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研究生:張國鍾
研究生(外文):Kuo-Chung Chang
論文名稱:線上取樣,稀釋,及注射裝置結合高效液相層析儀分析固定化纖維酵素水解紙張纖維素之醣類產物
論文名稱(外文):Analysis of Carbohydrate Products in Immobilized Cellulase Hydrolysate of Paper Cellulose by On-line Sampling, Dilution, and Injection Device Coupled High-performance Liquid Chromatography
指導教授:鄭建業鄭建業引用關係
指導教授(外文):Cheanyeh 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:2006
畢業學年度:94
語文別:中文
論文頁數:79
中文關鍵詞:酵素水解紙張纖維素木糖甘露糖纖維雙糖葡萄糖固定化纖維酵素稀釋及注射阿拉伯糖液相層析質譜儀半乳糖高效液相層析儀分析固相萃取線上取樣
外文關鍵詞:and injectionhigh-performance liquid chromatography aon-line samplingdilution
相關次數:
  • 被引用被引用:1
  • 點閱點閱:255
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  • 下載下載:4
  • 收藏至我的研究室書目清單書目收藏:1
本研究利用一種自行設計的線上直接取樣、稀釋及注射之裝置結合懸掛式固定化纖維酵素反應器與高效能液相層析儀,成功的對紙張纖維素之纖維酵素水解液中三種主要醣類產物,即葡萄糖、木糖與纖維雙糖進行分析。以此系統,能對紙張纖維素酵素水解反應進行96小時穩定的監測。並藉由對三種主要醣類產物分析的準確度、精確度、偵測極限值及系統誤差的減少來比較四種定量分析方法,證明以線上取樣稀釋之內部標準品校正曲線分析方法是最佳的定量方法。此方法在分析葡萄糖產物的準確度可達到99.9 %,相對標準偏差值可低於2.4 %,而其偵測極限值可達到2.1 ppm。除此之外,亦發現在使用折射率偵測器製作標準品校正曲線時,其與分析試樣的間質相配性是相當重要的。
我們也發現在紙張纖維酵素水解反應過程中,亦會有包括半乳糖、阿拉伯糖、甘露糖等之微量醣類產生。待反應結束後,水解液中此些微量醣類產物可透過非線上固相萃取去除其中的鹽類與間質,即可以液相層析質譜儀配合離子排斥層析管柱予以分析。液相層析質譜儀的連接介面及離子化方式為電噴灑法,其微量醣類的液相質譜儀定量分析是成功的,並證明出水解反應液內確實含有半乳糖、阿拉伯糖、甘露糖等微量糖產物。
A specific designed device featured with on-line direct sampling, dilution, and injection has been utilized to couple a vertically hanging immobilized cellulase bioreactor with the high-performance liquid chromatography (HPLC) system to make a successful analysis of the three major carbohydrate products, glucose, xylose, and cellobiose in the cellulase hydrolysate in this research. This analysis system could successively monitor the enzymatic hydrolysis of paper cellulose over a period of 96 hours and the monitoring stability was very good. Four different quantification methods for analysis of the three major carbohydrate products were compared and the internal standard calibration method with on-line dilution procedure was proved to be the best quantitative method in terms of the accuracy, precision, limit of detection, and reduction of systematic error. With this method, the analysis for glucose had an accuracy of 99.9%, the relative standard deviation (RSD) below 2.4%, and the limit of detection of 2.1 ppm. In addition, matrix matching between the reaction sample and standard solution was found extremely important in the preparation of standard calibration curves with the use of refractive index detection.
We also found that the trace carbohydrate products including galactose, arabinose, and mannose were produced during the enzymatic hydrolysis of paper cellulose. When the hydrolysis was ended, the salt and matrix in the enzymic hydrolysate was eliminated by the solid-phase extraction (SPE). Then, these trace carbohydrates were analyzed by liquid chromatography-mass spectrometry (LC-MS) coupled with an ion exclusion chromatographic column. The interface and ionization method of LC-MS was the eletrospray ionization (ESI). The concentration of the three trace carbohydrates was successfully determined by the LC-MS method, and the trace carbohydrate products galactose, arabinose, and mannose were demonstrated that they were existed in the hydrolysate indeed.
本文目次
中文摘要 I
英文摘要 II
誌謝 IV
目錄 V
圖目錄 VIII
表目錄 IX
第一章 緒論 1
1.1 前言 1
1.2 文獻回顧 2
1.2.1 紙張之成分 2
1.2.2 纖維素之纖維酵素水解作用 3
1.2.3 以結合性高效液相層析分析醣類產物 7
1.2.4 醣類產物之前處理 10
1.2.5 醣類產物之定量分析 11
1.3 研究目標 13
第二章 實驗 15
2.1 實驗材料 15
2.1.1 藥品及試劑 15
2.1.2 消耗器材 16
2.2 儀器設備 16
2.3 實驗步驟 20
2.3.1 廢棄紙張之前處理 21
2.3.2 纖維酵素之固定化 21
2.3.3懸掛式固定化纖維酵素水解紙張纖維素發酵槽反應 22
2.3.3.1 反應前廢紙纖維素溶液的處理 22
2.3.3.2 懸掛式固定化纖維酵素與廢紙纖維素反應 22
2.3.4 固定化纖維酵素水解紙張纖維素之醣產物分析 25
2.3.4.1 醣產物之線上取樣、稀釋與注射裝置的設計 25
2.3.4.2 醣類產物分析的標準品校正曲線製作 25
2.3.4.2.1 線上稀釋之內部標準品校正曲線製作 28
2.3.4.2.2 非線上稀釋之內部標準品校正曲線製作 30
2.3.4.2.3 線上稀釋之外部標準品校正曲線製作 30
2.3.4.2.4 非線上稀釋之外部標準品校正曲線製作 30
2.3.5醣產物HPLC分析的精密度 31
2.3.6 醣產物之折射率偵測器偵測極限 31
2.3.7醣產物HPLC分析的準確度 32
2.3.8 纖維酵素水解液除鹽及反應間質之前處理 33
2.3.9 纖維酵素水解液之微量醣產物液相層析質譜儀分析 33
第三章 結果與討論 35
3.1 特別設計之線上取樣、稀釋及注射裝置 35
3.2 反應間質在標準品校正曲線上的影響 35
3.3 四種校正曲線定量方式的醣產物偵測極限 36
3.4 紙張纖維素之酵素水解液主要醣產物之間續性監測 42
3.5 三種主要醣產物之定量分析結果比較 48
3.5.1 主要醣產物產量以四種校正曲線定量結果之比較 48
3.5.2 四種校正曲線定量方式之精確度比較 48
3.5.3 四種校正曲線定量方式的準確度 49
3.5.4 線上稀釋與非線上稀釋對定量分析結果的影響 51
3.6 纖維酵素水解液的微量次要醣產物液相層析質譜儀分析 53
3.6.1 纖維酵素水解液固相萃取前處理與HPLC分析 53
3.6.2 液相層析電噴灑游離質譜儀之微量醣類產物分析 58
第四章 結論 60
參考文獻 63
簡歷 69

圖目次
圖一、纖維酵素水解纖維素之β-1,4-葡萄配糖鍵產生β-D-葡萄糖。 5
圖二、纖維酵素對纖維素水解的作用機序。 6
圖三、醣類產物與diol固相吸附體之作用方式。 12
圖四、HPLC系統裝置簡圖。 17
圖五、發酵槽裝置簡圖。 19
圖六、懸掛內含固定化纖維酵素膠囊之尼龍網袋裝置。 23
圖七、懸掛式固定化纖維酵素反應器之裝置圖 24
圖八、特別設計之線上直接取樣、稀釋及注射之注射裝置。 26
圖九、以特別設計之注射裝置結合HPLC分析系統與懸掛式固定化纖維酵素水解纖維素反應系統裝置圖。 27
圖十、四種標準品校正曲線之製作流程圖。 29
圖十一、連續稀釋之標準品校正曲線圖。 37
圖十二、稀釋一倍之外部標準品校正曲線圖。 38
圖十三、稀釋一倍之內部標準品校正曲線圖。 39
圖十四、紙張纖維素水解反應液之線上稀釋監測層析圖譜。 43
圖十五、紙張纖維素水解之醣類產物間續性監測定量曲線圖。 44
圖十六、未去除水解液內鹽類及間質之層析圖譜。 54
圖十七、以乙氰去除水解液內大部分鹽類及間質之層析圖譜。 55
圖十八、以乙氰去除水解液內大部分鹽類及間質及固相萃取前處理後之樣品濃縮液層析圖譜。 56
圖十九、以乙氰去除水解液內大部分鹽類及間質及固相萃取前處理後以蒸餾水洗出滯留醣產物層析圖譜。 57

表目次
表一、線上稀釋與非線上稀釋之校正曲線的線性回歸方程式及其線性相關係數值(r2)。 40
表二、HPLC分析系統對醣類產物及內部標準品(木糖醇)的四種校正曲線定量方式偵測極限值。 41
表三、(A)非線上稀釋與(B)線上稀釋之外部標準品校正曲線定量結果。 46
表四、(A)非線上稀釋與(B)線上稀釋之內部標準品校正曲線定量結果。 47
表五、HPLC分析系統對醣類產物及內部標準品(木糖醇)之標準品添加的回收百分率。 50
表六、醣產物外部標準品與內部標準品校正曲線法之線上稀釋與非線上稀釋的定量結果配對t-測試。 52
表七、微量醣類產物之液相層析電噴灑游離質譜儀定量結果。 59
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