以質譜儀、毛細管電泳儀和高效液相層析儀分析幾丁寡醣

Analysis of Chitooligosaccharides by Mass Spectrometry, Capillary Electrophoresis, and High Performance Liquid Chromatography

蔡心琪(Xin-Qi Cai)

指導教授 ： 吳俊弘

淡江大學/理學院/化學學系碩士班/碩士(2020年)

https://doi.org/10.6846/TKU.2020.00290

摘要

幾丁聚醣經化學(過硫酸鈉)或酵素(胃蛋白酶)降解的主要產物是低分子量幾丁聚醣和幾丁寡醣(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產物的去乙醯化程度(DDA)和分子量相關電泳遷移率。而COS產物的組成和平均DDA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)分析。我們的分析方法具有快速檢測和微量分析的特性，有利於探討幾丁聚醣降解之反應性。
以CZE在鹼性(pH 9.35)與酸性(pH 2.00)電泳緩衝溶液下進行降解空白實驗之分析，經實驗結果得知，在分離純化降解產物時，胃蛋白酶會殘留於COS產物中。但因反應所使用的胃蛋白酶的濃度很低，且其具有較大分子量，所以幾乎不影響COS產物於CZE和MALDI-TOF/MS之分析。
此外，我們探討以NH MB100 40/75 μm Silica Gel作為吸附劑，應用於COS產物之純化和除鹽。經由CZE與MALDI-TOF/MS的分析，可獲得最佳吸附劑使用量。我們以酸性溶液(0.1% TFA)活化吸附劑，使其胺基(-NH2)質子化而具較強正電性，可排斥具正電性的幾丁寡醣分子，並吸附鹽類陰離子。此純化方式可以達到除鹽的效果，不會有明顯的樣品流失，因此有利於樣品之偵測和分析。
最後，利用HPLC初步純化COS產物，以MALDI-TOF/MS鑑定經HPLC分離回收COS之組成，比對原始COS產物與HPLC分離的COS分子之CZE析出時間以標示電泳吸收峰之組成。目前我們已經在CZE電泳圖上標示出產量較高的主要COS產物之組成。建構此完整的高解析度COS-CZE電泳圖，會更進一步提升幾丁聚醣降解所得幾丁寡醣產物之定性和定量分析之效率。

關鍵字

幾丁聚醣 ； 幾丁寡醣 ； 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 (sodium persulfate, NaPS) or enzyme (pepsin) 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 related electrophoretic mobility of the low Mw chitosan product. Moreover, the composition and average DDA of the COS product were determined by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Our analytical method features fast measurement and microanalysis, and thus is advantageous to the study of degradation reactivity of chitosan.
According to the basic (pH 9.35) and acidic (pH 2.00) CZE results obtained from the blank experiment of chitosan degradation reaction, pepsin was found left in the COS after the separation and purification process of degradation products. However, the CZE and MALDI-TOF/MS analyses of the COS product will not be affected due to the low concentration and high molecular weight of pepsin.
Furthermore, we investigated the application of NH MB100 40/75 μm Silica Gel as the adsorbent to purify and desalt the COS product. Through CZE and MALDI-TOF/MS analyses, the optimal amount of adsorbent was determined. An acidic aqueous solution (0.1% TFA) was used to activate the adsorbent by protonating the amine functional group, generating positively charged sites to repel the cationic COS and attract the salt anions. This purification method can achieve desalting without significant sample loss, and thus facilitates the sample detection and analysis.
Lastly, the COS product was preliminarily purified by HPLC, and the compositions of the HPLC separated and recovered COS molecules were determined by MALDI-TOF/MS. The CZE elution times of the original COS product and that of the HPLC separated COS molecules were compared to assign the peak compositions of the CZE electropherogram. Currently we have identified the compositions of the major peaks of the electropherogram for the original COS product. The construction of the complete high resolution COS-CZE electropherogram can further enhance the efficiency of the qualitative and quantitative analysis of COS product obtained from chitosan degradation reaction.

並列關鍵字

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

參考文獻

1.9 本章參考文獻

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