改善三氟醋酸於毛細液相層析電灑法質譜胜肽訊號抑制之研究

Improving Signal Intensity in the Analysis of Peptides with Trifluoroacetic Acid-containing Mobile Phase by Reversed-Phase Capillary Liquid Chromatography-electrospray Mass Spectrometry

李宛俐(Wan-Li Lee)

指導教授 ： 何國榮

國立臺灣大學/理學院/化學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.01635

摘要

在逆相液相層析分離胜肽上常會添加少量的三氟醋酸(TFA)，利用它與帶正電的胜肽有離子對效應，會提升胜肽的疏水性，進而增加胜肽與靜相之間的作用力，使分析物層析峰較對稱，解析度較高。但是在液相層析與質譜儀的銜接上，三氟醋酸會造成胜肽在質譜訊號上的抑制，其原因可能是來自於胜肽與三氟醋酸間很強的離子對關係，使在電噴灑時無法被有效的離子化，加上含三氟醋酸的溶液因具高表面張力和高導電度，會使噴灑不穩定而造成訊號的抑制。
因此，本實驗目的希望達到保有三氟醋酸在液相層析中分離的好處，而又能兼顧在質譜訊號偵測上的靈敏度。首先對三氟醋酸造成胜肽訊號抑制之程度做評估，研究的第二部分則使用實驗室所發展之液體結合式-低流速鞘流界面來嘗試克服三氟醋酸對訊號之抑制。利用在液體結合式界面和低流速鞘流界面分別施加一電壓，探討所產生之電壓差是否可使三氟醋酸停留在液體結合式界面之容槽中而不會進入質譜端，並且對液相層析峰之完整性作一評估。將此界面應用於胜肽的分析上，可以達到好的分離效率並同時保有胜肽在質譜儀上的靈敏度。

關鍵字

三氟醋酸 ； 液相層析 ； 電灑法質譜 ； 訊號抑制 ； 胜肽液體結合式界面 ； 低流速鞘流界面

並列摘要

Trifluoroacetic acid (TFA) is a commonly used ion-pair reagent in RP-HPLC to improve peak shape and enhance separation efficiency of peptides. However, TFA forms very strong ion pairs with peptides that are not broken apart in the process of electrospray ionization and consequently reduces the ionization efficiency of peptides. Moreover, TFA also leads to spray instability and signal reduction due to high conductivity and high surface tension of the mobile phase.
In this work, a low flow-liquid junction interface was used to alleviate the adverse effect of TFA on the peptide signal in ESI/MS. Different voltages are applied to the make-up solution and sheath liquid to create an electrical field between liquid junction and low flow interface. Under acidic conditions, EOF velocity will be lower than the velocity of TFA anion. TFA anions thus turn out to move away from ESI/MS and the sensitivity loss caused by TFA can be reduced. By using this low flow-liquid junction interface, it is possible to employ TFA in LC/MS for peptide separation.

並列關鍵字

TFA ； LC-ESI/MS ； signal suppression ； peptide ； liquid junction interface ； low flow interface

參考文獻

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