幾丁聚醣降解產物之分析

Analysis of degradation products of chitosan.

陳玨瑄(Chueh-Hsuan Chen)

指導教授 ： 吳俊弘

淡江大學/理學院/化學學系碩士班/碩士(2018年)

https://doi.org/10.6846/TKU.2018.00264

摘要

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。

關鍵字

幾丁聚醣 ； 幾丁寡糖 ； 毛細管電泳 ； 高效能液相層析儀 ； 基質輔助雷射脫附游離飛行時間質譜儀

並列摘要

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.

並列關鍵字

chitooligosaccharide ； capillary electrophoresis ； HPLC ； MALDI-TOF/MS ； chitosan

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