臺灣博碩士論文加值系統

本研究使用毛細管電層析分離胜肽。首先使用不同的緩衝溶液探討其對胜肽分離的影響，其中，三氟醋酸常添加於液相層析靜相，可提升胜肽的分離效率，但在電灑法質譜上卻會影響胜肽的離子化效率而抑制其訊號，使用在毛細管電層析上時，也因為添加在鞘流溶液中的三氟醋酸在電灑過程中會一起進入質譜儀而使得胜肽訊號被抑制。因此研究第二部份使用液體結合式界面，藉由改變噴灑電壓及噴頭口徑大小控制噴灑流速，使補償溶液不致進入質譜，進而降低三氟醋酸對胜肽訊號的抑制。然而，此界面的噴灑流速遠大於毛細管電層析的流速，使用在毛細管電層析上時很難達到電灑的最佳流速，或是必須將口徑縮到非常小，造成操作上的不易。所以第三部份使用液體結合式界面加上低流速鞘流界面，將噴灑電壓獨立出來，如此可以同時控制液體結合式界面的電壓使三氟醋酸不會往質譜方向移動並保有電灑的最佳流速，將此界面應用在以三氟醋酸分離胜肽上，可以同時達到好的分離效率並保有胜肽在質譜儀上的靈敏度。

Trifluroacetic acid (TFA) is a commonly used ion-pair reagent in HPLC to improve peak shapes and hence the separation efficiency of peptides. However, TFA is known to cause significant signal suppression when analyzed by electrospray ionization mass spectrometry (ESI-MS). In this study, we try to use TFA as mobile phase additive for capillary electrochromatography (CEC) in the analysis of peptides. Liquid junction interface is used and by manipulating the diameter of the spray tip and the ESI voltage applied, the introduction of make-up solution and TFA within into ESI-MS can be prevented successfully. However, the above interface has limited utility when applied in CEC because the flow rate of the spray tip is much higher than CEC flow rate. In order to match the flow rate of the tip and CEC column the optimized flow rate of ESI will not be achieved. Therefore, a low flow interface is introduced and placed in front of liquid junction interface to separate the ESI voltage, and the voltage applied on the make-up solution can be easily adjusted to match the flow rate of both sides of liquid junction without compromising the ESI optimized flow rate. By using liquid junction-low flow interface, it is possible to employ TFA in CEC for peptide separation and alleviate the suppression effect by TFA at the same time.

第一章 序論 ………………………………………………………1

1-1毛細管電層析發展與原理 …………………………………………1
1-2毛細管電層析/電灑法質譜儀界面…………………………………8
1-3毛細管電層析於胜肽分析…………………………………………13
1-4三氟醋酸於液相層析及毛細管電泳分離胜肽……………………19
1-5三氟醋酸對質譜訊號的抑制………………………………………22
1-6研究目的與方法……………………………………………………25

第二章 實驗 ………………………………………………………35

2-1 藥品…………………………………………………………………35
2-2 實驗裝置……………………………………………………………36
2-2-1 毛細管電層析/質譜儀 ……………………………………36
2-2-2 毛細管電層析管柱製作……………………………………36
2-2-3 低流速鞘流界面製作………………………………………37
2-2-4 液體結合式界面製作………………………………………37
2-2-5 低流速鞘流電灑基座製作…………………………………38
2-3 實驗方法 …………………………………………………………39
2-3-1 實驗前處理…………………………………………………39
2-3-2 低流速鞘流界面實驗條件…………………………………39
2-3-3 液體結合式界面操作………………………………………39
2-3-3-1 實驗條件…………………………………………39
2-3-3-2 加電壓方式………………………………………40
2-3-4 液體結合式-低流速鞘流界面操作 ……………………40
2-3-4-1 實驗條件…………………………………………40
2-3-4-2 加電壓方式………………………………………41

第三章 結果與討論 ……………………………………………49

3-1 以低流速鞘流界面探討不同緩溶液對胜肽分離的影響 ……49
3-2 液體結合式界面 ………………………………………………52
3-2-1 界面測試 …………………………………………………53
3-2-2 以液體結合式界面降低三氟醋酸抑制之概念 …………54
3-2-3 以電滲流直接注入法(EOF infusion)測試概念可行性 56
3-2-4 毛細管電泳分析胜肽 ……………………………………57
3-2-5 毛細管電層析分析胜肽 …………………………………58
3-3 液體結合式-低流速鞘流界面…………………………………60
3-3-1 以液體結合式-低流速鞘流界面降低三氟醋酸抑制之概念60
3-3-2 測試概念的可行性 ………………………………………61
3-3-3 毛細管電層析分析胜肽 …………………………………64

第四章 結論…………………………………………………92

參考文獻 ……………………………………………………93

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