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

Analysis of Carbohydrate Products in Immobilized Cellulase Hydrolysate of Paper Cellulose by On-line Sampling, Dilution, and Injection Device Coupled High-performance Liquid Chromatography

張國鍾(Kuo-Chung Chang)

指導教授 ： 鄭建業

中原大學/理學院/化學系/碩士(2006年)

https://doi.org/10.6840/cycu200600634

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

and injection ； high-performance liquid chromatography a ； on-line sampling ； dilution

參考文獻

. 鄭建業，廢紙的前處理及發酵研究，中國化學會，1990年9月第48卷第3期，中華民國，台北市。

. 吳敬謙，懸掛式固定化纖維酵素反應器之廢紙纖維素水解與醣類產物分析，中原大學化學研究所碩士論文，2003年7月。

. C.-C. Wu and C. Cheng. A study of the hydrolysis of waste paper cellulose with a vertically hanging immobilized cellulase reactor and the reuse of the immobilized cellulase. Journal of Chinese Chemical Society. 2005;52:85-95.

. M. Aliyu, M.J. Hepher. Effect of ultrasound energy on degradation of cellulose material. Ultrasonics Sonochemistry. 2000;7:265-268.

. R.C. Sun, J. Tomkinson, P.L. Ma , S.F. Liang. Comparative study of hemicelluloses from rice straw by alkali and hydrogen peroxide treatments. Carbohydrate Polymers. 2000;42:111-122.