Determination of tannic acid based on on-line optical detection system

摘要 本研究針對茶品中單寧酸總量檢測與茶品中單寧酸與蛋白質凝集造成澀味感受進行探討，分別建立了螢光消光流動系統與光散射流動系統。 在螢光消光流動系統中，能有效避免維他命C 與溫度變化的干擾，並擁有對單寧酸高度選擇性與檢測靈敏等優點。在茶品實測的應用面上，本系統的線性範圍為濃度0.0-1.0 mg/ml，R2 ＝ 0.9928，且分析結果與日本公定法具有高度相關性（R2 ＝ 0.9803）。 在光散射流動系統中，SDS 洗劑清洗程序能有效的改善訊號之背景值漂移與重複性不佳等實驗誤差，徹底清除吸附於管壁的單寧酸與BSA 之凝集分子，成功建立一套能即時檢測與即時清洗的線上感測系統。系統檢測下限為20 μg/ml，上限為300 μg/ml。此系統能有效以單寧酸與蛋白質的凝集情形得知澀味等生理活性。Two on-line systems have been setup in this research for determination of tannic acid content in green tea infusion and biological activation of protein and tannic acid aggregation in tea. In fluorescence quenching method with flow-injection system which has been applied for detection of real samples within a linear concentration range of 0.0-1.0 mg/ml and a R2 of 0.9928. The method is immune from the interference of the coexisting ascorbate in green tea infusion and temperature changing, and the analytical results were verified by Japanese official analytical method, ferrous tartrate method. In light scattering method with flow-injection system, experiment error such as drift of baseline and lacking of reproducibility can be solved effectively by cleaning procedure with SDS reagent for the effective removal of residual aggregation particles by tannic acid and BSA. Therefore, an on-line detection system with real time detection and cleaning ability have been setup successfully. The range of detection is 20-300 μg/ml, and astringent activation in tea can be known by aggregation of tannic acid and BSA目錄 封面····················································································································i 口試委員簽名單·······························································································ii 誌謝··················································································································iii 摘要··················································································································iv 英文摘要··········································································································· v 目錄 圖目錄 表目錄 一、前言··········································································································· 1 二、文獻探討 2.1 單寧酸的化學結構··············································································· 3 2.2 單寧酸的定量法··················································································· 8 2.2.1 縮合單寧定量法··········································································· 8 2.2.2 水解單寧定量法········································································· 10 2.2.3 單寧總量測定法·················································································11 2.2.4 單寧酸生物活性測定法······································································12 II 2.2.5 單寧酸之流動注射分析法··································································13 2.2.6 本研究之實驗原理··············································································14 三、材料與方法 3.1 實驗藥品製備與實驗儀器································································· 18 3.1.1 實驗藥品····················································································· 18 3.1.2 緩衝液與藥品製備····································································· 18 3.1.3 儀器設備····················································································· 19 3.2 系統架設與實驗方法········································································· 20 3.2.1 螢光消光流動分析系統架設與實驗方法································· 20 3.2.2 光散射流動分析系統架設與實驗方法······································ 22 3.2.3 日本公定綠茶單寧分析法························································· 24 四、結果與討論 4.1 螢光消光法························································································· 25 4.1.1 系統典型響應與系統線性範圍················································· 25 4.1.2 最適化探討················································································· 28 4.1.2.1 不同濃度發光胺對檢測之影響········································· 28 4.1.2.2 pH 值最適化探討································································ 30 4.1.3 溫度與干擾物影響····································································· 32 4.1.3.1 溫度對消光效應之影響····················································· 32 4.1.3.2 維生素C 之影響································································ 34 III 4.1.4 市售綠茶支單寧酸檢測····························································· 36 4.1.4.1 螢光消光法與日本公定法的比對····································· 36 4.1.4.2 茶葉浸置時間與單寧酸釋放量之探討····························· 39 4.2 光散射法····························································································· 41 4.2.1 非自動化檢測系統觀察單寧酸與BSA 之凝集過程················· 41 4.2.2 訊號漂移效應與改善方案·························································· 44 4.2.3 實驗參數探討············································································· 48 4.2.3.1 清洗管長最適化································································· 48 4.2.3.2 系統之典型響應································································· 49 4.2.3.3 BSA 濃度對檢測所造成之影響········································· 51 4.2.3.4 pH 值對檢測所造成之影響················································ 55 4.2.4 茶品實測····················································································· 56 五、結論········································································································· 59 六、參考文獻································································································· 6