Analytical protocols development for quality assessment of chitin and chitosan | |
| Author | San Hein |
| Call Number | AIT Diss. no.BP-03-06 |
| Subject(s) | Chitin Chitosan |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. BP-03-06 |
| Abstract | The lack of a reliable and standardized quality assessment system for chitin and chitosan is a major obstacle for the large-scale commercialization of chitomaterials. It has caused an increasing shortage of high purity grade chitosan for application in the biomedical, cosmetic and food sector. Although proposals of limited scope have been made for the standardization of various chitosan parameters, an integral and generally accepted quality assessment scheme is not available. A logical and systematic approach for a chitosan quality assessment scheme has been presented at the 8“1 International conference on chitin and chitosan, Yamaguchi, Japan (2000) to obtain an overview of the functional properties of chitosan in a logical sequence in which contaminated samples are rejected early and only high value samples are eligible for the full analytical flow sheet. The proposal for a chitosan quality assessment scheme mentioned above was further justified and discussed. The underlying research was augmented to improve important issues of the quality assessment scheme: the purity and the functional analyses of the chitomaterials. An important commercial parameter for chitosan is the clarity after dissolution in acetic acid. Turbidity in solutions of pure chitosan is attributed to insoluble materials resulting from incomplete blockwise-deacetylated chitin. The presence of insolubles in chitosan after dissolution severely limits the application of chitosan in medical and cosmetic applications. In other words, the purity of acid soluble chitosan is very important in the applications. It is therefore important to ascertain that a chitosan preparation does not contain or is very low in insoluble. It is however not easy to assess a small amount of insoluble. Because of low filterability and the low sedimentation velocity of the insolubles in highly viscous chitosan solutions, it is not practical use conventional filtration and centrifugation methods. Although the viscosity of a chitosan solution can be reduced by the nitrous acid treatment and the insoluble can be easily collected by filtration, nitrous acid cleaves the [5- glyosidic linkages of glucosamine moieties of chitosan resulting in an underestimation of the amount of insoluble. An alternative method using an enzyme with a chitinolytic action that can easily attack chitosan in solution but encounters satirical hindrance to penetrate into the insoluble particle was proposed. As a result the chitosan solution becomes filterable but the insoluble are hardly affected. A choice was made from 14 commercially available enzymes. It was found that commercial transglucosylase (EC 2.4.1.24) can degrade chitosan in solution efficiently but cannot degrade chitin. Then, further experiments with transglucosylase were camped out and compared with nitrous acid deamination method for determination of insoluble in chitosan solution. Insoluble from chitosan commonly produced by high temperature deacetylation (90°C) can be recovered 70-90% by the enzymatic method whereas only 25% recovery was achieved by the nitrous acid method. The insoluble material recovered after enzyme treatment had a higher degree of deacetylation and a lower degree of crystallization than that after nitrous acid treatment as shown by FTIR, XRD and HPLC analysis. The results are explained by the difference between enzyme and nitrous acid in penetration into the insoluble particle. The analytical protocols for quality assessment was further developed by investigating the influence of analytical methods used for determination of the degree of deacetylation and the degree of crystallinity since these parameters are directly affecting in the functionality of the biopolymer. Many methods used in determination of the degree of deacetylation are limited due to the insolubility of the sample. In this study. FTIR spectroscopy, solid state l3C CPMAS NMR spectroscopy and acid hydrolysis-HPLC methods were compared. A good relation was observed between two absolute methods: solid state 13C CP/MAS NMR spectroscopy and acid hydrolysis-HPLC. The most advocated FTIR methods were compared with the absolute methods and gave underestimation of degree of deacetylation. Although time consuming sample preparation is needed in the HPLC method, the advantages are far outweighed than the other two in terms of instrument cost, availability and insensitivity to protein contamination. Crystallinity is a physical parameter that governs the tendency of adsorption and mechanical strength of chitin and chitosan. XRD and DSC are the methods primarily recommended for the determination of crystallinity. Because of the lack of standard material, relative degree of crystallinity of chitin/chitosan was determined. The ratio of crystalline to amorphous separation method was found to correlate well with the enthalpy of decomposition as measured by Differential Scanning Calorimetry. Moreover, degree of deacetylation was also in good agreement with enthalpy of decomposition. Finally, the quality assessment scheme for chitosan was extended, improved and justified including the data of this thesis project and valuable comments from internationally recognized scientists. In addition a proposal was made for minimal and maximal testing. Minimal in case many reliable data are already available, maximal in case of a chitosan batch of unknown origin and unknown quality status. The quality assessment scheme, completed with all underlying protocols (Appendix C) was presented for a formal discussion to scientist and governmental officials to establish international standard for chitin and chitosan quality. This standardization can be barely revised in the further full- scale exploitation of chitosan as unique biocompatible polycationic compound with a plethora of opportunities for the benefits of mankind. |
| Year | 2003 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. BP-03-06 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Bioprocess Technology (BP) |
| Chairperson(s) | Stevens, Willem F.; |
| Examination Committee(s) | Suwalee Chandrkrachang;Athapol Noomhorm;Preeda Parkpian;Suwabun Chirachanchai;Roberts, George A. F.; |
| Scholarship Donor(s) | Asian Institute of Technology Fellowship; |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2003 |