利用毛細管電泳及MALDI-TOF mass研究幾丁聚醣之降解反應

Abstract

In this thesis, we used capillary electrophoresis (CE) technique to establish the respective calibration curves for the measurements of degree of deacetylation (DDA) and molecular weight (Mv) for chitosan samples. When measuring the electrophoretic mobility (μ) of chitosan samples with DDA values ranging from 26% to 95% in CE run buffers (tris-phosphate buffer, pH = 2.00) of various concentrations (25 mM~1.6M), we found that the μ-versus-DDA (NMR measured) calibration curves possess different linearity in the higher DDA region (55%~95%) and in the lower DDA region (26%~55%). Moreover, the linearity of the two regions gradually approach to each other with increasing buffer concentrations, and finally become one linear relation in the DDA range of 26%~95% when raising the CE buffer concentration up to 1.5 M. In addition, the UV absorption peak (at 200 nm) width at half maximum (W1/2, DDA) in the CE electropherogram enable us to obtain the DDA distribution in the chitosan samples. We found that chitosan with unchanged DDA but narrower DDA distribution can be generated by designed deacetylation and acetylation reactions. The high efficiency and microanalysis features of our CE based method also facilitate the kinetic studies of the deacetylation reactions for chitin and chitosan samples. We also used CE to measure the viscosity of the chitosan samples so as to calculate the intrinsic-viscosity defined molecular weight (Mv) by the Mark-Houwink equation, and to establish the Log(μ)-versus-Mv calibration curve by measuring the electrophoretic mobility of chitosan samples with varied Mv’s in capillary gel electrophoresis (CGE, with separation medium of 0.3% PEO in 100 mM tris-phosphate buffer, pH = 2.00). By using the CGE technique we were able to study the Mv degradation reactions of chitosan by the chemical degradation method, which is mainly initiated by potassium persulfate (KPS), and by the enzymatic digestion approach, which is based on the biological enzymes such as pepsin, lipase, and trypsin. Besides CE, we also used MALDI-TOF mass to detect the chitooligosaccharide product of the chitosan degradation reaction. According to the MALDI-TOF mass spectrums and data, we can analyze and make a comparison among the monomer compositions of the chitooligosaccharide obtained from the abovementioned different fragmentation methods.