利用毛細管電泳測量幾丁聚醣之去乙醯化程度及其分子量

Determination of degree of deacetylation and molecular weight of chitosan by capillary electrophoresis

陳世峰(Shih-Feng Chen)

指導教授 ： 吳俊弘

淡江大學/理學院/化學學系碩士班/碩士(2007年)

https://doi.org/10.6846/TKU.2007.00798

摘要

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。

關鍵字

幾丁聚醣 ； 去乙醯化程度 ； 分子量 ； 毛細管電泳

並列摘要

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.

並列關鍵字

chitosan ； degree of deacetylation ； molecular weight ； capillary electrophoresis

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