Determination of Chitin Based on the Colorimetric Assay of Glucosamine in Acidic Hydrolysate
Determination of Chitin Based on the Colorimetric Assay of Glucosamine in Acidic Hydrolysate
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DOI：
10.2116/analsci.32.701
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发表时间：
2016-06-01
影响因子：
1.6
通讯作者：
Kimoto, Hisashi
中科院分区：
文献类型：
--
作者：
Katano, Hajime;Takakuwa, Masahiro;Kimoto, Hisashi
氨基葡萄糖（GlcN）比色法测定甲壳素，它可以水解产生GlcN。将10 mg样品与10 mL 5 mol/L HCl水溶液混合，并将混合物在100 ℃下保持12 h。在这些条件下，甲壳素完全解聚和脱乙酰化产生GlcN，即使样品是蟹壳。将20 μ L等份水解产物与20 μ L 5 mol/L NaOH水溶液和200 μ L 50 mmol/L Na 2SiO 3、600 mmol/L Na 2 MoO 4、1.5 mol/L CH 3COOH和30%（v/v）二甲亚砜溶液混合。将混合物在70 ℃下保持30 min。在混合物中，GlcN还原Mo（VI）物质以形成蓝色的正硅酸根阴离子，其在约750 nm处产生最大吸光度。由于N-乙酰氨基葡萄糖和甲壳素寡糖不能使反应混合物呈蓝色，因此可以高选择性地比色测定水解物中的GlcN。当检查标准甲壳质样品时，水解产物中的GlcN浓度测定为0.97 +/-0.02g/L（作为盐酸盐），表明样品含有10.0 +/-0.2mg甲壳质（作为N-乙酰葡糖胺均聚物）。钙离子、氨基酸和蛋白质不干扰GlcN测定。该方法已成功应用于蟹壳样品中甲壳素的测定。
A colorimetric method for the glucosamine (GlcN) assay was applied for the determination of chitin, which can be hydrolyzed to produce GlcN. A 10-mg sample was mixed with 10 mL of a 5 mol/L HCl aqueous solution, and the mixture was kept at 100 degrees C for 12 h. Under these conditions, chitin was completely depolymerized and deacetylated to produce GlcN, even when the sample was a crab shell. A 20-mu L aliquot of the hydrolysate was mixed with 20 mu L, of a 5 mol/L NaOH aqueous solution and 200 mu L of a 50 mmol/L Na2SiO3, 600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH and 30% (v/v) dimethyl sulfoxide solution. The mixture was kept at 70 degrees C for 30 min. In the mixture, GlcN reduced the Mo(VI) species to form a blue molybdosilicate anion, which gave an absorbance maximum at around 750 nm. Since N-acetylglucosamine and chitin oligosaccharides could not render the reaction mixture blue, GlcN in the hydrolysate could be assayed colorimetrically with high selectivity. When a standard chitin sample was examined, the GlcN concentration in the hydrolysate was determined to be 0.97 +/- 0.02 g/L (as hydrochloride salt), indicating that the sample contained 10.0 +/- 0.2 mg chitin (as an N-acetylglucosamine homopolymer). Calcium cation, amino acids, and proteins did not interfere with the GlcN assay. Thus, the proposed method was successfully applied to determine chitin in a crab shell sample.