛行式質譜法(MALDI-TOFMS)中,經常使用到的基質:氰基-4-羥基肉桂酸(α-cyano-4-hydroxycinnamic acid, CHCA),直接添加於電泳溶液(同前述條件)中,作為為背景溶液,以間接吸收法進行偵測。當以氙燈經截光器後取出波長337 nm的紫外線作為吸收光源,且CHCA的濃度為10 mM時,葡萄糖的偵測極限為~1.3 × 10-3 M,線性範圍10-1 M ~10-3 M。
    我們使用三種方法偵測葡萄糖,其中拉曼光譜法可以進行定量以及定性分析,未來如果克服了靈敏度的限制,拉曼光譜法將會是非常便利的偵測方式。

    第一章 緒論 1-1研究目的.................................................1 1-2分析物簡介...............................................3 第二章 分析方法及原理........................................5 2-1 毛細管電泳層析法之發展歷程................................5 2-2 毛細管電泳法之基本原理...................................9 2-2-1 電泳分離與電泳遷移率...................................9 2-2-2 電滲流(EOF)........................................11 2-2-3 管柱分離效率.........................................14 2-3 毛細管電泳層析法之分離模式...............................15 2-3-1 毛細管區帶電泳(CZE).................................16 2-3-2 微胞電動層析法(MEKC)................................19 2-4 拉曼散射...............................................23 2-4-1 拉曼散射歷史簡介......................................23 2-4-2 拉曼散射原理介紹......................................25 2-5 間接偵測法.............................................29 第三章 儀器、藥品與實驗方法..................................31 3-1自組式毛管電泳/拉曼分析儀.................................31 3-2 自組式毛細管電泳/螢光分析儀..............................33 3-3自組式毛細管電泳/間接吸收分析儀...........................35 3-4 儀器及周邊設備列表......................................37 3-5 使用藥品列表...........................................42 第四章 結果與討論...........................................46 4-1 拉曼光譜法.............................................47 4-1-1 標準光譜的建立.......................................47 4-1-2 葡萄糖進行毛細管電泳之探討.............................53 4-1-3 毛細管區帶電泳(CZE)電泳條件的確立與配製.................55 4-2 螢光衍生法.............................................60 4-2-1葡萄糖螢光化衍生反應之探討..............................60 4-2-2 葡萄糖衍生物之製備....................................62 4-2-3 光譜性質之測量.......................................63 4-2-4 衍生物質譜建立.......................................65 4-2-5 微胞電動層析法(MEKC)電泳條件的配製與確立................66 4-2-6 MEKC的檢量線製作.....................................73 4-3 間接UV吸收法...........................................76 4-3-1 間接法電泳之探討......................................77 4-3-2 背景溶液的UV吸收光譜..................................78 4-3-3 間接法電泳液的條件確立與配製...........................79 4-3-4 間接UV吸收法檢量線製作................................84 4-3-5背景電解質濃度對偵測極限的影響..........................86 4-3-6 真實樣品的應用:蜂蜜中葡萄糖的偵測......................88 4-4 三種方法的比較與討論....................................96 第五章 結論................................................98 參考文獻...................................................99 附錄

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