Mechanism And Kinetics Study On Low Temperature Deacetylation And Pyrolysis In Process Of Preparing Natural Water-soluble Chitosan Using Freezing-thawing Cyclic Treatment

Chitosan, a unique natural alkaline polysaccharide, a derivative of the biopolymer chitin, which has been aroused widespread concerns in many fields for its special properties, such as favorable biocompatibility, broad-spectrum antimicrobial activity, biodegradability, chelating of heavy metals, film forming ability, fiberizability and other special features. Actually, partial structure and functions, which has been reported in most previous study, was obtained from the chitosan salts, because of chitosan can only soluble in some special acid solution. This is disadvantageous for the in-depth theoretical and applied research of chitosan. Therefore, preparing a higher molecular weight, non-derivative and natural water-soluble chitosan (WSC), has a great significance on not only the related basic research but also the applied research.In this study, we prepared a WSC by using freezing-thawing cyclic (FTC) treatment and homogeneous deacetylation reaction without introduce any other groups, with degree of deacetylation (DDA) about 50 %, intrinsic molecular weight approximately 300 kDa, and could be soluble in deionized water at room temperature. The method broke through the traditional methods' defects, such as long time-consuming, low-yield, high cost, serious degradation, poor water-solubility. Kinetics of low temperature deacetylation was studied by using determination of DDA. XRD, FT-IR, Raman, DSC, CD, and AFM were used to investigate the effects of freezing-thawing treatment on the structure of chitin. Which revealed the formation mechanism of the WSC, Meanwhile the FTC treatment was explored as a simple, controllable, green, efficient method for pre-processing of chitin. Thermal stability, pyrolysis mechanism, pyrolysis kinetics and cytotoxicity was also investigated by multiple scan rate thermal analysis, FT-IR and MTT assay, respectively. The main results indicated as follows:1. The formation and growth of ice crystal in the slowly freezing process can break the inter-molecular hydrogen bonds interaction, disordered the molecules ordered structure, and destructed the condensed matter structure and make the crystallinity of chitin decline. However, the structural damage and crystallinity reducing effect is relatively weak only by the freezing treatment, it is not sufficient to make chitin formation a homogeneous solution completely, and the crystallization damage is somewhat partial irreversible, with heating treatment, it forms a new crystalline structure again. However, the slowly freezing and low temperature thawing cycle process can destructed the condensed matter especially the crystalline state structure more thoroughly, because of the ice crystals' repeatedly formed and recrystallization. Furthermore, via three times FTC treatment at -18℃, chitin can be completely dissolved in NaOH concentration aqueous solution and form a homogeneous solution with a chitin concentration about 4 wt% and a final NaOH concentration approximately 10.67 wt%. After standing for 48h-72h at 25℃, a WSC can be prepared and almost dissolved in water.2. Kinetics of low temperature deacetylation of chitin showed that the effects of the alkaline concentration, reaction time, freezing-thawing method, and their interactions played the dominant role on influencing the process of deacetylation. For most conditions, semi-logarithmic plots between the amount of acetyl glucosamine residues and the deacetylation time had well correlation coefficients (R²) above 0.9000, the deacetylation process followed the pseudo-first-order kinetics under low temperature. The apparent rate constants of the reaction ranged from 3.3×10^-3 h^-1 to 22.8×10^-3h^-1. And the apparent activation energy was 9.76 kJ/mol at aqueous 35 wt% NaOH solution in the temperature range of -5℃--35℃, which was lower than that reported at room or high temperature. That meant it had much higher reactively by using FTC treatment.3. XRD results indicated that slowly freezing and low temperature thawing cycle treatment could reduce the strong intra- and inter- hydrogen bonds, and destroy the dense crystal structure of chitin, make it possible to soluble in water. FT-IR analysis showed that the bands due to O-H stretching vibration absorption at 3448 cm^-1 in WSC or the similar water-soluble samples shifted significantly to low wavenumbers. And the better water solubility (WS) it is, the more shift and the smaller area of this region. As for WSC samples, the absorption appeared at about 3265 and 3103 cm^-1 were concealed, which indicated the reduction of N-H stretching vibration and intra- or inter-molecular hydrogen bonds, respectively. The DSC results showed that the parameters such as peak area, peak height and so on, were not only influenced by DDA, but also influenced by WS. In other words, the DSC parameters also affected by crystalline, and sometimes the latter was even more than the impact of DDA. The CD spectra also showed that FTC treatment broke the strong molecular hydrogen bonds, made WSC have more stretching spatial structure, less helix structure, and led it soluble in water more easily. The molecular morphology of WSC was studied by AFM, the results indicated that WSC soluble in water with neutral, non-protonated form, it means WSC prepared in this study was a kind of natural chitosan, but not its derivatives.4. In WSC aqueous solution, there had a linear relationship between absorbance and wavelength in the range of 208 nm - 212 nm, when DDA was as constant. And there was also a good linear relationship between the value of the slope and the concentration of WSC. As a result, a novel multiwavelength linear regression UV spectrophotometry was established for determining WS, and overcame the traditional methods' shortcomings. For example, filter paper constant weight method, which was cumbersome and inaccurate.5. FT-IR analysis of the pyrolysis products showed the thermal decomposition process of chitosan, chitin and WSC might be a random degradation of polymer main chains. The decomposition started from the scission of C-O-C bond at a weak point. Furthermore, the start barrier for the pyrolysis of WSC was higher, and more complex reactions occurred simultaneously with the decomposition process. The pyrolysis of chitosan hydrochloride (CHC) was a typical multi-step reaction, the ammonium polymer decomposed at first, and then the main polymer chain started decomposition.6. Multiple scan rate nonisothermal DSC curves were employed to investigate the pyrolysis kinetics of chitosan, chitin, WSC and CHC. The apparent activation energy (E) and frequency factor (lnA) for thermal decomposition were 153.02±2.67, 162.96±5.47, 188.20±1.98, 169.40±1.52 (1^st stage), 126.52±1.62 kJ/mol (2^nd stage) and 23.46±1.47, 23.09±1.14, 37.82±1.12, 35.28±0.62 (1^st stage), 24.69±0.44 min^-1 (2^nd stage), respectively. And the most probable mechanism functions were obtained by Achar's differential method. The functions of chitosan and chitin were f(α)=3/2(1-α)^2/3[1-(1-α)^1/3]^-1 and f(α)=3/2(1-α)^4/3[1/(1-α)^1/3-1]^-1, which was considered as 3D diffusion, ball symmetry mechanism and 3D diffusion mechanism, respectively. The function of WSC was f(α)=2(1-α)^3/2, belonged to a 2/3-order chemical reaction mechanism. For CHC, the most probable mechanism functions of two stages of decomposition were same, the equation expressed as f(α)=1-α, which conformed to a random nucleation and subsequent growth mechanism with only one core of each particle.7. MTT assay was employed to evaluate the cytotoxicity of WSC, the results indicated that WSC was non-cytotoxicity, furthermore, it could protect the L929 cell for the presence of non-protonated free amino groups. The dynamic coagulation test showed that the acetic acid solution of WSC had a certain coagulation effects, but the water solution of WSC had none.