- 學位論文
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
On-line Desalting and Successive Analysis of Carbohydrate Products from Immobilized Enzyme Hydrolysis of Waste Cellulosic Materials by Elution-time-difference 2-D HPLC
摘要
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。
並列摘要
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.