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研究生:陳其嵩
研究生(外文):Chi-Sung Chen
論文名稱:線上除鹽及洗沖時間差之二維高效液相層析持續分析固定化酵素水解廢棄纖維素之醣產物
論文名稱(外文):On-line Desalting and Successive Analysis of Carbohydrate Products from Immobilized Enzyme Hydrolysis of Waste Cellulosic Materials by Elution-time-difference 2-D HPLC
指導教授:鄭建業鄭建業引用關係
指導教授(外文):Chean-Yeh Cheng
學位類別:碩士
校院名稱:中原大學
系所名稱:化學研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:102
中文關鍵詞:線上除鹽及分析纖維素枯樟樹枝葡萄糖纖維雙糖固定化酵素廢紙二維高效液相層析半纖維素木糖半乳糖阿拉伯糖甘露糖
外文關鍵詞:immobilized enzymecellobiosexylosegalactosemannosearabinosehemicellulasewaste camphor tree branchcellulasewaste paperglucosetwo dimensional high-performance liquid chromatographyon-line desalting and analysis
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富含纖維素及半纖維素之廢紙與枯樟樹枝分別收集後,使用纖維酵素與半纖維酵素予以酵素水解產生如葡萄糖或木糖等幾種醣產物,進而可發酵生成有用之生質燃料或化學儲料酒精。因水解產生之醣產物結構相似不易以傳統之液相層析技術同時乾淨的分離回收,故建構聯結氫離子型陽離子交換管柱與鉛離子型離子排斥管柱之二維高效液相層析系統,並與混合固定化酵素反應器線上結合,再發展核心切割與洗沖時間差之管柱轉換技術,配合無汙染之去離子水移動相,進行線上除鹽與六種水解醣產物(纖維雙糖、葡萄糖、木糖、半乳糖、甘露糖與阿拉伯糖)之持續分析與回收,且有效縮短分析時間。
六種醣產物之定量方法為使用木醣醇當作內標準品之內部標準品檢量線法,六種醣類標準品濃度皆為0至2000 μg mL-1,所得醣產物檢量線之線性迴歸係數範圍為0.9984至0.9999之間。六種醣產物(纖維雙糖、葡萄糖、木糖、半乳糖、甘露糖與阿拉伯糖)之偵測極限值分別為0.8、1.9、3.6、4.9、0.1與1.4 μg mL-1,以標準品添加回收實驗所得分析準確度分別為96.9%、99.0%、102.2%、95.1%、94.2%和90.4%,而主要之醣產物-葡萄糖有很好的精確度(RSD)範圍為0.06% – 3.56 %,其餘之醣產物(木糖、纖維雙糖、甘露糖與阿拉伯糖)之精確度(RSD) 範圍為0.01 – 11.43%。因此所發展的洗沖時間差二維高效液相層析具高精確性、準確性、靈敏性、可進行產物回收,並符合綠色環保之要求。定量分析結果顯示廢棄紙張中所含之纖維素較枯樟樹枝為多,而枯樟樹枝中則含較多之半纖維素。
Waste paper and camphor tree branch which contain abundant cellulose and hemicellulose were collected separately and hydrolyzed enzymatically to produce several carbohydrate products such glucose and xylose. These carbohydrate products can be further fermented to produce the biomass fuel like ethanol and other chemical feedstocks. Because the structures of the carbohydrate products are all similar that is difficult to separate and recover with traditional liquid chromatography technique. Therefore, a two dimensional high-performance liquid chromatography (2D-HPLC) which was formed by connecting a H+ ion cation-exchange column and a Pb2+ ion ion-exclusion column was developed for on-line desalting and successive analysis of the six carbohydrate products (cellobilose, glucose, xylose, galactose, mannose, and arabinose) by using the combination of the heart-cut technique and the elution-time-difference technique. Together with the use of deionized distilled water as the mobile phase, the six carbohydrate products can be recovered cleanly and the analysis time can also be reduced by this on-line 2D-HPLC system. The 2D-HPLC system was also on-line hyphenated to a combined immobilized cellulase and immobilized hemicellulase bioreaction system.
The quantitative analysis was made by the internal standard calibration method and xylitol was used as the internal standard. The linear correlation coefficient of the six internal standard calibration curves were in the range 0.9984-0.9997 that corresponds to a concentration range of 0-2000 μg mL-1. The limit of detection obtained from the six calibration curves of cellobiose, glucose xylose, galactose, mannose, and arabinose are 0.8, 1.9, 3.6, 4.9, 0.1, and 1.4 μg mL-1, respectively. The analysis accuracy from spike experiments for cellobiose, glucose, xylose, mannose, and arabinose of were 96.9%, 99.0%, 102.2%, 94.2%, 95.1% and 90.4%, respectively. The quantification precision (RSD) of glucose was from 0.06% to 3.56% and the quantification precision (RSD) of other carbohydrate products (xylose, cellobiose, galactose, mannose, and arabinose) were from 0.01% to 11.43%. This elution-time-difference 2-D HPLC exhibits high precision, accuracy, sensitivity, and can recover the carbohydrate products cleanly that fulfills the requirement of the green technology The quantitative results demonstrate the major component of the waste paper fiber is cellulose. The amount of cellulose in the waste paper is more than that in the waste camphor tree branch fiber. The waste tree branch fiber contains more hemicelluloses than that in the waste paper fiber.
目錄
中文摘要
英文摘要
誌謝
目錄
圖目錄
表目錄
第一章 緒論
1.1 前言
1.2高等植物之枝幹成份
1.3 纖維酵素與半纖維酵素之水解作用
1.4 高效液相層析技術分析醣類
1.5 二維液相層析與洗沖時間差技術
1.6 醣類的定量分析
1.7 研究目標
第二章 實驗部份
2.1 實驗材料
2.1.1 藥品及試劑
2.1.2 消耗器材
2.2 儀器設備
2.3 實驗步驟
2.3.1 廢紙之前處理步驟
2.3.2 枯樹枝之前處理步驟
2.3.3 酵素固定化
2.3.4 固定化酵素水解廢紙或枯樹枝纖維素之搖瓶反應
2.3.4.1 反應前廢紙纖維素或枯樟樹枝纖維素溶液之處理
2.3.4.2 固定化酵素水解枯樹枝纖維素之理想pH值
2.3.4.3 固定化酵素水解枯樹枝纖維素之理想溫度
2.3.5 懸掛式固定化酵素之批次反應水解廢紙纖維素或枯樟樹枝纖維素
2.3.5.1 反應前廢紙纖維素或枯樟樹枝纖維素溶液之處理
2.3.5.2懸掛式固定化酵素之廢紙纖維素或枯樟樹枝纖維素水解反應
2.4 水解醣產物之分析
2.4.1 醣產物之線上取樣、過濾與注射裝置
2.4.2 二維高效液相層析系統及分離的條件
2.4.3 二維高效液相層析系統最適管柱切換時間
2.4.4 線上內部標準品檢量線法之檢量線製備
2.4.5 持續線上二維高效液相層析對固定化酵素水解廢紙及枯樹枝之醣產物之分析
2.4.6 折射率偵測器對醣類產物之偵測極限值(LOD)與定量極限值(LOQ)
2.4.7 醣類產物分析之精確度
2.4.8醣類產物之二維高效液相層析系統分析準確度
第三章 結果與討論
3.1 氫離子型陽離子交換層析與鉛離子型離子排斥層析之醣產物分離分析
3.2氫離子型陽離子交換層析管柱串聯鉛離子型離子排斥層析管柱之二維高效液相層析系統
3.3氫離子型陽離子交換層析管柱結合鉛離子型離子排斥層析管柱之洗沖時間差二維高效液相層析
3.4洗沖時間差二維高效液相層析系統切換時間之找尋與比較
3.5 pH值與溫度對固定化混合酵素活性之影響
3.6 不同基質效應對內部標準品檢量線的影響
3.7 折射率偵測器對不同基質之醣產物偵測極限值與定量極限值
3.8混合固定化酵素水解廢紙纖維素之線上二維層析醣產物持續性監測
3.9混合固定化酵素水解枯樟樹枝纖維素線上二維層析之醣產物持續性監測
3.10洗沖時間差之二維液相層析系統對混合固定化酵素水解纖維素質定量分析之精確度
3.11洗沖時間差之二維液相層析系統對混合固定化酵素水解纖維素質定量分析之準確度
第四章 結論與展望
參考文獻
簡歷

圖目錄
圖一、β-D-葡萄糖之結構與以其為單體聚合之纖維素結構。
圖二、纖維酵素對纖維素水解作用之反應機制
圖三、二維高效液相層析系統
圖四、桌上型固定化酵素反應系統
圖五、懸掛於壓克力環之內含褐藻酸鈣固定化混合酵素膠囊的尼龍網袋裝置
圖六、線上取樣、稀釋及試樣承載裝置。
圖七、以特別設計之注射裝置結合二維高效液相層析系統與懸掛式固定化酵素水解纖維素反應系統裝置圖
圖八、氫離子型陽離子交換層析分離六種糖標準品之圖譜
圖九、鉛離子型離子排斥層析分離六種糖類標準品之圖譜
圖十、管柱串聯之二維高效液相層析系統分離六種糖標準品之圖譜
圖十一、氫離子型陽離子交換層析管柱結合鉛離子型離子排斥層析管柱之洗沖時間差二維高效液相層析系統示意圖
圖十二、洗沖時間差之氫離子型陽離子交換層析管柱串聯鉛離子型離子排斥層析管柱之二維高效液相層析圖譜
圖十三、固定化混合酵素與廢紙纖維素水溶液在45℃及不同pH值搖瓶反應四天之結果
圖十四、固定化混合酵素與廢紙纖維素水溶液在pH 5.5及不同溫度搖瓶反應四天之結果
圖十五、廢紙纖維素基質之內部標準品檢量線圖
圖十六、枯樟樹枝纖維素基質之內部標準品檢量線圖
圖十七、廢紙纖維素水解反應32小時之線上二維高效液相層析監測圖譜
圖十八、木糖醇之外標準品檢量線圖。x軸為濃度,y軸為波峰面積。
圖十九、混合固定化酵素水解廢紙纖維素反應四天之線上二維層析持續監測各醣產物定量曲線圖
圖二十、枯樟樹枝纖維素水解反應24小時之線上二維高效液相層析監測圖譜
圖二十一、混合固定化酵素水解廢紙纖維素反應四天之線上二維層析監測各醣產物定量曲線圖

表目錄
表一、二維高效液相層析系統轉換閥切換時間之ANOVA比較
表二、氫離子型陽離子交換層析管柱結合鉛離子型離子排斥層析管柱之洗沖時間差二維高效液相層析系統兩轉換閥切換時間
表三、固定化混合酵素與廢紙纖維素水溶液在45℃以不同pH值搖瓶反應四天之醣產物產量及酵素活性分析結果
表四、固定化混合酵素與廢紙纖維素水溶液在pH = 5.5以不同溫度搖瓶反應四天之醣產物產量及酵素活性分析結果
表五、廢紙纖維素基質與枯樟樹枝纖維素基質之內部標準品檢量線的線性廻歸方程式及其線性相關係數值(r2)
表六、廢紙纖維素基質與枯樟樹枝纖維素基質之內部標準品檢量線的偵測極限值(LOD)及定量極限值(LOQ)
表七、混合固定化酵素水解廢紙纖維素反應四天之線上二維層析監測定量結果
表八、混合固定化酵素水解枯樟樹枝纖維素反應四天之線上二維層析監測定量結果
表九、洗沖時間差之二維液相層析系統對醣產物定量分析之標準添加的百分回收率
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