臺灣博碩士論文加值系統

目前幾丁質的應用層面相當廣泛，但有鑒於研究者利用幾丁質(chitin)及幾丁聚醣(chitosan)之前，必需先了解原料之特性，包括：乙醯化程度(degree of N-acetylation)及純度，且目前測定乙醯化程度之測定方法雖然繁多，但仍各有缺點。因此本實驗乃就以一次微分紫外光光譜法測定乙醯化程度之應用性提升及設計一套利用染料與幾丁質之吸附計算純度之方法，並建立利用13C-NMR測定幾丁質純度之新方法進行探討。

以一次微分紫外光光譜法測定乙醯化程度，除使用藥品配製簡單、省時之優點外，使用一般實驗室常見之分光光度計測定，因此其普及化程度可大大提升，而其缺點在於醋酸無法溶解乙醯化程度較高之樣品，因此本研究將比較其應用於測定酸酐法回接之較高乙醯化程度之水溶性幾丁聚醣乙醯化程度之準確性，經比較改良式高效能液相層析法與傅立葉紅外光光譜法，結果顯示具良好之相似性。

幾丁質類樣品之純度即幾丁質類樣品中所含幾丁質及幾丁聚醣之總重量佔樣品重量之百分比。將幾丁聚醣溶解於0.5 M HCl glycine buffer，利用幾丁聚醣上所帶胺基與染劑Cibacron brilliant red 3B-A上之SO3¯吸附，經過濾後以分光光度計測定未吸附之染劑含量。計算被吸附之染劑含量、幾丁聚醣上胺基之含量，再配合傅立葉紅外光光譜法測定樣品之乙醯化程度，即可求得樣品之純度。比較染劑吸附法及扣除法所得之純度，其結果並無顯著差異。

另一方面，利用13C-NMR法亦可測定幾丁質類產品之純度，然此法僅可使用於殘留蛋白質含量低於2% 之樣品。

Abstract

The applications of chitin are quite extensive nowdays. Researchers need to know the purity and the degree of acetylation of the raw materials before utilizing them. There are many methods to determine the degree of acetylation of chitn and chitosan at present, but none of them are flawless. The objectives of this study are to seek the possibile application of the first derivative UV spectroscopy on determining the degree of acetylation, to design a dye-binding method for purity determination, and to establish a new method by using 13C-NMR spectroscopy for purity determination.

Besides the advantages of saving time and simplifying chemical preparation, the UV first derivative method uses the UV spectrophotometer which can be found in most laboratories. Therefore, its popularization can be improved greatly. The shortcoming of the method lies in the fact that chitin with higher degrees of acetylation can not be dissolved in acetic acid. The method is found useful to determine the degree of acetylation of water-soluble chitinous products which are made by acetylation with acetic anhydride. The degrees of acetylaion obtained by using the UV first derivative method are close to those obtained by the improved HPLC method and the FTIR method.

The purity of chitin is defined as the ratio of the total weight of chitin and chitosan in the chitinous sample to the weight of the whole sample. When chitosan dissolved in 0.5 M HCl-glycine buffer, the amine group on chitosan chain can bind with SO3¯ of dye Cibacron brilliant red 3B-A. The amount of non-bounded dye can be determined by spectrophotometer after filtration. The purity of the sample is then calculated from the amount of the bounded dye, the glucosamine content in the chitosan chain, and the degree of acetylation which is determined by the IR method. Compare the results obtained from the dye-binding method with that from the deduction method, no significant difference was found.

Another purity-determination method is also established by the use of 13C-NMR spectroscopy. However, the method is useful only when the protein residue in the sample is below 2%.

目錄
摘要…………………………………………………………………………....I
Abstract………………………………………………………………………III目錄……………………………………………………………………….......V
圖目錄……………………………………………………………………….IX
表目錄……………………………………………………………………….XI
第一章 前言………………………………………………………………...1
第二章 文獻整理………………………………………………………….3
一、幾丁質與幾丁聚醣之來源與結構………………………………..3
（一） 幾丁質與幾丁聚醣之來源……………………………….3
（二） 幾丁質與幾丁聚醣之結構……………………………….4
（三） 幾丁質與幾丁聚醣之製備……………………………….4
二、幾丁質類產品之應用…………………………………………….6
（一） 化工方面…………...……………………………………..6
（二） 生醫方面……………………………………………….....6
（三） 食品方面……………………………………………...…..7
1. 保健功能…………………………………………………..7
2. 可食性膜…………………………………………………..8
3. 抗菌能力…………………………………………………..8
4. 食品添加物………………………………………………..9
5. 膳食纖維…………………………………………………..9
（四） 廢水處理方面…………………………………………….9
三、幾丁聚醣與染劑之吸附作用……………………………………11
（一） 廢水中染劑之脫除方法…………………………………11
1. 化學方法…………………………………………………11
2. 物理方法…………………………………………………11
3. 生化方法…………………………………………………12
（二） 幾丁聚醣與染劑吸附效能之影響因子…………...……12
（三） 幾丁聚醣吸附染劑之反應模式……………………...…13
（四） 幾丁聚醣吸附染劑於分析上之應用……………...……15
四、幾丁質與幾丁聚醣N-乙醯化程度之測定………………..…....16
（一） 酸水解－高效能液相層析法…………………...………16
（二） 核磁共振光譜法……………………………………...…18
1. H1-NMR法………………………………………………18
2. C13-NMR法……………………………………………...19
3. N15-NMR法……………………………………………...20
（三） 紅外光光譜法………………………………………...…20
（四） 一次微分紫外光光譜法……………………………...…22
（五） 膠體滴定法…………………………………………...…22
（六） 電位滴定法…………………………………………...…23
五、幾丁質與幾丁聚醣純度之測定………………………………….24
（一） 以碳元素含量分析………………………………………24
（二） 以氮元素含量分析………………………………………25
六、結語……………………………………………………………..26
第三章 材料與方法……………………………………………………..27
一、材料……………………………………………………………..27
（一） 樣品……………………………………………………...27
（二） 標準品…………………………………………………...27
（三） 藥品………………………………………...……………27
（四） 層析管柱………………………………………………...28
（五） 透析膜…………………………………………………...28
（六） 儀器……………………………………………………...28
二、方法……………………………………………………………….30
（一） 製備不同乙醯化程度之幾丁質、幾丁聚醣………............30
（二） 酸酐乙醯化法製備不同乙醯化程度水溶性幾丁聚醣.......30
（三） 測定幾丁質、幾丁聚醣之乙醯化程度……………………30
1. 一次微分紫外光光譜法…………………………………30
2. 固態核磁共振光譜法……………………………………31
3. 改良式高效能液相層析法………………………………32
4. 紅外光光譜法…………………………………………....33
（四） 幾丁質及幾丁聚醣純度分析…………………………...33
1. 染劑吸附法………………………………………………33
2. 13C-NMR法……………………………………………...38
3. 扣除法……………………………………………………39
第四章 結果討論………………………………………………………...41
一、不同乙醯化程度幾丁質類產品之製備………………………...41
（一） 熱鹼處理法所得之不同乙醯化程度之幾丁質類產品…41
（二） 酸酐乙醯化法所得之不同乙醯化程度幾丁聚醣………41
二、測定樣品之乙醯化程度…………………………………………41
（一）固態核磁共振光譜法……….……………………………41
（二）改良式高效能液相層析法….…...……………………….42
（三） 紅外光光譜法……….…………………………………...42
（四）一次微分紫外光光譜法...……………………….………43
三、樣品純度之測定…………………………………………………44
（一） 染劑吸附法……………………………………………...44
（二） 扣除法…………………………………………………...49
（三） C13-NMR法…………………………………….............49
第五章 結論……………………………………………………………...51
圖…………………………………………………………………………...52
表………………………………………………………………………...…90
參考文獻…………………………………………………………………...95
附錄A：Eqn. 20 之推導…………………………………………………107






















圖目錄
圖一、纖維素、幾丁質及幾丁聚醣之構造………………………………..52
圖二、樣品A之13C-核磁共振光譜圖……………………………………..53
圖三、樣品B之13C-核磁共振光譜圖……………………………………..54
圖四、樣品C之13C-核磁共振光譜圖……………………………………..55
圖五、樣品D之13C-核磁共振光譜圖……………………………………..56
圖六、樣品E之13C-核磁共振光譜圖…………………………………......57
圖七、樣品F之13C-核磁共振光譜圖……………………………………..58
圖八、樣品G之13C-核磁共振光譜圖………………………………….....59
圖九、樣品H之13C-核磁共振光譜圖……………………………………..60
圖十、幾丁聚醣樣品之CP-MAS 13C-NMR光譜圖……………………....61
圖十一、樣品C之改良式高效能液相層析圖………………………….....62
圖十二、樣品W1之改良式高效能液相層析圖………………………......63
圖十三、樣品W2之改良式高效能液相層析圖………………………......64
圖十四、樣品W3之改良式高效能液相層析圖………………………......65
圖十五、樣品W4之改良式高效能液相層析圖……………………….......66
圖十六、樣品W5之改良式高效能液相層析圖…………………………..67
圖十七、幾丁質類產品之傅立葉紅外線轉換光譜圖I…………………….68
圖十八、幾丁質類產品之傅立葉紅外線轉換光譜圖II………………......69
圖十九、不同濃度N-acetyl glucosamine溶液一次微分紫外光光譜圖….70
圖二十、N-acetyl glucosamine溶液vs. H value之標準曲線…………......71
圖二十一、染劑Cibacron Brilliant Red 3B-A吸光値之標準曲線………..72
圖二十二、染劑Cibacron Brilliant Red 3B-A之結構……………………..73
圖二十三、不同pH對幾丁聚醣與染劑Cibacron Brilliant Red 3B-A
作用吸光値之影響………………………………………….74
圖二十四、不同作用時間下幾丁質吸附染劑之含量……………………..75
圖二十五、幾丁質顆粒大小對染劑吸附量之影響……………………..…76
圖二十六、作用溫度對幾丁聚醣吸附染劑含量之影響…………………..77
圖二十七、幾丁聚醣染劑吸附Cibacron Brilliant Red 3B-A以
Langmuir isotherm所繪製之線性方程式…………………......78
圖二十八、幾丁聚醣與染劑Cibacron Brilliant Red 3B-A之吸附………..79
圖二十九、膠原蛋白質之吸光値標準曲線………………………………...80
圖三十、純度測定所用葡萄糖胺標準品之13C-核磁共振光譜圖………81
圖三十一、純度測定之樣品A之13C-核磁共振光譜圖…………………82
圖三十二、純度測定之樣品B之13C-核磁共振光譜圖…………………83
圖三十三、純度測定之樣品C之13C-核磁共振光譜圖…………………84
圖三十四、純度測定之樣品D之13C-核磁共振光譜圖…………………85
圖三十五、純度測定之樣品E之13C-核磁共振光譜圖…………………86
圖三十六、純度測定之樣品F之13C-核磁共振光譜圖………………….87
圖三十七、純度測定之樣品G之13C-核磁共振光譜圖…………………88
圖三十八、純度測定之樣品H之13C-核磁共振光譜圖…………………89











表目錄
表一、以13C-核磁共振光譜法及紅外光光譜法測定幾丁質類樣品
之N-乙醯化程度…………………………………………………….90
表二、以改良式高效能液相層析法、一次微分紫外光光譜法及紅外
光光譜法測定幾丁質類樣品之N-乙醯化程度……………………91
表三、以扣除法及染劑吸附法測定幾丁質類樣品之純度…………...…...92
表四、幾丁質與幾丁聚醣中水分、灰分與殘留蛋白質量，並以扣除
法計算樣品純度…………………………………………………….93
表五、以13C-NMR法及扣除法測定幾丁質類樣品之純度………………94

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