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研究生：	石浚旻 Chun-Min Shih
論文名稱：	大體積進樣之毛細管電泳法的開發與研究 Large-Volume Sample Injection in Capillary Electrophoresis
指導教授：	林震煌 Lin, Cheng-Huang
學位類別：	碩士 Master
系所名稱：	化學系 Department of Chemistry
論文出版年：	2005
畢業學年度：	93
語文別：	中文
論文頁數：	136
中文關鍵詞：	毛細管電泳、大體積進樣
英文關鍵詞：	capillary electrophoresis, large-volume sample injection
論文種類：	學術論文
相關次數：	點閱：148 下載：0
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為了更加提高毛細管電泳法的偵測靈敏度，本研究開發出兩種線上濃縮技術，分別為大小管結合線上掃集法（Coupled-capillary/ sweeping-MEKC, CC/sweeping-MEKC）及整管進樣堆積結合線上掃集法（Full-capillary sample stacking/sweeping-MEKC, FCSS/sweeping- MEKC）。在大小管結合線上掃集方法中，藉由管徑較大部分為進樣端，樣品進樣量可達1.8 L。其中，因為毛細管大小內徑的不均等，會造成電場的不均勻性（大管徑部分：低場強；小管徑部分：高場強）。當SDS微胞在大管徑部分進行掃集時，由於電場不均勻性的影響下，使分析物以非常緩慢的速率逐漸被微胞掃集起來，因此可進一步增加濃縮效率。另一方面，整管進樣堆積結合線上掃集法結合了stacking、sweeping、dynamic pH-junction、MEKC四種技術，而達成整管樣品進樣（最大體積）之目的，其中樣品進樣量可達2.7 L。與MEKC做比較，以上兩種方法在偵測靈敏度上分別有500倍及350倍的改進效果。

In an attempt to improve the sensitivity of detection in capillary electrophoresis, two novel online sample concentration methods were developed, coupled-capillary/sweeping-MEKC (CC/sweeping-MEKC) and full-capillary sample stacking/sweeping-MEKC (FCSS/sweeping- MEKC). In the former case, compared to a single capillary, a larger volume of sample solution can be injected (up to 1.8 L), since the ID of capillary used is wider. Furthermore, when the coupled-capillary is used, an unequal-electric field (wide portion: lower field strength; narrow portion: higher field strength) along with the capillary axis is created. When the SDS micelles passed through the coupled-capillary, the analytes are gradually and slowly swept to the wide portion. Hence, the analytes can be swept more efficiency. In the latter case, four techniques, including stacking, dynamic pH-junction, sweeping, MEKC, were combined to acquire a larger sample injection. As a result, a full column injection could be achieved (up to 2.7 L). Compared to the normal MEKC mode, the limit of detection can be improved by 500- and 350-fold, respectively, when the CC/sweeping-MEKC and FCSS/
sweeping-MEKC methods are applied.

中文摘要..………………………………………………………… I
英文摘要…………………………………………………………………… II
目錄………………………………………………………………… III
圖目錄…………………………………………………………… VI
表目錄…………………………………………………………… VIII

第一章  緒論…………………………………………………………… 1
1-1   研究目的………………………………………….………….… 1
1-2   分析物簡介…………..……………………………….………. 3
      1-2-1  多巴胺（Dopamine）……………………………… 3
      1-2-2  正腎上腺素（Norepinephrine）………………….. 4
      1-2-3  色氨酸（Tryptophan）與異白氨酸（Isoleucine）….………. 5
1-3   發光二極體簡介…………………………………….……….. 6

第二章  分析方法及原理…………………………………….…..…. 9
2-1   毛細管電泳之發展歷程…...………………..……….…... 9
2-2   毛線管電泳之基本原理…..…………………………………. 13
      2-2-1  電泳遷移率……………………………………….... 13
      2-2-2  電滲流（EOF）………………………………………………. 15
      2-2-3  管柱分離效率…………………………………………...…. 19
2-3   毛細管電泳法的分離模式..………………………………….…...…. 20
      2-3-1  毛細管區帶電泳（CZE）……………………………..…...…. 21
      2-3-2  微胞電動層析法（MEKC）………………………...……….. 23
2-4   毛細管線上濃縮技術……..………………...……………………….. 27
      2-4-1  毛細管電泳線上堆積法（stacking）……………………...…. 29
      2-4-2  毛細管電泳線上掃集法（sweeping）…………...…………. 31
      2-4-3  陽離子選擇完全注射掃集法（CSEI-sweeping）………….. 33
2-5   大體積進樣技術之改良…..…………………………………………. 35
      2-5-1  大小管結合線上掃集法
 （Coupled-capillary/sweeping-MEKC）..………….….…….. 36
      2-5-2  冰溫輔助大小管線上掃集法
 （LTB/coupled-capillary/sweeping-MEKC）…..…….….…. 39
      2-5-3  整管進樣堆積結合線上掃集法
 （FCSS/sweeping-MEKC）………...…………….….………. 41
2-6   NDA（naphthalene-2,3-dicarboxaldehyde）衍生反應……….……... 44

第三章  儀器、藥品與實驗方法…………………………………………... 46
3-1   自組式毛細管電泳/紫光LED誘導螢光分析儀..…………………... 46
3-2   儀器與周邊設備列表…………..……………………………………. 48
3-3   藥品列表………..……………………………………………………. 50
3-4   NDA衍生物製備…………..……………………………………….... 52

第四章  研究過程與結果討論…………...................................................... 53
4-1   NDA衍生反應之探討……..…........................................................... 53
4-2   光源及衍生物之光譜……..……….………………………………… 55
      4-2-1  衍生物光譜性質之測量…….................................................. 55
      4-2-2  紫光LED發光光譜之測量…................................................. 55
      4-2-3  NDA衍生物紫光LED光譜之比較...................................... 56
Part I  大小管結合線上掃集法
（Coupled-capillary/sweeping-MEKC）…………......................... 61
4-3   單一形式之毛細管（normal capillary）………….............................. 61
 4-3-1  微胞電動層析法（MEKC）之電泳條件探討…………......... 61
4-3-1-1  最佳化MEKC電泳條件確立…………..................... 61
4-3-1-2  MEKC檢量線製作…………...................................... 67
      4-3-2  線上掃集法（sweeping-MEKC）之電泳條件探討……….... 70
4-3-2-1  最佳化sweeping-MEKC電泳條件確立.................... 70
4-3-2-2  Sweeping-MEKC檢量線製作..................................... 75
4-4   大小管徑接合之毛細管（coupled-capillary）..................................... 78
 4-4-1  大小管之製作.......................................................................... 78
 4-4-2  線上掃集法（sweeping-MEKC）之電泳條件探討................ 79
4-4-2-1  最佳化sweeping-MEKC電泳條件確立.................... 79
4-4-2-2  Sweeping-MEKC檢量線製作..................................... 86
 4-4-3  MEKC檢量線製作.................................................................. 89
4-5   Single capillary與coupled-capillary在掃集（sweeping）模式下
      之比較.................................................................................................. 92
4-6   不同比例coupled-capillary在掃集（sweeping）模式下之比較....... 94
4-7   大小管之效應……….…………….…………………………………. 96
4-7-1  電場不均勻效應（unequal electric field effect）…………….. 96
4-7-2  大小管誘導堆積效應
（wide-to-narrow induced stacking effect）…………….…… 99
4-8   不同管徑組合之掃集（sweeping）測試……………….………….. 102
Part II  冰溫輔助大小管線上掃集法
（LTB/coupled-capillary/sweeping-MEKC）………….……...... 106
4-9   掃集技術應用於DA-CBI和NE-CBI……........................................ 106
      4-9-1  單管（single capillary/sweeping-MEKC）………………... 106
      4-9-2  大小管（coupled-capillary/sweeping-MEKC）……............ 109
      4-9-3  混合界面活性劑（mixed surfactant/coupled-capillary/ sweeping-MEKC）……………………………………….... 111
      4-9-4  冰溫輔助（LTB/coupled-capillary/sweeping-MEKC）……. 113
          4-9-4-1  冰溫槽（low temperature bath）之裝置及操
                  作流程…………………………………………….... 113
          4-9-4-2  最佳進樣長度之測量…………………………….... 114
          4-9-4-3  LTB/coupled-capillary/sweeping-MEKC
                  檢量線製作……………………………………….... 117
Part III  整管進樣堆積結合線上掃集法
（Full-capillary sample stacking/sweeping-MEKC）............... 121
4-10   CZE模式下不同pH值之影響……….………….……………….. 121
4-11   FCSS（full-capillary sample stacking）模式之測試....................... 124
4-12   Normal-MEKC與FCSS/sweeping-MEKC之比較.......................... 126

第五章  結論…………………………………………………………….... 129

論文發表………............................................................................................ 131
參考文獻........................................................................................................ 132
                                

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