由於元素間不同的物種型態具有不同的特性，對於生物體來說其作用也大不相同，因此真實樣品中的物種分析是相當重要的，它可以幫助我們了解不同物種的分布，避免對樣品的性質造成誤判。感應耦合電漿質譜儀（Inductively coupled plasma mass spectrometry，ICP-MS）對於元素分析來說是一靈敏度高、線性範圍廣的分析儀器，適合應用於真實樣品的分析，然而樣品進入ICP後會先經過原子化的步驟，所以在ICP-MS 中無法分辨不同物種，因此做物種分析時必須搭配分離工具進行分離。本研究利用毛細管電泳（Capillary electrophoresis，CE）、液相層析（Liquid chromatography，LC）分別對汞物種及鈷物種進行分離及分析應用。
毛細管電泳具有分離效率高、樣品及試劑量少等優點，因此被廣泛應用於生化、藥物分析及臨床醫學等領域上。第一部分研究係利用毛細管電泳系統透過CEI-100介面結合感應耦合電漿質譜儀於魚肉中的汞物種型態分析之應用，研究使用內徑75 μm、長80 cm的毛細管，以含0.01% m/v L-cysteine之10 mmol L-1Glycine（pH 10.5）作為分離電解質，在17 kV的分離電壓下，於800秒內成功分離甲基汞、乙基汞與無機汞，其滯留時間、波峰面積及波峰高度之再現性皆低於6.6%，甲基汞、乙基汞及無機汞的偵測極限分別為4.0、3.8及3.5 μg L-1。另外，本研究在CEI-100霧化器的輔助溶液中加入甲酸，除了保持電通路外，其碳基質能扮演電荷轉移的角色幫助汞游離，提升訊號，降低偵測極限。在最適化的甲酸濃度下，對汞訊號的S/N約可提升2倍。將本系統應用於樣品上的分析，分別使用兩種標準參考樣品（DORM-2、DOLT-3）及旗魚樣品，萃取方式採用微波輔助萃取，萃取試劑為2% m/v L-cysteine溶液，三個樣品的萃取回收率可達95~99%，添加回收率則在95~106%之間，證實本方法的實用性。
第二部分研究是以液相層析結合薄膜去溶劑系統（Membrane desolvation sample introduction system，ARIDUS）及感應耦合電漿質譜儀，對無機鈷、氰鈷胺素及氫氧鈷胺素等鈷物種分析之應用，本研究並搭配液相層析結合電噴灑質譜儀（Electrospray ionization spectrometry，ESI-MS）對樣品中的鈷物種結構進行結構鑑定。分離管柱採用逆相層析管柱（Reverse phase column），而鈷胺素與管柱作用力強，因此沖提液必須使用高濃度甲醇，但過高的有機溶劑濃度會壓抑分析訊號並使電漿不穩定，所以本篇在LC管柱末端與ICP-MS的連接上採用ARIDUS來解決使用高濃度甲醇的問題，此裝置藉由低流速的霧化器搭配加熱的薄膜系統以及逆向的掃除氣體（Sweep gas），藉以去除溶劑蒸氣，只讓分析物及少量溶劑進入電漿中，因此可以於高有機溶劑環境下穩定的操作ICP-MS。本實驗對動相流速、甲醇濃度及醋酸銨濃度等LC分離條件及ARIDUS系統中操作條件進行最適化後，無機鈷、氰鈷胺素及氫氧鈷胺素等三物種能在六分鐘之內分離完成，滯留時間、波峰面積及波峰高度之再現性皆低於4.2%，偵測極限的範圍於0.008-0.014 μg L-1之間。將以建立的系統應用於綠藻錠及維生素B群補給品中鈷物種之測定，萃取方式採用微波輔助萃取，萃取試劑為0.5% HNO3溶液，萃取效率皆可達98%以上，物種的添加回收率介於91~108%。最後利用液相層析結合電噴灑質譜儀對樣品進行分析，以樣品的二次質譜圖與標準品二次質譜圖比對也可以成功鑑定樣品中鈷物種的結構。

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目錄
論文摘要..............................................................................Ⅰ
謝誌......................................................................................Ⅲ
目錄......................................................................................Ⅳ
圖表目錄..............................................................................Ⅵ
第一章 毛細管電泳結合感應耦合電漿質譜儀於魚肉中汞物種型態分析應用

壹、汞.............................................................................................1
貳、毛細管電泳.....................................................................1
參、毛細管電泳與ICP-MS介面...........................................4
肆、汞物種分析的方法.........................................................7
伍、碳基質對元素增益的機制.............................................8
陸、實驗部分
一、儀器裝置...................................................8
二、試藥與溶液的配製.................................10
三、實驗操作.................................................11
柒、結果與討論
一、甲酸濃度對汞訊號的影響.....................14
二、毛細管電泳分離條件最適化.................17
三、毛細管電泳對甲基汞與無機汞分離條件再次探討..............................................................................32
四、真實樣品分析應用.................................38
捌、結論..............................................................................47
玖、參考文獻......................................................................48

第二章 液相層析結合感應耦合電漿質譜儀於鈷物種型態分析應用

壹、鈷..........................................................................................52
貳、鈷物種分析方法..........................................................56
參、薄膜去溶劑進樣裝置及其與液相層析之介面..........57
肆、電噴灑質譜儀..............................................................59
伍、實驗部分
一、儀器裝置......................................................................61
二、試藥與溶液的配製......................................................63
三、實驗過程......................................................................64
陸、結果與討論
一、液相層析與ARIDUS之介面設計..........70
二、液相層析分離條件之最適化.................70
三、薄膜去溶劑進樣系統操作的最適化.....76
四、重複性、檢量線與偵測極限.................81
五、電噴灑質譜儀操作參數最適化.............88
六、真實樣品的分析.....................................94
柒、結論............................................................................105
捌、參考文獻....................................................................106
 
圖表目錄
第一章 毛細管電泳結合感應耦合電漿質譜儀於魚肉中汞物種型態分析應用

圖1-1 毛細管電泳系統儀器示意圖...........................3
圖1-2 Laminar flow和flat flow流動模式的比較......5
圖1-3 CEI-100於CE與ICP-MS之間連接介面之儀器示意圖.................................................................................6
圖1-4 輔助溶液中甲酸濃度對無機汞訊號的變化.15
圖1-5 分離甲基汞及無機汞時，照射UV光對訊號的影響......................................................................................16
圖1-6 汞物種與L-cysteine之錯合反應..................18
圖1-7 不同種類之電解質對分析物分離之影響.....19
圖1-8 分離電解質之pH值對電泳分離之影響........21
圖1-9 分離電解質之濃度對電泳分離之影響.........22
圖1-10 毛細管長度對電泳分離之影響.....................25
圖1-11 分離電壓對電泳分離之影響.........................26
圖1-12 進樣時間對電泳分離之影響.........................27
圖1-13 經最適化條件後，CE-ICP-MS 系統對乙基汞、甲基汞及無機汞之電泳圖..........................................28
圖1-14 毛細管長度對甲基汞及無機汞的分離影響..........................................................................................33
圖1-15 分離電壓對甲基汞及無機汞分離之影響.....34
圖1-16 進樣時間對甲基汞及無機汞分離之影響.....35
圖1-17 經最適化條件後，CE-ICP-MS系統對甲基汞及無機汞分離之電泳圖......................................................36
圖1-18 魚肉標準參考樣品（NRCC DORM-2）的電泳分離圖..............................................................................42
圖1-19 魚肝標準參考樣品（NRCC DOLT-3）的電泳分離圖..............................................................................43
圖1-20 旗魚樣品的電泳分離圖.................................45

表1-1 ICP-MS與CE系統之實驗條件參數..............29
表1-2 汞物種的遷移時間及訊號的再現性.............30
表1-3 汞物種之校正曲線參數及偵測極限.............31
表1-4 ICP-MS與CE系統對甲基汞和無機汞分離之實驗條件參數......................................................................37
表1-5 甲基汞及無機汞之校正曲線參數及偵測極限..........................................................................................39
表1-6 以CE-ICP-MS測定標準參考樣品中汞物種含量及其回收率的結果..........................................................40
表1-7 以CE-ICP-MS對旗魚樣品進行汞物種定量..........................................................................................44
表1-8 實驗結果與其他文獻之比較.........................46

第二章 液相層析結合感應耦合電漿質譜儀於鈷物種型態分析應用

圖2-1 維生素B12的結構..........................................54
圖2-2 （A）維生素B12結構（B）偽維生素B12結構..........................................................................................55
圖2-3 薄膜去溶劑進樣系統之構造圖.....................58
圖2-4 電噴灑過程示意圖.........................................60
圖2-5 HPLC-ARIDUS-ICP-MS之系統示意圖.......62
圖2-6 實驗流程圖.....................................................67
圖2-7 微波輔助萃取的流程圖.................................69
圖2-8 改變分流比例對訊號的影響.........................71
圖2-9 動相流速對鈷物種分離之影響.....................73
圖2-10 甲醇濃度對鈷物種分離之影響.....................74
圖2-11 NH4OAc濃度對鈷物種分離之影響.............75
圖2-12 使用ARIDUS進樣系統探討Sweep gas flow rate對鈷分析訊號的影響...................................................77
圖2-13 使用ARIDUS進樣系統探討Nitrogen gas flow rate對鈷分析訊號的影響..........................................78
圖2-14 使用ARIDUS進樣系統探討霧化器溫度對鈷分析訊號的影響.................................................................79
圖2-15 使用ARIDUS進樣系統探討薄膜溫度對鈷分析訊號的影響.....................................................................80
圖2-16 HPLC-ARIDUS-ICP-MS系統經最適化後對三個鈷物種之層析圖..........................................................84
圖2-17 毛細管施加電壓的改變對OH-Cbl訊號的影響..........................................................................................89
圖2-18 毛細管施加電壓的改變對CN-Cbl訊號的影響..........................................................................................90
圖2-19 HPLC-ESI-MS之EIC及二次質譜圖.............92
圖2-20 綠藻錠樣品在不同萃取試劑之萃取效率.....95
圖2-21 綠藻錠樣品之層析圖.....................................97
圖2-22 維生素B群＃1樣品之層析圖........................99
圖2-23 維生素B群＃1的二次質譜圖......................100
圖2-24 維生素B群＃2樣品之層析圖......................102
圖2-25 維生素B群＃2之二次質譜圖......................103

表2-1 微波消化設定條件.........................................68
表2-2 ICP-MS、LC、ARIDUS系統之實驗條件參數..........................................................................................82
表2-3 鈷物種的滯留時間及訊號的再現性.............84
表2-4 鈷物種之校正曲線參數及偵測極限.............85
表2-5 實驗結果和其他文獻之比較.........................87
表2-6 ESI-MS系統最適化操作條件........................91
表2-7 三種營養補給品樣品中鈷之萃取效率.........96
表2-8 以LC-ICP-MS測定市售營養補給品中鈷物種含量及回收率的結果........................................................104

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