Chemical process of chitin extraction
Demineralization is commonly accomplished through the application of acid treatments, which effectively dissolve the calcium carbonate and calcium chloride components present in shrimp shells. Specifically, a 4% concentration of hydrochloric acid is utilized for this purpose. The demineralization process entails subjecting the shrimp shells to a 24-hour treatment duration, conducted at a room temperature of 25 °C, with a solid to solvent ratio of 15:1 (ml/g). Notably, the emission of carbon dioxide gas (CO2) during demineralization serves as a reliable indicator of the mineral content level within the process (Mohammed et al., 2013). Following the demineralization process, the dried shrimp powder is subjected to a 10% sodium hydroxide (NaOH) solution for a duration of 2 hours, maintained at a temperature of 70 °C, with a solvent to solid ratio (v/w) of 15:1 ml/g. This step serves the purpose of eliminating any remaining proteins and other organic materials. Subsequently, the mixture is filtered under vacuum and subsequently washed with tap water for a period of 30 minutes until the solution attains a neutral pH level (pH = 7). Consequently, the resulting product obtained from this process is chitin(Mohanasrinivasan et al., 2014) .To eliminate the pigment present in crustacean shell chitin, reagents such as ethanol, ether, sodium hypochlorite (NaOCl), or hydrogen peroxide (H2O2) are employed. However, it is crucial to ensure that the chosen bleaching reagent does not impact the physicochemical properties of chitin. Hence, in this study, H2O2 was selected for this purpose (Kumari and Rath, 2014). The deacetylation process is employed to eliminate the acetyl group from chitin. Typically, this is accomplished by treating chitin with a concentrated sodium solution (40-50%). The deacetylation is conducted at 100°C, with a solvent-to-solid ratio of 10/1 (ml/g), and a reaction time of 12 hours. Once deacetylation is complete, chitosan is obtained, followed by washing and rinsing with hot distilled water at 90°C. Subsequently, the material is filtered and oven-dried at 50°C for 16 hours (Benhabiles et al., 2012). The degree of deacetylation (% DD) of the extracted chitosan can be obtained using Equation (1). Chitosan is a copolymer consisting of N-acetylglucosamine and D-glucosamine units, and % DD is calculated as the molar fraction of N-acetylglucosamine units in its chain.
% DD = (n1 / (n1 + n2)) × 100 (1)
For the determination of % DD, both titration method and Fourier Transform Infrared Spectroscopy (FTIR) approach are employed, using Equations (2) and (3) respectively.
% DD = (c1V1 × c2V2) / (M × (100 - W)) × 0.016× 100/9.94 (2)
% DD = 100 × (1 - A1655 / A3450 × 1/1.33) (3)
In Equations (2) and (3), n1 and n2 represent the average numbers of D-glucosamine and N-acetylglucosamine units, c1 and c2 represent the concentrations of HCl and NaOH, V1 and V2 represent the volumes of HCl and NaOH added, M represents the mass of the sample, and W represents the moisture content. The factor 0.016 in Equation (2) corresponds to the amount of amino group in 1 M HCl aqueous solution, and the theoretical NH2 percentage in chitosan is 9.94%. The absorbance values A1655 and A3450 at wavelengths 1655 cm-1 and 3450 cm-1, respectively, are calculated independently using Equation (4):
A = 2 × log (%T) (5)
where A and T represent the absorbance and transmittance, respectively.