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研究生: 謝敏琦
Min-Chi Hsieh
論文名稱: 毛細管微胞電泳層析掃集濃縮法結合77 K低溫螢光光譜分析對紅葡萄酒中微量逆轉醇即時光譜的鑑定與研究
On-line identification of trace trans-resveratrol in red wine using a sweeping technique combined with capillary electrophoresis/77 K fluorescence spectroscopy
指導教授: 林震煌
Lin, Cheng-Huang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2004
畢業學年度: 92
語文別: 中文
論文頁數: 95
中文關鍵詞: 毛細管微胞電泳層析77 K低溫螢光光譜
論文種類: 學術論文
相關次數: 點閱:131下載:4
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  • 本研究完成首次結合線上濃縮和毛細管電泳層析低溫螢光光譜偵
    測法,並同時確認紅葡萄酒中逆轉醇77 K低溫的光譜。
    利用sweeping-MEKC毛細管電泳層析法進行線上濃縮並分離逆轉
    醇,當逆轉醇到達偵測窗口,瞬時將溫度降至77 K,測量on-line螢光光
    譜,並和標準品的77 K低溫螢光光譜作指紋比對。
    偵測紅酒萃取物中的逆轉醇最佳分離條件為matrix 20 mM
    H3PO4,分離緩衝溶液內含30 mM H3PO4、150 mM SDS,溶液中甲醇
    和水的比例為25:75 (v/v)。和normal-MEKC毛細管電泳層析法相比較,
    使用sweeping-MEKC的毛細管電泳層析法,可以在偵測靈敏度上增加
    1500倍。最低偵測極限是5 ppb (S/N = 3),偵測條件為激發螢光波長313
    ± 8 nm,吸收螢光波長400 ± 16 nm,使用濾光片濾掉320 nm以下的光
    線。
    利用毛細管微胞電泳層析濃縮掃集,結合on-line 77 K 螢光光譜偵
    測法,不只可在紅酒萃取物中分離並偵測逆轉醇,且77 K低溫的線上
    螢光光譜,更可確認逆轉醇在紅葡萄酒中的真實存在。

    The feasibility of combining the techniques of on-line concentration
    and capillary electrophoresis/low temperature fluorescence spectroscopy
    (CE/LTFS) in the detection and identification of trans-resveratrol in red
    wine at 77 K is demonstrated for the first time.
    This technique, involving sweeping-micellar electrokinetic chromatog
    -raphy (sweeping-MEKC), was used for the initial on-line concentration
    and separation, after which, a cryogenic molecular fluorescence experiment
    was performed at 77 K.
    The complete, optimal separation of trans-resveratrol from a red wine
    extract (in a sample matrix (an aqueous 20 mM H3PO4 solution)) could be
    achieved with phosphate buffer(30 mM) containing SDS (150 mM) in a
    methanol -water solution (25:75 v/v) , and a 1500-fold improvement in
    detection sensitivity was obtained compared with the normal-MEKC
    method. The limit of detection is 5 ppb (LOD). (S/N = 3). The excitation
    wavelength was 313 ± 8 nm; the emission was measured at 400 ± 16
    nm(with 320 nm cut filter).
    The proposed method permits, not only the separation and detection
    of trans-resveratrol from red wine extracts, but also ensures that the on-line
    spectrum is readily distinguishable and can be un-ambiguously assigned at
    77 K.

    第一章緒論…………………………………………………….……….01 1-1 分析物簡介…………………………………………….…………..01 1-1.1 Resveratrol 結構……………………..…………...…..…..03 1-1.2 逆轉醇為什麼能抗癌?.…….……………………...……….04 1-1.3 紅葡萄酒的抗氧化力與地區有關?…..………………...….06 1-2 研究目的…………………………………………………..……….08 第二章研究原理和方法………………………...………..........……….11 2-1 毛細管電泳層析法的發展…………………...……………………11 2-2 毛細管電泳層析法的分離原理………………...…………………16 2-2.1 電泳的分離與遷移率……………………..…………..…....16 2-2.2 Zeta 電位與電滲流…………...…….….…….……...…....18 2-2.3 電分散作用…………………………..…………...…….......24 2-2.4 分離效率及解析度……………………...…………….…....26 2-3 毛細管電泳層析法的分離模式…………..…...……………….….27 2-3.1 毛細管區帶電泳層析法(CZE)…………………………....27 2-3.2 微胞電動毛細管層析法(MEKC)…………….……….....28 2-4 毛細管電泳線上濃縮技術……………………………………..….32 2-4.1 毛細管電泳掃集法…………………......…………………..34 2-5 77 K 低溫螢光光譜分析法…………………….…………………..36 第三章儀器及藥品………………………….………………………….40 3-1 自組式毛細管電泳螢光分析儀………….………………………..40 3-2 自組式螢光光譜儀……………………….………………………..42 3-3 毛細管電泳/低溫(77 K)螢光偵測系統……….………………..44 3-4 使用儀器及週邊設備列表……………………….………………..47 3-5 藥品……………………………………………….………………..49 3-5.1 使用藥品列表…………………………………..………….49 3-5.2 藥品配製……………………………………………..….…50 3-5.2.1 標準品的配製…………………………………..…50 3-5.2.2 緩衝液(running buffer)的配製………………….50 3-5.2.3 樣品基質溶液的配製(sample matrix)….………...50 3-5.3 葡萄酒樣品前處理(液-液萃取過程)..……………………51 第四章……………………………………………………………...…...52 4-1 逆轉醇標準品毛細管電泳掃集法(sweeping-MEKC)條件分析...52 4-1.1 界面活性劑濃度對掃集效果的影響………….…………...52 4-1.2 有機溶劑比例對掃集效果的影響………………………..54 4-1.3 matrix 中磷酸濃度對掃集效果的影響…...……...….……...56 4-1.4 進樣長度對掃集效果的影響………………………...…….58 4-1.4.1 虹吸時間的測量………………....……………...…58 4-1.4.2 最佳進樣長度的測量……………………………...58 4-1.5 Normal-MEKC 與sweeping-MEKC 偵測靈敏度比較…..…62 4-1.6 逆轉醇檢量線的製作………………………………………64 4-1.7 Normal-MEKC 和sweeping-MEKC 再現性探討…..………67 4-2 葡萄酒中逆轉醇毛細管電泳掃集法(sweeping-MEKC)條件分 析………………………………………………………………………..69 4-2.1 界面活性劑濃度對掃集效果的影響………………………69 4-2.2 有機溶劑對掃集效果的影響………..……………….…….72 4-2.3 磷酸濃度對掃集效果的影響………………………...…….75 4-2.4 電壓對掃集效果的影響………………………...………….77 4-2.5 葡萄酒中逆轉醇的定量實驗………………………...…….79 4-2.6 葡萄酒中逆轉醇的萃取回收率…………………………....82 4-3 葡萄酒中逆轉醇毛細管電泳掃集法結合on-line 77 K 螢光光譜法 研究…...…………………............................……………………...83 第五章結論與展望………………………………………………..........85 論文發表………………………………………………………………..86 參考資料………………………………………………………………..87 附錄:期刊論文

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