幾丁質(chitin)富藏於蝦蟹殼等甲殼類和昆蟲類外殼中，為一結構類似纖維素之天然高分子，纖維素單體葡萄糖的2’碳之氫氧基(-OH)以乙醯基(-NHCOCH3)取代，即為幾丁質。幾丁質經強鹼加熱反應後，可將其分子上的乙醯基轉變成胺基(-NH2)而形成幾丁聚醣(chitosan)。幾丁聚醣的胺基含量通常以去乙醯化程度(Degree of Deacetylation，簡稱DDA)表示。幾丁聚醣可應用於醫藥、保健食品、環境保護等方面，其去乙醯化程度和分子量大小，是幾丁聚醣的重要性質，會影響其應用性。本研究主要是利用毛細管電泳技術開發快速簡便的幾丁聚醣之去乙醯化程度和分子量之測定方法。
  幾丁聚醣因為胺基的質子化而帶正電荷，故所帶正電大小與其胺基含量成正比，利用毛細管電泳測量幾丁聚醣的電泳遷移率，可以測得其去乙醯化程度。我們以600 MHz NMR測定一系列具不同去乙醯化程度的幾丁聚醣樣品(由強鹼加熱法製備，或是所購得之標準品)，再以毛細管電泳在pH 2，濃度為100 mM 的tris-phosphate 緩衝溶液中測量個別樣品的電泳遷移率(μ)，可得到一電泳遷移率與去乙醯化程度的線性關係，利用此校正曲線與電泳實驗，可偵測未知去乙醯化程度之幾丁聚醣。在此實驗中我們也探討了樣品溶劑和濃度，緩衝溶液的組成、pH 值和離子強度對幾丁聚醣電泳行為的影響。此外，為了提高電泳遷移率測定的準確度和再現性，我們也選擇了鈷胺離子(Hexamminecobalt ion，Co(NH3)63+)作為電泳實驗的內標準品。在進行幾丁聚醣毛細管電泳實驗時，因為帶正電的幾丁聚醣樣品容易吸附毛細管壁，而導致測量誤差，因此我們在以電泳測量不同樣品的實驗之間，會分別以0.1 N 醋酸、1 N 鹽酸、0.03%聚環氧乙烯(PEO，Mwt=6×105)溶液沖洗毛細管內壁，以獲得較佳的再現性以及較長的毛細管使用壽命。此外，具有相同去乙醯化程度，但不同分子量的幾丁聚醣樣品，在適當的高分子分離介質中之電泳遷移率具有與其分子量成反比的特性。我們以過硫酸鉀(Potassium Persulfate)分別對幾個具有不同去乙醯化程度的幾丁聚醣樣品做不同程度的分子量降解反應，如此可得到幾組具相同去乙醯化程度(80%~95%)但不同分子量的幾丁聚醣樣品。這些樣品的黏度平均分子量可由毛細管電泳儀所測得的極限黏度(intrinsic viscosity)以及Mark-Houwink 方程式求得，而其電泳遷移率可在填充0.3%PEO(Mwt=8×106)的分離介質中測得，由此，我們可以得到幾丁聚醣的電泳遷移率和黏度平均分子量的線性關係，以做為測量未知樣品的依據。根據本實驗校正曲線所測量的兩種市售幾丁聚醣產品之去乙醯化程度及黏度平均分子量分別為甲：83.05%，5.22×105 和乙︰81.61%，7.84×105。另外，我們也測量了幾個經由蝦蟹殼及魷魚軟骨萃取所得幾丁聚醣之去乙醯化程度及分子量。

Chitosan is a polysaccharide derived by deacetylation of chitin, a rich component found in the shells of crustaceans such as shrimp and crab. The degree of deacetylation (DDA) and the molecular weight of chitosan greatly influence its chemical and physical properties, and also their applicability in biomedicine, health food, and environmental protection. We have developed a capillary
electrophoresis (CE) based method for the fast determinations of chitosan DDA and molecular weight.
  Chitosan can be viewed as a positively charged polyelectrolyte due to the protonation of the amine functional group, which is resulted from the deacetylation of N-acetyl-D-glucosamine of chitin. The positive charge density of a chitosan molecule is thus proportional to its DDA. Since the electrophoretic mobility of a polyelectrolyte in free solution is governed by its charge density, the DDA of a chitosan sample can be readily calculated according to its electrophoretic mobility
measured by CE. In this study, chitosan samples with different DDA were prepared by alkali treatment. The mobility measurements were performed in 100mM tris-phosphate (pH 2) buffer by CE. A linear calibration curve in the plot of electrophoretic mobility versus DDA (determined by NMR) was obtained, and therefore the DDA of unknown chitosan samples could be determined. We also
investigated the effects of sample concentration and solvent, and the compositions, pH value and ionic strength of buffer solutions on the electrophoretic behaviors of the chitosan samples. Moreover, in order to raise the accuracy and reproducibility of the mobility measurements, hexamminecobalt III (Co(NH3)63+) was chosen as the internal standard to decrease the run-to-run fluctuations in CE
conditions. Rinsing capillary column with 0.1N acetic acid, 1N HCl, and 0.03% polyethylene oxide (PEO, Mwt = 6×105) before each CE run could largely improve the problem caused by chitosan adsorption onto capillary inner wall. On the other hand, we also utilized CE as an automatic viscometer to measure the intrinsic-viscosity defined molecular weights of chitosan samples. For chitosans having the same DDA, i.e., the same charge density, but different molecular weights, the electrophoretic mobilities measured in the hydrophilic polymer solution were inversely proportional
to the molecular weights. By using the CE separation medium of 0.3% PEO (Mwt = 8×106), a good calibration curve which correlated mobility to molecular weight for chitosan samples could be obtained.

目錄
中文摘要..................................................Ⅰ
英文摘要..................................................Ⅱ
目錄......................................................Ⅲ
圖表索引..................................................Ⅵ
第一章 緒論................................................1
1.1 前言...................................................1
1.2 研究動機...............................................2
1.3 幾丁質、幾丁聚醣簡介...................................3
1.3.1 幾丁質、幾丁聚醣的來源及結構.........................3
1.3.2 幾丁質與幾丁聚醣之製備...............................5
1.3.3 幾丁質與幾丁聚醣之應用...............................8
1.3.4 幾丁聚醣基本性質的測定...............................9
1.4 毛細管電泳簡介........................................15
1.4.1 電泳基本原理........................................15
1.4.2 毛細管電泳介紹......................................16
1.4.3 分離介質介紹........................................24
1.4.4 電泳遷移模式........................................27
1.5 本章參考資料..........................................31
第二章 實驗...............................................35
2.1 實驗藥品..............................................35
2.1.1 各種緩衝溶液配製....................................38
2.2 實驗條件..............................................38
2.2.1 毛細管電泳儀........................................38
2.2.2 毛細管處理方式......................................39
2.2.3 利用毛細管電泳測量幾丁聚醣之去乙醯化程度之條件......40
2.2.4 利用毛細管電泳測量幾丁聚醣黏度分子量之條件..........40
2.2.5 利用毛細管電泳測量不同分子量幾丁聚醣之條件..........41
2.3 實驗步驟..............................................41
2.3.1 利用膠體滴定法測定幾丁聚醣之去乙醯化程度............41
2.3.2 利用紫外線光譜法測定幾丁聚醣之去乙醯化程度..........42
2.3.3 利用核磁共振法測量幾丁聚醣之去乙醯化程度............43
2.3.4 幾丁質樣品之去乙醯化反應............................45
2.3.5 以過硫酸鉀降解幾丁聚醣之分子量......................45
2.4 本章參考資料..........................................47
第三章 結果與討論 ........................................48
3.1 偵測幾丁聚醣之去乙醯化程度............................48
3.1.1 利用毛細管電泳測量幾丁聚醣之去乙醯化程度............49
3.1.2 不同去乙醯化程度偵測方法之比較......................66
3.2 測定幾丁聚醣之分子量..................................68
3.2.1 利用毛細管電泳儀測定幾丁聚醣之黏度平均分子量........68
3.2.2 利用毛細管電泳分離不同分子量幾丁聚醣................71
3.3 總結..................................................94
3.4 本章參考資料..........................................95

圖表索引
圖1-1、纖維素、幾丁質與幾丁聚醣化學結構式..................................4
圖1-2、自然界中幾丁質的排列方式......................................................5
圖1-3、以蝦蟹殼製備幾丁質流程簡圖..................................................6
圖1-4、毛細管內的電滲透流................................................................17
圖1-5、流體動力及毛細管電泳的拋物線流與平板流........................19
圖1-6、高分子溶液濃度與其糾結情形................................................27
圖2-1、幾丁聚醣1H 光譜......................................................................44
圖3-1、胺基酸電泳圖............................................................................96
圖3-2、染料分子電泳圖........................................................................97
圖3-3、氯化鈷胺和硫酸鋁的電泳圖....................................................98
圖3-4、鈷胺離子對幾丁聚醣電泳行為的影響....................................99
圖3-5、不同分子量幾丁聚醣樣品(Aldrich)之電泳圖.......................100
圖3-6、樣品濃度效應..........................................................................101
圖3-7、具不同分子量樣品在無濃度效應時之電泳圖......................102
圖3-8、不同緩衝溶液組成的影響(1).................................................103
圖3-9、不同緩衝溶液組成的影響(2).................................................104
圖3-10、緩衝溶液pH 值對電泳圖的影響.........................................105
圖3-11、觀察緩衝溶液離子強度效應................................................106
圖3-12、內徑100 微米毛細管之樣品濃度效應................................107
圖3-13、毛細管內徑對於氯化鈷胺吸收峰形狀的影響....................108
圖3-14、氯化鈷胺濃度的影響............................................................109
圖3-15、進樣條件對氯化鈷胺吸收峰形狀的影響............................110
圖3-16、以壓力推動樣品觀察不同進樣條件的影響........................111
圖3-17、不同進樣條件的影響............................................................112
圖3-18、不同去乙醯化程度幾丁聚醣電泳圖....................................113
圖3-19、電場強度對解析度影響Ⅰ(pH=2, I=50 mM, Ld=10
cm)..........................................................................................114
圖3-20、電場強度對解析度影響Ⅱ(pH=2, I=50 mM, Ld=20
cm)..........................................................................................115
圖3-21、電場強度對解析度影響Ⅲ(pH=2, I=100 mM, Ld=10
cm)..........................................................................................116
圖3-22、電場強度對解析度影響Ⅳ(pH=2, I=100 mM, Ld=20
cm)..........................................................................................117
圖3-23、建立去乙醯化程度校正曲線之電泳圖................................118
圖3-24、電泳遷移率對去乙醯化程度校正曲線................................119
圖3-25、不同方法測定幾丁聚醣去乙醯化程度之比較....................120
圖3-26、(a)樣品流速測定圖；(b)ηred 和ηinh 對濃度之線性關係...121
圖3-27、壓力與電動進樣條件之比較................................................122
圖3-28、分離介質HEC(Mwt=9×104)之濃度效應.............................123
圖3-29、分離介質HEC(Mwt=1.3×106)之濃度效應..........................124
圖3-30、分離介質HPMC 之濃度效應...............................................125
圖3-31、分離介質Dextran 之濃度效應.............................................126
圖3-32、分離介質PEO 之濃度效應..................................................127
圖3-33、添加CTAB 觀察Chitosan 10 之電泳圖...............................128
圖3-34、添加CTAC 所得結果............................................................129
圖3-35、CTAC 系統下改變幾丁聚醣濃度結果.................................130
圖3-36、CTAC 系統下觀察有無減少電荷差異性.............................131
圖3-37、CTAC 對新活化毛細管影響.................................................132
圖3-38、不同四級胺鹽緩衝溶液組成所得電泳圖............................133
圖3-39、改變TEA 濃度結果..............................................................134
圖3-40、TEA-phosphate 和tris-phosphate 電泳結果比較.................135
圖3-41、CTAC 添加於TMA-phosphate 所得電泳圖........................136
圖3-42、沖洗PEO(Mwt=6×105)改善CTAC 吸附之結果.................137
圖3-43、CTAC 添加於TMA-phosphate 和tris-phosphate 電泳圖...138
圖3-44、觀察CTAC 系統下不明吸收來源........................................139
圖3-45、觀察添加CTAC 的TMA-phosphate 幾丁聚醣濃度改變結
果............................................................................................140
圖3-46、不同進樣方式對添加CTAC 之tris-phosphate 結果...........141
圖3-47、添加TDAPS 結果.................................................................142
圖3-48、TDAPS 濃度效應..................................................................143
圖3-49、Aldrich chitosans 在PEO 中之電泳圖(1).............................144
圖3-50、Aldrich chitosans 在PEO 中之電泳圖(2).............................145
圖3-51、高溫酸解樣品之電泳圖........................................................146
圖3-52、相似DDA 不同分子量之電泳圖.........................................147
圖3-53、幾丁聚醣在毛細管膠電泳中之樣品濃度效應....................148
圖3-54、KPS 降解Chitosan 1000(DDA~80%)...................................149
圖3-55、KPS 降解TCI Chitosan(DDA~82%)....................................150
圖3-56、KPS 降解Chitosan high(DDA~85%)....................................151
圖3-57、不同去乙醯化程度系列校正曲線........................................152
表3-1、DDA 校正曲線之電泳遷移率的相對標準偏差......…...…….65
表3-2、DDA measured by CE method for the commercial products….66
表3-3、不同方法測定幾丁聚醣去乙醯化程度之比較………………66
表3-4、計算黏度平均分子量的各種黏度數據……………...……….70
表3-5、不同來源幾丁聚醣樣品之DDA 及Mv 測定值……...…….71
表3-6、不同DDA 之幾丁聚醣經去乙醯化反應前後之
DDA 和Mv…………………………………………………....89
表3-7、經KPS 降解前後幾丁聚醣之Mv 及DDA…………………90
X
表3-8、不同PEO 組成所得到樣品電泳析出的時間差………….…91
表3-9、市售幾丁聚醣樣品之DDA 及Mv 測定值…………….…....94

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第二章 參考資料
1. Tôei, K.; Kohara, T. Anal. Chim. Acta 1976, 83, 59-65.
2. Muzzarelli, R. A. A.; Rocchetti, R. Carbohydr. Polym. 1985, 5, 461-472.
3. Lavertu, M.; Xia, Z.; Serreqi, A. N.; Berrada, M.; Rodrigues, A.; Wang, D.; Buschmann, M. D.; Gupta, A. J. Pharm. Biomed. Anal. 2003, 32, 1149-1158.
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5. Mima, S.; Miya, M.; Iwamoto, R.; Yoshikawa, S. J. Appl. Polym. Sci. 1983, 28, 1909-1917.
第三章參考資料
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