- 學位論文
氣相層析電灑質譜法於矽烷化衍生物之研究與低電滲流壓力輔助雙槽式介面之開發
The Investigation of GC-ESI/MS of TMS Derivative and the Development of Low EOF Hydrodynamic Flow Assisted Double Junction Interface
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摘要
氣相層析與毛細管電泳為成熟之分離技術，具有高分離效率與低樣品消耗等特性，當與質譜銜接時便可成為具有高分離效率與高定性能力之分析技術。本研究探討氣相層析電灑質譜法於矽烷化物之研究以提升其應用潛力，以及開發低電滲流壓力輔助雙槽式介面用於毛細電泳電灑質譜，以改善非揮發性鹽類於電噴灑質譜分析之訊號抑制問題。 常見用於銜接氣相層析與質譜之游離化方法為電子游離法。相較於電子游離法，使用電噴灑游離化法作為氣相層析與質譜銜接之游離化方法時，可得到碎片離子較少之圖譜，因此氣相層析電噴灑游離質譜除了高分離效率與高定性能力之外，可讓訊號集中於分子離子，以提高靈敏度。氣相層析質譜多用於分析低極性之化合物，欲分析高極性的化合物時，分析物需先進行衍生化，使其適合氣相層析分析。眾多衍生化方法中矽烷化為常使用的衍生化方法之一。由於矽烷化衍生物具有易水解的特性，在潮濕的環境中，矽烷化衍生物會水解回未衍生化型態。因此，當氣相層析與電噴灑游離質譜銜接時，矽烷化衍生物於游離化過程中將會水解回到未衍生化高極性形態，更適合電噴灑游離化，進而提升靈敏度。本研究利用氣相層析電灑游離質譜分析不同種類的矽烷化衍生物，包括不利於液相層析分離之有機酸、酚類化合物與結構較複雜之乙型受體素，並探討矽烷化衍生物的水解現象與應用，以提升氣相層析電灑質譜法之應用潛力。 研究發現有機酸的矽烷化衍生物水解效率較低，無法於電噴灑游離過程完全水解。於正離子模式偵測下可以看到未水解訊號，而負離子模式偵測下可以看到水解訊號。從多質子酸的結果中可發現，越多矽烷化衍生基團的有機酸，越不易水解完全。雖然矽烷化有機酸無法水解完全，但部分水解的特性，可於正、負離子掃描模式下提供更多的定性資訊，搭配氣相層析的滯留時間可以提升定性結果之可靠性。 在酚類的探討中得知，不同取代基之酚類衍生物有不同的水解效率，如甲基酚與硝基酚於正離子模式可測得未水解訊號，負離子模式可測得水解之訊號，而氯酚則只有偵測到水解訊號。為了得知氣相層析電灑游離質譜分析矽烷化物之靈敏度，選擇訊號集中於水解訊號之氯酚進行二次質譜分析，除了碎片離子訊號不明顯的氯酚之外，其他氯酚之偵測極限介於 0.25 至 5 ng/mL 之間。 分析結構較為複雜的乙型受體素時，只能偵測到矽烷化乙型受體素之未水解正離子訊號，因此推測其水解效率較有機酸與氯酚來得的低，即便增加噴灑溶液流速，依然無法測到水解訊號。從質譜圖中可得知電噴灑游離化法可明顯降低分子離子被打碎的情形。選擇未水解分子離子訊號以二次質譜進行靈敏度測試，其偵測極限範圍介於 0.5 至 10 ng/mL 之間，也說明即使矽烷化物無法水解，亦可以達到合理的靈敏度。 除了氣相層析之外，毛細管電泳也具有高分離效率。毛細管電泳中常使用非揮發性鹽類做為緩衝溶液添加物來提升分離效率，然而在毛細管電泳電灑質譜分析時，非揮發性鹽類會抑制訊號。本研究利用壓力輔助雙槽式介面避免非揮發性鹽類進入電噴灑游離，以改善訊號抑制的情形。壓力輔助雙槽式介面其組成為液體接合溶液槽以及導電溶液槽，中間利用 1 公分之毛細管進行銜接。操作為利用進樣注射器施加壓力流於分離管與銜接管之間，調控壓力流大小與施加電場方向僅讓分析物隨壓力流往電噴灑頭移動，而非揮發性鹽類往液體接合槽方向回退。 為了提高壓力輔助雙槽式介面之泛用性，將原先設計中所使用的銜接管由熔融石英毛細管更換為聚乙烯醇塗佈毛細管，藉由聚乙烯醇塗佈毛細管電滲流趨近於零的特性，可使銜接管之電滲流降低，讓銜接管中的分析物與添加物只受本身之電淌度與壓力流影響，進行添加物與分析物移動方向的調控，以簡化操作。由於壓力流不受銜接管內電滲流的影響，因此只要掌握添加物與分析物之淌度，便可以利用此介面使其以不同方向移動，以改善訊號抑制的問題。
並列摘要
Gas chromatography (GC) and capillary electrophoresis (CE) are the common analytical techniques, both of them provide high separation efficiency and low sample consumption. The coupling of GC (or CE) with mass spectrometry (MS) provides a technique with high separation efficiency and high selectivity. Among the ionization techniques, electron ionization (EI) is the most commonly used ionization technique in GC/MS. Unlike EI, little fragmentation is observed under electrospray ionization (ESI). Therefore, the GC–ESI/MS combination provides a technique with high separation efficiency, high selectivity and abundant information on the molecular weight of the analyte. GC is an effective separation technique for volatile and nonpolar compounds. However, for polar analytes, derivatization is often required, because it can enhance the separation efficiency of polar compounds. Among the derivatization methods, trimethylsilylation is one of the most widely adopted approaches. A drawback of trimethylsilyl (TMS) derivatives is that they are sensitive to moisture. In aqueous environments, TMS derivatives may hydrolyze back to their native form. After GC separation, the TMS derivatives eluted from the GC column interact with the aqueous charged droplets during ESI analysis. The TMS derivatives may hydrolyze back to their native form and thus become more suitable for ESI analysis. In this work, several types of compounds (organic acids, phenols and beta-agonists) were trimethylsilylated before GC–ESI/MS analysis. The hydrolysis efficiency and application of GC-ESI/MS in TMS-derivatives were studied. Analyzing TMS-organic acids by GC–ESI/MS indicated that hydrolysis was incomplete. Both the TMS derivative ([M+TMS+H]+) and the hydrolysis product ([M-H]−) were detected. According to the results of polyprotic acids, the hydrolysis efficiency was lower with more TMS groups. Although the hydrolysis is incomplete, the detection of a TMS derivative and its hydrolysis product at the same retention time may facilitate compound confirmation. The analysis of TMS-phenols indicated that the hydrolysis efficiency was affected by the functional group attached to phenol. For example, partial hydrolysis was observed for methylphenols and nitrophenols. Both [M+TMS+H]+ and [M-H]− were detected. However, only the hydrolysis product, [M-H]−, was detected for CPs. This result suggested that TMS-CPs eluted from the GC were hydrolyzed back to their native form during the ESI process. To evaluate the limit of detection of GC-ESI/MS analysis of TMS-CPs, collision-induced dissociation (CID) was used. The LOD of TMS-CPs was estimated to be in the range of 0.25–5 ng/mL except for TMS-CPs with poor fragmentation efficiency. In GC–ESI/MS analysis of beta-agonists, only the protonated molecules of TMS-beta agonists were detected. The results suggested that the hydrolysis efficiency of TMS-beta agonists was considerably lower than that of TMS-organic acids and TMS-CPs. No hydrolysis product was detected even after the flow rate of the spray solvent was increased. The ESI mass spectra were dominated by protonated molecule signals with little fragmentation. Under CID, the LODs were estimated to be in the range of 0.5–10 ng/mL, although they did not hydrolyze back to their polar native form during ESI. Besides GC, CE is another separation technique with high separation efficiency. CE often involves adding nonvolatile additives or salts to improve the separation. However, nonvolatile additives are not suitable for ESI-MS, primarily because of their ion suppression effect. In this work, a hydrodynamic flow assisted double junction interface was used to prevent nonvolatile additives from entering the ESI source, to alleviate ion suppression caused by nonvolatile additives in CE-ESI/MS analysis. The hydrodynamic flow assisted double junction interface was fabricated with a liquid junction reservoir and conducting liquid reservoir and a short transfer column (1 cm). The hydrodynamic flow was introduced by a syringe into between the separation column and transfer column. By adjusting the hydrodynamic flow and electric field, the analytes, but not additive, were pushed to the ESI sprayer. To improve the versatility of the hydrodynamic flow assisted double junction interface, a conventional uncoated fused silica transfer column was replaced with a polyvinyl alcohol (PVA) column. Because of PVA-coated column has an extremely low electroosmotic flow (EOF), the velocity of an ion was determined by its electrophoretic mobility and the hydrodynamic flow applied to the interface. EOF no longer plays a role in the velocity of an ion. The migration behavior of the analyte and additive in transfer columns could be controlled by adjusting the hydrodynamic flow applied to the interface to alleviate the problem of ion suppression.
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