臺灣博碩士論文加值系統

化學發光可以廣泛地應用在檢測金屬離子、抗氧化劑、以及有機化合物上，因此我們針對Luminol發光系統使用氧化劑N–溴丁二內亞醯胺，並以停止流光譜儀加以偵測，同時也利用�牷V環糊精來增強整個化學發光系統。N–溴丁二內亞醯胺與其他無機氧化物相較下，具有無色及較高靈敏度的優點。
本研究發現�牷V環糊精能有效地增強N–溴丁二內亞醯胺氧化Luminol之化學發光。在pH 10.5，N-溴丁二內亞醯胺（12.5μM）氧化Luminol（10μM）呈現化學發光非常微弱；添加2.5mM的�牷V環糊精可以使化學發光強度增加73倍。推測應是化學發光中間體或者產物在過程中與�牷V環糊精生成的穩定錯合物造成化學發光增強。我們也針對pH值、各種試劑濃度、溫度對化學發光的影響做系統性的探討。pH值對化學發光呈現鐘形曲線關係，而在pH 10.5有最強的化學發光。當Luminol濃度為0μM ~ 140μM，N-溴丁二內亞醯胺濃度為0μM ~ 20μM，及�牷V環糊精濃度在0mM ~ 7mM時，化學發光強度明顯皆呈現增加，直到高濃度時才出現飽和而偏離。而當溫度從15℃~35℃對於化學發光強度影響增加了3.8倍。
將化學發光系統應用到抗氧化劑L-ascorbic acid、trolox以及3-異丁基-4-羥基苯甲醚（BHA）的偵測。抗氧化劑會在化學發光反應中消除自由基，而造成化學發光強度減弱。Trolox與BHA濃度在0μM ~ 65μM時，化學發光皆呈現線性遞減。L-ascorbic acid在濃度0μM ~ 35μM時，化學發光呈現線性遞增，而75μM ~ 125μM時呈現線性遞減。大多數胺基酸在濃度為50μM時化學發光有明顯的抑制效果，所以本發光系統經過層析分離後可以用來偵測各種胺基酸，半胱胺酸（Cys）濃度在0μM ~ 50μM時，與其它胺基酸有明顯的不同。

Chemiluminescence（CL） has wide analytical applications for the determination of metal ions, antioxidants, and organic compounds. Stopped–Flow spectrometry was employed to determine the CL of luminol system using N-bromosuccinimide（NBS）as the oxidizing agent. We have also investigated the enhancement of CL emission by �牷V cyclodextrin（�牷VCD）. The main advantages of NBS over other common inorganic oxidants are that these oxidants are colorless, provides higher sensitivity.
In this study, we have found that the CL emission from the Oxidation of luminol with NBS is dramatically enchanced by the presence of �牷VCD. Oxidation of luminol（10μM）with NBS（12.5μM）at pH 10.5 exhibited only extremely weak CL emission; however, the CL intensity was increased by 73 folds upon addition of 5 mM �牷VCD. The CL enhancement is probably due to the stabilization of the CL intermediate(s) and/or product through complex formation with �牷VCD, thereby increasing the overall CL efficiency. We have also investigated the effects of pH, concentrations of various reagents, and temperature on the CL emission. The pH dependence of CL emission is bell-shaped with a maximum intensity occurred at pH 10.5. The CL intensity increased significantly with the concentration of luminol（0 ~ 140 μM）, NBS（0 ~ 20 μM）, and �牷VCD（0 ~ 7 mM）and leveled off at high concentrations of these reagents. The CL intensity increased by 3.8 folds over the temperature range of 15℃to 35℃.
The CL system has been applied to the determination of antioxidants L-ascorbic acid, trolox, and 3-tert-butyl-4-hydroxyanisole（BHA）. The antioxidants scavenge the radicals involved in the CL reaction, resulting in a decrease in CL emission. The linear dynamic range for trolox and BHA is 0 ~ 65 μM. For L-ascorbic acid, a linear increase in CL intensity was observed over 0 ~ 35 μM and linear reduction of CL intensity occurred at 75 ~ 125 μM. Most amino acid also showed a significant inhibition of CL emission at a concentration 50μM. This CL system allows the determination of the amino acid after a chromatographic separation. On the other hand, cysteine can be determined over concentration of 0 ~ 50μM without significant interferences form other amino acids.

目 錄…………………………………………………………...1
圖 目 錄……………………………………………...……….4
表 目 錄……………………………………………………...12
胺基酸中英對照………………………………….………....13
中文摘要……………………………………………………...14
Abstract………………………………………….........……...16
第一章 緒論………………………………………………..18
第一節 化學發光(Chemiluminescence，CL)簡介……………18
1.1.1 簡述化學發光………………………….………..……18
1.1.2 化學發光原理…………………………….…………..20
1.1.3 以N-Bromosuccinimide為氧化劑之化學發光簡介..22
第二節 Luminol發光系統之反應機制……………………....24
第三節 環糊精與化學發光……………………….…………..28
1.3.1 環糊精……………………………………..………….22
1.3.2 環糊精的結構……………………………….………..29
1.3.3 環糊精的性質………………………………...………32
第四節 研究動機………………………………...……………33
第二章 實驗部分…………………………...……….…….35
第一節 實驗藥品與溶液配製……………………..………….35
2.1.1 實驗藥品…………………………………………...…35
2.1.2 溶液的配製…………………………………………...37
第二節 實驗儀器……………………………………………...38
第三節 實驗方法……………………………………………...40
2.3.1 停止流光譜儀的原理………………………………...40
2.3.2 實驗儀器SF-61 DX2簡介…………………………..42
第三章 實驗結果………………………………………….47
第一節 N-Bromosuccinimide氧化Luminol產生化學發光...47
3.1.1 酸鹼度對發光強度的影響…………………………...48
3.1.2 環糊精對發光強度的影響………………………..….50
3.1.3 氧化劑NBS濃度對發光強度的影響……………….56
3.1.4 Luminol濃度對發光強度的影響…………………..59
3.1.5 溫度對發光強度的影響……………………………...61
第二節 其他環糊精對本化學發光系統之影響……………...63
第三節 方法應用……………………………………………...66
3.3.1 偵測胺基酸…………………………………………...67
3.3.2 偵測抗氧化劑………………………………………...73
第四章 結論與未來展望………………………………...85
第一節 結論…………………………………………………..85
第二節 未來展望……………………………………………..88
第五章 參考文獻…………………………………………89

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