本研究目的是建立一個利用含釕赤血鹽(ruthenium hexacyanoferrate)來偵測咖啡因的系統。利用催化劑在pH 1環境中，將原本咖啡因的氧化電位約在1.54V下降至1.10V(vs.Ag/AgCl)。而催化劑的電位會受到環境pH值影響造成偏移，最低還可以下降至0.945V
(vs.Ag/AgCl)。此系統經最佳化探討後，操作電位1.15V下，其偵測線性範圍從0.2μM至10μM，靈敏度為 298.05nA/μM，本系統偵測極限為 42.27nM (S/N=3)，對於1μM的咖啡因重複操作的相對標準偏差為2.86%(n=20)。最後利用HPLC將星巴克的美式咖啡分離出咖啡因，偵測器為本研究的催化電極， HPLC的分離條件為，流動相為75% 100mM pH 1 PBS和25%的70%乙腈混和，分離管柱為InertSustain AQ-C18 5um 4.6 x 250mm，流速為1ml/min，約7.7分鐘分離出來。

The purpose of this study was to establish a system that uses catalysts of ruthenium hexacyanoferrate to detect caffeine. The oxidation potential of caffeine with ruthenium hexacyanoferrate was reduced to about 1.10 V at about 1.54V (vs. Ag/AgCl) in pH 1. And the potential of ruthenium hexacyanoferrate is affected by the environmental pH, which can be reduce to 0.945V (vs. Ag/AgCl). After optimization of this system, the operating potential is 1.15V, the linear range is 0.2μM to 10μM, the sensitivity is 298.05nA/μM, and the detection limit is 42.27nM (S/N=3). The relative standard deviation of the 1 μM caffeine repeat operation was 2.86% (n=20). Finally, the caffeine was separated in sample of caffé Americano(starbucks) by HPLC. And the detector use the system of this study. The caffeine was separated by a mobile phase of 75% 100 mM pH 1 PBS and 25% 70% acetonitrile, the column was InertSustain AQ-C18 5um 4.6 x 250m and flow rate was 1 ml/min by HPLC. And the caffeine was separated in 7.7 minutes.

總目錄
總目錄	iii
圖目錄	v
表目錄	vii
第一章 緒論..............................................1
1-1偵測咖啡因之重要性....................................1
1-2咖啡因(caffeine)(1,3,7-Trimethylpurine-2,6-dione)....2
1-3咖啡因的分離方法......................................5
1-4咖啡因的偵測方法......................................8
1-5高效液相層析系統.....................................14
1-6 混價化合物之赤血鹽..................................23
1-7 本研究之目的........................................25
第二章 實驗步驟.........................................25
2-1儀器................................................25
2-2藥品................................................26
2-3實驗步驟與設計.......................................27
2-3-1系統操作電位的探討.................................30
2-3-2初步電解質酸鹼值的探討.............................30
2-3-3電解質的濃度的探討.................................30
2-3-4修飾時KCl濃度的探討................................31
2-3-5修飾時pH值的探討...................................31
2-3-6分析特性評估.......................................31
2-3-7偵測咖啡因在HPLC上之應用評估........................32
第三章 結果與討論.......................................32
3-1工作原理的探討.......................................32
3-2最佳化條件的探討.....................................36
3-2-1系統操作電位.......................................43
3-2-2電解質酸鹼值.......................................45
3-2-3電解質的濃度.......................................47
3-2-4 修飾時KCl濃度.....................................48
3-2-5修飾時pH值.........................................49
3-2-6轉速的探討.........................................51
3-3分析特性的探討.......................................53
3-4偵測咖啡因在HPLC上之應用..............................61
3-5結論................................................66
參考文獻................................................68
 
圖目錄
圖 1咖啡因之化學結構.....................................4
圖 2電化學氧化咖啡因可能的氧化機制........................12
圖 3六向注射閥原理......................................17
圖 4滯留時間關係圖......................................19
圖 5分析物在分離管柱中之分散.............................20
圖 6解析度與波峰之關係圖.................................23
圖 7 ruthenium hexacyanoferrate的生成情形...............28
圖 8 ruthenium hexacyanoferrate玻璃碳電極的循環伏安法....29
圖 9 ruthenium hexacyanoferrate玻璃碳電極偵測咖啡因的循環伏安法...................................................29
圖 10 空白玻璃碳電極偵測咖啡因的循環伏安法................34
圖 11 ruthenium hexacyanoferrate玻璃碳電極的循環伏安法，在不同pH環境下..............................................35
圖 12 pH值對催化劑氧化電位作圖...........................35
圖 13 本系統所依據的偵測原理及反應機制....................36
圖 14 ruthenium hexacyanoferrate旋轉圓盤玻璃碳電極，pH 1.0環境下.................................................38
圖 15 ruthenium hexacyanoferrate旋轉圓盤玻璃碳電極，pH 2.0環境下.................................................39
圖 16 ruthenium hexacyanoferrate旋轉圓盤玻璃碳電極，pH 3.0環境下.................................................40
圖 17 ruthenium hexacyanoferrate旋轉圓盤玻璃碳電極，pH 4.0環境下.................................................41
圖 18  ruthenium hexacyanoferrate旋轉圓盤玻璃碳電極，pH 5.0環境下.................................................42
圖 19 ruthenium hexacyanoferrate旋轉圓盤玻璃碳電極，pH 7.0環境下.................................................43
圖 20咖啡因偵測系統之操作電位的探討......................45
圖 21電解質酸鹼值探討...................................46
圖 22咖啡因偵測系統之磷酸鹽電解質濃度的探討...............48
圖 23 探討修飾電極時不同KCl之濃度所形成的ruthenium hexacyanoferrate對咖啡因催化電流的影響...................49
圖 24探討修飾電極時不同pH值所形成的ruthenium hexacyanoferrate對咖啡因催化電流的影響...................51
圖 25 轉速的二分之一對電流作圖的探討......................52
圖 26圖轉速的負二分之一開根號對電流的倒數作圖的探討........53
圖 27 咖啡因偵測系統校正曲線.............................54
圖 28咖啡因偵測系統之再現性..............................55
圖 29 偵測系統之干擾物測試...............................57
圖 30偵測系統之混和干擾物測試............................58
圖 31偵測系統之混和干擾物測試長條圖.......................59
圖 32 咖啡因在干擾物中的偵測.............................62
圖 33 咖啡在HPLC中的偵測................................65
圖 34 咖啡和混和干擾物的疊圖在HPLC中.....................66
表目錄
表 1偵測咖啡因之氧化電位、線性及偵測極限比較...............56
表 2於最佳化條件下所得分析結果...........................60

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