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研究生:陳詠志
研究生(外文):chen,yung-chih
論文名稱:以震盪輔助-深共熔溶劑-液液微萃取法快速檢測環境水樣中微囊藻毒素殘留
指導教授:丁望賢丁望賢引用關係
指導教授(外文):Wang-Hsien Ding
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:122
中文關鍵詞:微囊藻毒素深共熔溶劑超高效液相層析串聯質譜儀震盪式輔助-DES-液液微萃取法
外文關鍵詞:MicrocystinDeep Eutectic SolventsUHPLC-QTOF-MSVA-DES-LLME
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優養化是水中植物、藻類過度繁殖所引起,不僅會造成水質惡化及
惡臭,大量繁殖的藻類甚至會產生微囊藻毒素 (Microcystin),而在多種異構體中 Microcystin-LR (MC-LR) 最為普遍且毒性最強。除了暴露於環境水體中,人類也可能透過食物鏈而接觸到微囊藻毒素,故有必要開發一套快速方法檢測水環境中 Microcystin 濃度。

本研究開發出一套快速、有效、低花費、且對環境友善的萃取方法
來檢測水中的微囊藻毒素濃度。利用簡單合成的方式,合成出新穎的
綠色化學溶劑-深共熔溶劑(Deep Eutectic Solvents, 簡稱 DES)作為萃取劑,再利用震盪輔助-DES-液液微萃取(Vortex assisted-DES-liquid-liquid microextraction (VA-DES-LLME)) 進行樣品前處理,並結合超高效液相層析串聯質譜儀 (UHPLC- qTOF-MS) 做定量檢測。並利用實驗設計 (statistical experimental design) 法中的 Box-Behnken design (BBD) 及變異數分析 (analysis of variance, ANOVA) 來做VA-DES-LLME 最佳化的條件探討。
萃取的最佳化條件為:將5 mL 去離子水或水樣放入尖底離心管中,
溶液調成酸性,加入 900 µL DES 和 1.4 mL THF,使用 vortex 震盪 50 s ,以 5000 rpm 的轉速離心 5 分鐘,取上層溶液加入甲醇定容為 1 mL,取其中 2 µL 進樣 UHPLC- qTOF-MS 進行檢測。
本實驗 MC-LR 及 MC-YR 線性範圍為 1-100 μg/L,決定係數皆
0.999 以上,定量極限 (LOQ) MC-LR 與 MC-YR 分別為 0.4 與 0.5
ng/mL,精密度以相對標準差 (RSD) 表示,皆小於 11%,準確度以回
收率表示,在 100 至 126 %間,顯示此方法穩定且具有良好的再現性。然在目前所收集的環境水樣檢測中,微囊藻毒素的含量皆低於本方法的偵測極限。
The eutrophication is caused by the over-breeding of plants and algae in the rivers and lakes, which not only causes deterioration of water quality and leading to malodor, but also produces microcystins in various isoforms. Among them, the most common and toxic microcystins is Microcystin-LR (MC-LR). In addition to exposure to aquatic system, humans may also be exposed to microcystins through the food chain, so it is necessary to develop a rapid and reliable method for the determination of microcystin residues in surface water. This study developed a fast, efficient, low-cost and environmentally friendly extraction method to detect two common detected microcystins: MC-LR and MC-YR, in surface water samples. Firstly, using a simple method to produce deep eutectic solvents (DESs), a group of novel “green” solvents, as an extractant, and then Vortex assisted-DES-liquid- liquid microextraction (VA-DES-LLME) was employed for sample preparation and combined with Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS) was used for quantitative detection. The parameters of VA-DES-LLME were optimized by multivariate statistic Box-Behnken design (BBD) and analysis of variance (ANOVA). The optimal extraction conditions were: 5 mL of water sample was placed in a sharp-bottomed centrifuge tube, and the solution was made acidic (pH = 2). DES (900 μL) and THF (1.4 mL) were added, and vortexed for 50 s, and then centrifuged at 5000 rpm for 5 minutes. The supernatant was reconstituted with methanol to a volume of 1 mL, and then 2 μL of extractant was directly injected into UHPLC-qTOF-MS for quantitative detection.
The linear ranges of MC-LR and MC-YR was 1-100 μg/L, the coefficient of determination (r2) was above 0.999, and the limit of quantitation (LOQ) of MC-LR and MC-YR were 0.4 and 0.5 ng/mL, respectively. The relative standard deviation (RSD) was less than 11%, and in terms of recovery, the accuracy were between 100% and 126%. This method displayed to be stable and has good reproducibility. Although, for the collected water samples, the content of MC-LR and MC-YR were lower than those of the LOQs of the method. The developed method is sensitive enough to meet the requirement of the World Health organization (WHO) proposed monitoring program. In this program, the maximum allowance concentration in drinking water of microcystins is 1.0 ng/mL
摘要 3
Abstract iii
謝誌 v
目錄 vii
圖目錄 xiii
表目錄 xv
第一章前言 1
1-1 研究起源 1
1-2 研究目標 3
第二章 文獻回顧 5
2-1微囊藻毒素簡介 5
2-1-1 微囊藻毒素來源結構與性質 5
2-1-2 毒性研究 9
2-1-3 相關檢測文獻 11
2-2超高效液相層析高解析串聯質譜儀簡介 20
2-2-1超高效液相層析高解析串聯質譜儀 20
2-2-2 層析管柱(column) 22
2-3 離子液體(Ionic liquid) 24
2-3-1離子液體簡介 24
2-3-2深共熔溶劑(Deep Eutectic Solvents) 25
2-4 Vortex assisted-DES-liquid-liquid microextraction (VA-DES-LLME) 30
2-4-1前言 30
2-4-2 原理 30
2-4-3分散式液液微萃取法流程 31
2-4-4 影響因素 32
2-4-5 分散式液液微萃取法改良 33
2-5實驗設計 35
2-5-1實驗設計(Design of experiments)簡介 35
2-5-2 反應曲面法 36
2-5-3 Box-Behnken Design 38
2-6 內標(Internal standard) 40
第三章 實驗步驟與樣品分析 41
3-1 實驗藥品與設備 41
3-1-1 實驗藥品 41
3-1-2 儀器設備 43
3-2 實驗步驟 44
3-2-1 標準品配置 44
3-2-2超高效液相層析串聯質譜儀參數設定 45
3-2-3質量校正 46
3-2-4 實驗流程 47
Vortex assisted-DES-liquid-liquid microextraction 47
固相萃取法 48
3-2-5深共熔溶劑的合成 49
3-3 樣品採集 49
第四章 結果與討論 51
4-1超高效液相層析高解析度串聯式質譜儀對微囊藻毒素待測物的檢測 51
4-1-1微囊藻毒待測物檢測 51
4-1-2待測物之質譜圖 52
4-1-3檢量線及偵測極限 53
4-2 VA-DES-LLME(Vortex Assisted DES-Liquid-Liquid Microextraction)最佳化條件探討 54
4-2-1 深共熔溶劑種類探討 54
4-2-2深共熔溶劑比例的探討 55
4-2-3震盪方式比較 56
4-2-4 pH值探討 57
4-2-5 深共熔溶劑體積探討 58
4-2-6 四氫呋喃體積探討 59
4-2-7 震盪時間探討 60
4-2-8 離心時間探討 61
4-2-9 單一因子最佳化結果 62
4-3震盪輔助液液微萃取法實驗設計最佳化探討 63
4-3-1 BBD實驗設計模型 63
4-3-2因子間交互作用力之探討 66
4-3-3殘差分布圖 68
4-3-4 Box-Behnkne Design實驗設計最佳化結果 71
4-4 Mandel test & The Lack-of-Fit test 72
4-4-1 Mandel test 72
4-4-2 The Lack-of-Fit test 73
4-5真實水樣樣品之檢測 74
4-5-1 環境水樣檢測結果 74
4-5-2 方法確效 83
4-5-3 檢測結果比較 84
第五章 結論 87
第六章 參考文獻 89
附錄 95
附錄A F table for α=0.05 95
附錄B 深共熔對於ESI-MS信號的影響 96
附錄C T test 98
附錄D DES結構鑑定 99
附錄E石門水庫採樣點 102
附錄F中央大學中大湖採樣點 103
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