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研究生: 李勻鑲
Li, Yun-Siang
論文名稱: 毛細管區帶電泳結合線上直接光解產生ABTS陽離子自由基後衍生系統進行掃除自由基偵測之研究
On-line UV Direct Photolysis of ABTS for Analysis of Radical Scavengers by Capillary Zone Electrophoresis
指導教授: 吳劍侯
Wu, Chien-Hou
口試委員:
學位類別: 碩士
Master
系所名稱: 原子科學院 - 生醫工程與環境科學系
Department of Biomedical Engineering and Environmental Sciences
論文出版年: 2009
畢業學年度: 98
語文別: 中文
論文頁數: 102
中文關鍵詞: ABTS陽離子自由基直接光解毛細管區帶電泳後衍生抗氧化能力
外文關鍵詞: ABTS radical cation, direct photolysis, capillary zone electrophoresis, post-column, antioxidant capacity
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  • ABTS•+(2,2’-azinobis-(3-ethylbenzothiazoline-6- sulfonate acid) radical cation)去色分析不僅被廣泛地使用在抗氧化能力分析上,自從ABTS•+與層析分離技術結合後,對於環境分析領域相關的應用更具重要價值。本研究主要的目的為提供一個線上產生ABTS•+試劑作為CZE後衍生系統用以分析樣品抗氧化能力。本實驗利用UV 254 nm光源直接光解ABTS產生非常穩定的ABTS•+,並嘗試建立一套線上光氧化裝置,利用PTFE tubing線圈形成流動式光反應器(flow-through photoreactor)作為後衍生分析系統。此系統除了可簡化操作程序,還可以快速地在線上產生ABTS•+以降低逆反應之情形。經由批次直接光解實驗,在pH 3,提供飽合溶氧或不特意供氧的條件下,結果皆得到產率大約70%的ABTS•+,因此本實驗採用後者條件應用於線上流動式光反應器進行線上直接光解產生ABTS•+,其產率也可達70%。CZE結合光氧化後衍生系統,可以簡單有效地分離五種混合樣品(L-ascorbic acid, D-isoascorbic acid, sorbic acid, benzoic acid, gallic acid)並同時偵測其抗氧化能力,電泳分離時間不超過18分鐘,其抗氧化能力分析的偵測極限約0.58 µM(進樣量~84 nL),經換算VCEAC值與文獻相符合。綜上所述,本研究建立之CZE-UV/ABTS系統具有快速、簡單且能夠線上即時產生ABTS•+試劑等優點,並已成功分析真實樣品之抗氧化能力。


    Abstract
    2, 2’-azinobis-(3-ethylbenzothiazoline-6- sulfonate acid) radical cation (ABTS•+)
    decolorization assay is one of the most widely used methods for analyzing the
    antioxidant capacity(AOC) in biological and environmental samples. A separation
    technique liquid chromatography combining with ABTS•+ post-column detection step
    has become a useful analysis approach. In this study, we present an on-line generated
    ABTS•+ as post-column with capillary electrophoresis for antioxidant capacity
    analysis. Ultraviolet (UV) irradiation at 254 nm was directly photolyed colorless
    ABTS to form blue-green colored ABTS•+. Furthermore, PTFE tubing coil was use to
    constitute a flow-through photoreactor as an on-line direct photolysis device. This
    system can simplify operating sequence and generate rapidly on-line ABTS•+ to
    reduce reverse reaction. Whether purging O2 or not, about 70% production of ABTS•+
    was obtained individually both in batch and on-line direct photolysis experiments.
    Based on the on-line assay results, the CZE-UV/ABTS system has good precision and
    post-column signal was very stable at various pHs. The system can successfully
    separate five compounds and measure their antioxidant capacities simultaneously. The
    migration time was smaller than 18 min and detection limitedwas about 0.58 μM
    (injection sample: 84 nL). The antioxidant capacity value of compounds shows a
    good agreement with reported data. In conclusion, the CZE-UV/ABTS system is
    feasible to determine the antioxidant capacity of real samples.

    中文摘要 ……………………………………………………………..…. I 英文摘要 ……………………………………………………………..…. II 謝誌 …………………………………………………………………..…. III 目錄 …………………………………………………………………..…. IV 圖目錄 …………………………………………………………………….. VII 表目錄 ………………………………………………………………………. X 第一章 前 言……….……………………………………………….…. 1 1.1 抗氧化能力..………………………………………..………….…. 1 1.1.1 抗氧化能力之量測………………………………….……… 2 1.1.1.1 評估抗氧化能力的方法………………………….…… 4 1.1.1.2 抗氧化能力之表示……………………………….…… 9 1.2 ABTS•+去色方法…………………………………………….….… 12 1.2.1 ABTS•+的產生方式…………………………………………… 13 1.2.2 光化學產生ABTS•+……………………..…………………… 15 1.2.3 ABTS•+在環境分析上之應用……………………………… 17 1.3 抗氧化物量測於層析(chromatography)之應用…………………… 19 1.3.1 應用液相層析進行線上抗氧化能力分析………………… 19 1.3.2 毛細管電泳在抗氧化物分析之發展……………………… 20 1.3.2.1 毛細管電泳簡介………………………………….…… 20 1.3.2.2 毛細管電泳原理………………………………….…… 21 1.3.2.3 毛細管電泳在抗氧化物分析上之應用………….…… 25 1.4 研究動機及目的…………………………………………….…… 27 第二章 實驗內容…………………………………………..……….…… 28 2.1 儀器設備與藥品配製……………………………….……….…… 28 2.1.1 儀器設備與材料……………………………………….…… 28 2.1.2 試劑藥品……………………………………………….…… 30 2.1.3 標的分析物簡介………………….………………………… 31 2.1.4 母液配製…………………………………………….……… 33 2.2 直接光解開放式、密閉式與線上實驗……………...…………… 34 2.2.1 開放式光反應(批次)裝置與實驗程序…………..……… 34 2.2.2 封閉式光反應(批次)裝置與實驗程序………………….. 36 2.2.3 線上光反應裝置與實驗程序…………………………...…… 37 2.3 毛細管電泳結合線上光氧化後衍生系統……………………….… 39 2.3.1 毛細管清洗方式………………..……………………...…… 44 第三章 結果與討論.……………………………………………………… 45 3.1 光譜分析…………………………………………………….……… 45 3.1.1 ABTS之光解與ABTS•+之生成……………………………… 45 3.1.2 ABTS質子化與初始ABTS濃度調整……………..………..… 46 3.1.3 ABTS•+生成濃度與被分解ABTS濃度之推估……………..… 47 3.2 直接光解實驗最佳化參數之探討………………………….……… 51 3.2.1 不同光源……………………………………………………… 51 3.2.2 光照強度……………………………………………………… 52 3.2.3 反應物初始濃度……………………………………………… 55 3.2.4 pH值…………………………………………………………… 56 3.2.5 溶氧………..………….……………………………………… 58 3.2.6 ABTS•+之穩定性探討………………………………………… 59 3.2.7 線上光反應線圈最佳長度之探討…………………………… 61 3.2.8 批次與線上光反應槽材質效率之探討……………………… 62 3.3 CZE結合線上光氧化後衍生系統之連線分析.…………………… 63   3.3.1 後衍生界面元件連接方式之改良…………………………… 63 3.3.2 pH對後衍生系統偵測之影響……….…..…………………… 64 3.3.3不同樣品注入量對系統偵測之影響…………….…………… 66 3.3.4 ABTS濃度對後衍生系統偵測之影響……..………………… 68 3.3.5最佳分析條件圖譜與後衍生偵測極限…………..………… 69 3.3.6系統再現性…………..……....……………..………………… 73 3.3.7真實樣品分析………………...………..……………………… 74 第四章 結論與展望…………………………..…………………..…..…… 82 參考文獻…………………………………………………………..…..…… 83 附錄……………………………………………………………………..…… 96 附錄一 不同光強度下,ABTS在340 nm和415 nm吸收峰之變化…...... 96 附錄二 調酸時使用HCl與HClO4之比較………………………………… 97 附錄三 直接光解產生之ABTS•+與CE buffer(pH 9.5)混合後穩定性之影 響……………………………………………...……………………………… 98 附錄四 定性五種抗氧化劑………………...……………………………… 99 附錄五 改變A牌保健食品分析之萃取方式…….……………………… 102

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