幾丁聚醣經化學或酵素降解後的主要產物是低分子量幾丁聚醣 (Low Mw chitosan) 和幾丁寡醣 (COS)，本論文分別以毛細管區帶電泳(CZE) 和毛細管膠體電泳 (CGE) 分析低分子量幾丁聚醣樣品的去乙醯化程度 (DDA) 和分子量。而幾丁寡醣樣品的組成分析則以高效能液相層析儀 (HPLC) 與基質輔助雷射脫附游離飛行時間質譜儀 (MALDI-TOF/MS)進行，並根據所得質譜訊號計算樣品的平均DDA值。
本論文利用過硫酸鈉 (NaPS) 降解幾丁聚醣，所得Low Mw chitosan之DDA與原始樣品相近，但其分子量有明顯變化，當降解反應時間達60分鐘後，產物CGE析出時間即不再明顯變化；而利用胃蛋白酶 (Pepsin) 降解幾丁聚醣，反應 72 小時內所得Low Mw chitosan的分子量則持續變小。此外，根據CGE分析結果發現，以NaPS降解幾丁聚醣時，反應短時間內會有具多分子量分佈的產物出現。本論文測量NaPS降解幾丁聚醣所得不同分子量Low Mw chitosan的CGE電泳遷移率及其極限黏度分子量(Mv)，並推導兩者之間的定量關係式，藉以測量經酵素降解所得Low Mw chitosan樣品的Mv值。
為了分析具有寬廣DDA分佈的幾丁聚醣樣品，我們將其CZE-UV吸收峰解析成數個較窄分布的吸收峰，並計算各個解析峰的積分面積與質心時間，再以其DDA和面積比或濃度比，計算具寬廣DDA分佈樣品的平均DDA，並與NMR測得結果比較。

The major products of chitosan degraded by chemical or enzyme are low molecular weight chitosan (Low Mw chitosan) and chitooligosaccharide (COS). In this thesis we utilized capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) to analyze the degree of deacetylation (DDA) and the molecular weight of the Low Mw chitosan products. Furthermore, the composition analysis of COS products were carried out by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and the average DDA were calculated on the basis of the relative mass signals of the COS components.
The DDA of Low Mw chitosan products obtained from sodium persulfate (NaPS) degradation of chitosan were close to the DDA of the original chitosan samples. However, obvious variations in the molecular weight of Low Mw chitosan products were observed. When the degradation time exceeded 60 minutes, the CGE elution time of Low Mw chitosan products remained virtually the same while that of pepsin digestion products kept decreasing within 72 hours. Moreover, the CGE analysis revealed some multi-molecular weight distributions of NaPS degradation products obtained in short reaction time. In this thesis, we measured both the CGE mobility and the intrinsic viscosity defined molecular weight (Mv) of some Low Mw chitosan samples derived from NaPS degradation reaction, and deduced their quantitative relation, which was then used to determine the Mv of Low Mw chitosan products of enzymatic digestion reaction.
In order to analyze the chitosan sample with wide DDA distribution, we deconvoluted the CZE-UV absorption peaks into several narrower peaks and calculated the integrated area and the center of mass for each deconvoluted peak. Subsequently, the average DDA was calculated according to the respective DDAs and area ratios or concentration ratios of all the deconvoluted peaks. The resulting DDA was then compared with the NMR measured DDA.

目錄
中文摘要 	I
英文摘要 	II
目錄 	IV
圖目錄 	VIII
表目錄 	XIV
簡寫表 	XVI
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
1.4	幾丁聚醣之降解反應	4
1.4.1酵素降解	4
1.4.2化學降解	6
1.5毛細管電泳簡介	7
1.5.1毛細管電泳介紹	7
1.5.2毛細管電泳原理	7
1.5.3電泳遷移率(electrophoretic mobility)	7
1.5.4電泳滲透流(electroosmotic flow, EOF)	8
1.5.5毛細管電泳的分離方法	10
1.5.6樣品注入方式	11
1.6 MALDI-TOF/MS簡介	12
1.7測量幾丁聚醣降解產物之去乙醯化程度與分子量	16
1.7.1利用毛細管電泳分析幾丁聚醣	16
1.7.2利用MALDI-TOF/MS 測量幾丁寡醣之乙醯化程度	18
1.7.3利用核磁共振法測量幾丁聚醣之去乙醯化程度	18
1.7.4利用黏度計測量幾丁聚醣的平均黏度分子量 (Mv)	20
1.8利用高效能液相層析儀分離幾丁寡糖	21
1.9本章參考文獻	24
第二章 實驗	30
2.1實驗藥品與器材	30
2.2實驗儀器	36
2.3實驗方法與步驟	38
2.3.1毛細管處理	38
2.3.2毛細管電泳緩衝溶液的配置	39
2.3.3以毛細管電泳測量幾丁聚醣去乙醯化程度之條件與步驟	39
2.3.4利用毛細管膠體電泳測定幾丁聚醣電泳遷移率	41
2.3.5利用毛細管電泳測定幾丁聚醣極限黏度分子量	42
2.3.6利用核磁共振法測量幾丁聚醣之去乙醯化程度	47
2.3.7利用MALDI-TOF/MS分析幾丁寡醣樣品	48
2.3.8利用高效能液相層析儀分離純化幾丁寡糖樣品	51
2.4實驗樣品備製	52
2.4.1幾丁聚醣的乙醯化反應	52
2.4.2幾丁聚醣的去乙醯化反應	53
2.4.3幾丁聚醣的降解反應	53
2.4.4以Silica Gel 純化酵素降解幾丁聚醣所得幾丁寡醣	56
2.5本章參考文獻	57

第三章 結果與討論	59
3.1幾丁聚醣之化學降解	59
3.1.1以NaPS 降解幾丁聚醣所得低分子量幾丁聚醣產物之
  分析	59
3.1.2以NaPS 降解幾丁聚醣所得幾丁寡糖產物之分析	61
3.1.3利用Silica gel純化酵素降解幾丁聚醣所得幾丁寡醣	61
3.2利用毛細管膠體電泳測定幾丁聚醣之分子量	77
3.3幾丁聚醣化學降解與酵素降解所得 Low Mw chitosan樣品之
比較	86
3.3.1兩種降解對幾丁聚醣分子量的影響之比較	86
3.4幾丁聚醣在CZE中的UV吸收	96
3.5幾丁寡糖在HPLC中的分離	103
3.5.1以折射率和紫外線吸收光偵測COS樣品訊號	103
3.5.2有機相比例對COS分離的影響	103
3.5.3利用HPLC分離前後之COS比較	104
3.6本章參考文獻	110
第四章 結論	111
 
圖目錄
圖1-1、幾丁質與幾丁聚醣化學結構式 	3
圖1-2、幾丁聚醣經過硫酸根離子降解之反應機構 	6
圖1-3、毛細管內壁及電滲透流生成示意圖 	9
圖1-4、流體動力的拋物線流及毛細管電泳的平板流 	10
圖1-5、MALDI-TOF/MS實驗示意圖 	12
圖1-6、飛行時間質析 (TOF) 示意圖 	14
圖1-7、直線型 (Linear Mode) MALDI-TOF/MS與反射型 (Reflector 
 Mode) MALDI-TOF /MS示意圖 	15
圖1-8、由14種幾丁聚醣標準品所建立之電泳遷移率對去乙醯化程
 度 (NMR測量結果) 的校正曲線 	17
圖1-9、NMR光譜中幾丁聚醣各個吸收與其代表名稱 	19
圖2-1、Chitosan 10 – De 黏度對濃度關係圖 	45
圖2-2、Chitosan 10-De - NaPS 10 min 黏度對濃度關係圖 	45
圖2-3、Chitosan 10-De - NaPS 60 min 黏度對濃度關係圖 	46
圖2-4、幾丁聚醣 NMR氫譜中各個H化學位移訊號及其在分子上
 的位置 	48
圖2-5、LMW-S  (DDA 34.2% ) 經 pepsin降解 72 小時所得 COS 
 之 MS 圖譜 	50
圖3-1、利用NaPS 降解四種不同分子量大小的幾丁聚醣 (DDA
 95%) 所得Low Mw chitosan樣品之DDA對時間的關係圖
 	63
圖3-2、Chitosan 10-De經 NaPS 降解後所得 Low Mw chitosan 樣
品之毛細管膠體電泳圖 	65
圖3-3、LMW-S-De 經 NaPS 降解後所得 Low Mw chitosan 樣品之
		毛細管膠體電泳圖 	66
圖3-4、MMW-De 經 NaPS 降解後所得 Low Mw chitosan 樣品之
毛細管膠體電泳圖 	67
圖3-5、HMW-De 經 NaPS 降解後所得 Low Mw chitosan 樣品之
毛細管膠體電泳圖 	68
圖3-6、四種幾丁聚醣經 NaPS 降解後所得Low Mw chitosan 樣品
之毛細管膠體電泳遷移率與降解時間的關係 	69
圖3-7、樣品具有多重峰CGE 與其CZE 電泳圖之比較 	70
圖3-8、利用 NaPS 降解 MMW-De 後所得COS 之質譜圖 	71
圖 3-9、利用CZE分析經 Silica gel 純化前、純化後的 COS 樣品 
(LMW-S-reA0.8-pepsin72hr-COS) 之電泳圖 	72
圖 3-10(a)、利用MALDI-TOF/MS分析經 Silica gel 純化前、純化
後的 COS 樣品 (LMW-S-reA0.8-pepsin72hr-COS) 之質譜
圖 	73
圖 3-10(b)、將圖 3-10(a) 中所偵測到的 COS 樣品訊號強度除以
m/z 608.2 (1D2A, Na) 訊號強度之整理圖譜 	74
圖3-11(a)、利用MALDI-TOF/MS分析經 Silica gel 純化前、純化
後的 COS 樣品 (MMW-De-NaPS10min.-COS) 之質譜圖 
 	75
圖3-11(b)、利用MALDI-TOF/MS分析經 Silica gel 純化前、純化
後的 COS 樣品 (MMW-De-NaPS60min.-COS) 之質譜圖 
 	76
圖3-12(a)、不同分子量幾丁聚醣起始物經一連串乙醯化反應所得
Low Mw chitosan產物之 -Log(CGE mobility) 對AA/amine
比作圖 	79
圖3-12(b)、根據圖3-12(a) 各別將九種樣品 - Log(CGE mobility) 對
AA/amine比作圖，並計算其線性關係 	80
圖3-13、不同DDA值幾丁聚醣之-Log(CGE mobility) 對Mv 作圖
 	82
圖 3-14、DDA53.64% 幾丁聚醣 (LMW-S-reA0.4) 經 Pepsin 降解
1~72 小時所得 Low Mw chitosan 樣品之毛細管膠體電泳圖 
	89
圖 3-15、DDA 53.64% 幾丁聚醣 (LMW-S-reA0.4) 經 NaPS 降解 
1~180 min 所得 Low Mw chitosan 樣品之毛細管膠體電泳圖 
	90
圖 3-16(a)、DDA 35% 幾丁聚醣 (LMW-S-reA0.8) 經 Pepsin降解 
0~72 小時所得 Low Mw chitosan 樣品之毛細管膠體電泳圖 
	91
圖 3-16(b)、DDA 35% 幾丁聚醣 (LMW-S-reA0.8) 經 Pepsin降解 
48和 72 小時所得 Low Mw chitosan 樣品與 COS 樣品之
毛細管膠體電泳圖 	92
圖 3-17、DDA 35% 幾丁聚醣 (LMW-S-reA0.8) 經 NaPS 降解 
0~180 min 所得 Low Mw chitosan 樣品之毛細管膠體電泳圖 
	93
圖 3-18、DDA 53.64% (LMW-S-reA0.4) 分別經 Pepsin 和 NaPS 降
解所得 Low Mw chitosan 樣品之毛細管膠體電泳遷移率對
數值與降解反應時間關係 	94
圖 3-19、DDA 35% (LMW-S-reA0.8) 分別經 Pepsin 和 NaPS 降解
所得 Low Mw chitosan 樣品之毛細管膠體電泳遷移率對數
值與降解反應時間關係圖 	94
圖 3-20(a)、DDA 84.14% (LMW-S) 經 Pepsin 降解 3 小時所得 
Low Mw chitosan 樣品的CGE mobility電泳圖 	95
圖 3-20(b)、DDA 84.14% (LMW-S) 經 Pepsin 降解 3 小時所得 
Low Mw chitosan 樣品的極限黏度 – 濃度關係圖 	95
圖3-21、MMW-De經不同AA/amine比的乙醯化反應後所得幾丁聚
醣樣品之CZE電泳圖 	99
圖3-22、多分布樣品之CZE電泳圖 	100
圖3-23、不同DDA值的幾丁聚醣之UV吸收度與 (GluNAc) 單體
 濃度關係圖 	101
圖3-24、多重峰解析後利用面積比與濃度比計算DDA值並與 CZE 
單質心計算法以及 NMR 測得 DDA 值比較 	102
圖3-25、幾丁聚醣 (DDA 35%) 經Pepsin降解後所得COS樣品之 
HPLC 層析圖 	105
圖3-26、HPLC動相中有機溶劑比例對COS樣品分離效果之比較
 	106
圖3-27、幾丁聚醣經酵素 (Pepsin) 降解後所得COS樣品與酵素降
解空白實驗層析圖譜之比較 	107
圖3-28、利用MALDI-TOF/MS分析經HPLC分離純化後的COS樣
品 	108 
表目錄
表1-1、常用基質整理表 	13
表1-2、利用胺基修飾管柱分析幾丁寡糖所使用的技術 	22
表2-1、Chitosan 10-De 各項黏度 	45
表2-2、Chitosan 10- De -NaPS 10 min 各項黏度 	46
表2-3、Chitosan 10-De-NaPS 60 min 各項黏度 	46
表2-4、LMW-S  (DDA34.2%) 經 Pepsin降解 72 小時所得 COS 
之 MALDI-TOF/MS 訊號 	50
表2-5、各種酵素降解幾丁聚醣之反應條件 	56
表3-1、以NaPS 降解四種幾丁聚醣樣品後所得Low Mw chitosan樣
品之DDA值與CGE結果整理表 	64
表3-2、所製備CGE分析標準品的 DDA 值，Mv 值，以及 CGE 
mobility 	78
表3-3、不同分子量幾丁聚醣起始物經一連串乙醯化反應所得產物之
- Log (CGE mobility) 對AA/amine比作圖之線性轉折點	81
表3-4、圖 3-13 所使用標準品的 DDA 值、Mv 值，以及 
–Log (CGE mobility) 	83
表3-5、章節3.4所使用樣品整理表 	98
表3-5、利用MALDI-TOF/MS分析經HPLC分離純化後的COS樣
品之訊號表 	109

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