GB/T 40173-2021Determination of reducing-end sugar in water-soluble chitosan—Spectrophotometric method (English PDF)

1 Scope

This document specifies the spectrophotometric method for the determination of reducing-end sugar in water-soluble chitosan.

This document applies to the detection of reducing-end sugar in water-soluble chitosan (content >=90%, degree of deacetylation >=90%).

The detection limit of this method is 0.006 mmol/g; the quantitation limit is 0.023 mmol/g.

2 Normative references

The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. For dated references, only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable to this document.

GB/T 6682, Water for analytical laboratory use - Specification and test methods

3 Terms and definitions

4 Principle

The reducing-end sugar at the end of the chitosan molecule reacts with 3-methyl-2-MBTH under alkaline conditions to form azine. Under acidic conditions, excess MBTH is oxidized by Fe3+ to cations; then, it reacts with azine to produce a cyan compound. The concentration of the reducing-end sugar is in a linear relationship with its absorbance at 590 nm within a certain range. Quantify by external standard method by measuring the absorbance value at 590 nm.

5 Reagents and materials

Unless otherwise specified, the reagents which are used in this method are analytical reagents. The water is grade-3 water that meets the requirements of GB/T 6682.

5.1 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH, C8H9N3S·HCl·H2O): CAS number: 38894-11-0, purity >=98%.

5.2 DL-Dithiothreitol [DTT, HSCH2CH(OH)CH(OH)CH2SH]: CAS number: 3483-12-3, purity >=98%.

5.3 D-Glucosamine hydrochloride (C6H13NO5·HCl): CAS number: 66-84-2, purity >=99%, or a standard substance that is certified by the country and awarded with a certificate of standard substance.

5.4 Concentrated hydrochloric acid.

5.5 Sodium hydroxide solution (0.5 mol/L): Weigh 2 g of sodium hydroxide; add water to dissolve; use water to dilute and fix volume to 100 mL; mix well.

5.6 MBTH solution: Weigh 0.3 g of MBTH; add water to dissolve and dilute to 100 mL; mix well; store in the dark at 4 °C; it is valid for 10 days.

5.7 DTT solution: Weigh 0.080 g of DTT; add water to dissolve and dilute to 100 mL; mix well; store in the dark at 4 °C; it is valid for 10 days.

5.8 MBTH reagent: Mix MBTH solution (5.6) and DTT solution (5.7) in equal volumes to obtain MBTH reagent. Prepare when necessary.

5.9 Ferric ammonium sulfate reagent: Weigh 5 g of ferric ammonium sulfate [NH4Fe(SO4)2·12H2O] and 5 g of sulfamic acid; add 41.8 mL of concentrated […]