GB/T 40173-2021.Determination of reducing-end sugar in water-soluble chitosan-Spectrophotometric method.
1 Scope
GB/T 40173 specifies the spectrophotometric determination method for reducing end sugar in water-soluble chitosan.
GB/T 40173 is applicable to the detection of reducing end sugar in water-soluble chitosan (content ≥90%, deacetylation degree ≥90%).
GB/T 40173 has a detection limit of 0.006mmol/g and a quantification limit of 0.023mmol/g.
2 Normative references
The contents of the following documents constitute essential clauses of this document through normative references in the text. Among them, for dated references, only the version corresponding to that date applies to this document; for undated references, the latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water for analytical laboratories
3 Terms and definitions
3.1
Chitosan
The derivative obtained by deacetylation of chitin, insoluble in water, soluble in most dilute acids, chemical name is β-1,4-2-amino-2-deoxy-D-glucan.
3.2
Deacetylation degree
The percentage of sugar residues in chitosan molecules that have been deacetylated to the total number of sugar residues in chitosan molecules.
3.3
Water-soluble chitosan
Low-polymerization chitosan that is soluble in acidic, neutral and alkaline solutions.
4 Principle
The reducing end sugar at the end of chitosan molecule reacts with 3-methyl-2-benzothiazolone hydrazone (MBTH) under alkaline conditions to form azine. Under acidic conditions, the excess MBTH is oxidized by Fe3+ to form cations, which then react with azine to form a cyan compound. The concentration of the reducing end sugar is in a positive linear relationship with its absorbance at 590nm within a certain range. The absorbance value at 590nm is measured and quantified by the external standard method.
This document specifies the spectrophotometric determination method of reducing end sugar in water-soluble chitosan. This document is applicable to the detection of reducing end sugar in water-soluble chitosan (content ≥90%, deacetylation degree ≥90%). The detection limit of this method is 0.006 mmol/g, and the quantification limit is 0.023 mmol/g.

ICS07.080
CCS A 40
National Standard of the People's Republic of China
GB/T40173—2021
Determination of reducing-end sugar in water-soluble chitosan-Spectrophotometric method
Issued on 2021-05-21
State Administration for Market Regulation
National Standardization Administration
Implementation on 2021-12-01
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GB/T40173—2021
This document is drafted in accordance with the provisions of GB/T1.12020% "Guidelines for Standardization Work Part 1: Structure and Drafting Rules for Standardization Documents".
Please note that some contents of this document may involve patents: the issuing organization of this document does not assume the responsibility for identifying patents. This document is proposed and managed by the National Technical Committee for Standardization of Biochemical Testing (SAC/TC387). The drafting units of this document: Qingdao University of Science and Technology, Institute of Biology, China Institute of Testing Technology, Qingdao Institute of Metrology Technology, Wuhan Xinhuayang Biological Co., Ltd., Zhengren Investment Co., Ltd. Qingdao Branch, Qingdao Weilan Biological Group Co., Ltd., Shenzhen Market Supervision and Administration Bureau License Inspection Center.
The main drafters of this document: Zhang Yongqin, Kou Xingshuang, Li Huaiping, Li Jian, Wu Faxing, Xu Li, Gan Fei, Shao Jing, Zhang Fengguo, Zhan Zhichun, Ma Lixia, Bi Yifen, Zhou Ying, Xia Gu, Jiang Zijing, Xu Wenting, Zhang Jing, Xing Mingxia, Tu Jialin, Li Yuli. rrKaeerkAca-
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1 Scope
Determination of reducing terminal sugars in water-soluble chitosan spectrophotometric method
This document specifies the spectrophotometric determination method for reducing terminal sugars in water-soluble chitosan: GB/T 40173—2021
This document is applicable to the detection of reducing terminal sugars in water-soluble chitosan (content 90%, deacetylation degree 90%). The detection limit of this method is 0.006mmol/g and the quantification limit is 0.023mmol/g2 Normative referenced documents
The contents of the following documents constitute the indispensable terms of this document through normative references in the text. For the referenced documents with a date, the version corresponding to that issue applies to this document: for the referenced documents without an issue II, the latest version (including all amendments) applies to this document.
Analysis and experimental air and water specifications and test methods GB/T 6682
3 Terms and definitions
Chitosan
Derivatives obtained after deacetylation of chitin: insoluble in water, soluble in most dilute acids, chemical name β-1.4-2-amino-2-deoxy-D-glucan.
Degree of deacetylation The number of sugar residues in chitosan fractions that have been deacetylated accounts for the total number of sugar residues in chitosan fractions. 3.3
Water-soluble chitosan
Water-soluble chitosan
Low-polymerization chitosan that is soluble in acidic, neutral and alkaline solutions. 4 Principle
The reducing sugar at the end of chitosan molecule reacts with 3-hydroxy-2-thiazolidine (MBTH) under acidic conditions to generate azine. Excess MBTH is oxidized by Fe\ to form cations under acidic conditions, and then reacts with azine to generate a cyan compound. The concentration of the reducing end group is proportional to the absorbance at 590nm within a certain range: By measuring the absorbance at 590nm, the external standard method is used for quantification:
5 Reagents and materials
Unless otherwise specified, the reagents used in this method are analytically pure. Water is grade _ water in accordance with GB/T6682. 5.13 Methyl 2-methoxythiazolinone hydrochloride monohydrate (VBTII.C.II.N,S·IICI·IIO): CAS No.: 38894110.1
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GB/T40173—2021
Purity 98%
5.2 DL-dithiothreitol [DTT,HSCH,CH(OH)CH(OH)CHSHJ: CAS No.: 3483-12-3, purity 98%. 5.3 Glucosamine hydrochloride standard (C,H.NO·HCI): CAS No.: 66-84-2, purity 99%, or standard substances certified by the state and awarded with standard substance certificates.
5.4 Concentrated hydrochloric acid.
5.5 Sodium hydroxide solution (0.5mol/L): Weigh 2g sodium hydroxide. Dissolve in water, dilute with water and make up to 100ml, mix, 5.6M13TH solution: Weigh 0.3gM13TH, dissolve in water, dilute with water and make up to 100ml, mix, store at 4℃ away from light, valid for 10 days.
5.7DTT solution: Weigh 0.080gITT, dissolve in water, dilute with water and make up to 100ml. Mix, store at 1℃ away from light. valid for 10 days.
5.8MBTII reagent: Mix equal volumes of MBTII solution (5.6) and DTT solution (5.7) to obtain MBTII reagent, freshly prepared before use. 5.9 Ammonium ferric sulfate reagent: Weigh 5g ammonium ferric sulfate [NH4Fe(S03)·12H03_ and 5g aminosulfonic acid. Add 41.8mL concentrated hydrochloric acid and wait until fully dissolved until clear. Dilute with water and make up to 1000ml. Mix. Store at 1 in dark place. The shelf life is 1 hour. 5.10 Glucosaminoglycan hydrochloride standard solution (0.1mg/mL): Weigh 0.1g Glucosaminoglycan hydrochloride (accurate to 0.0001g). Dissolve in water, dilute with water and make up to 100ml. Mix (concentration is 1mg/mL); accurately aspirate 10ml of the above solution. Dilute with water and make up to 100ml. Mix well and prepare fresh before use.
6 Instruments and equipment
6.1 Analytical balance: sensitivity is 0.01g and 0.0001g: 6.2 Constant temperature oscillating water bath: temperature control accuracy is ±0.2℃. 6.3
Vortex oscillator.
6.4 Spectrophotometer: equipped with 1cm colorimetric mL
7 Preparation of sample solution
7.1 Preparation of sample
Accurately weigh about 0.1g of sample (accurate to 0.0001g), add about 50ml of water, vortex to dissolve it, add water to make up to 100ml, let it stand, and take the supernatant as the sample solution. 7.2 Preparation of standard working solution
Take 0mL, 1mL, 3mL, 5mL, 7mL, and 9mL of 0.1mg/mL aminoinulin hydrochloride standard solution. Dilute to 10ml with water respectively. Prepare standard working solutions with concentrations of 0mg/ml, 0.01mg/ml, 0.03mg/ml, 0.05mg/ml, 0.07mg/ml, and 0.09mg/ml.
8 Determination steps
8.1 Preparation of standard curve
List Pipette 500ul of glucosamine hydrochloride standard T solution, add 500ul 0.5mol/L sodium hydroxide solution and 500ul MBTH reagent into a colorimetric tube, mix well, place in 80° water bath for 13min, quickly add 1000uL ammonium ferric sulfate reagent while hot, mix well, and let stand for 1h to develop color: adjust the zero point with water, and measure the absorbance at a wavelength of 590nm: draw a curve with the mass concentration of glucosamine hydrochloride as the horizontal axis and the difference between the absorbance of the standard working solution and the absorbance of the blank tube with zero mass concentration as the vertical axis. 2
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2 Determination of sample solution
GB/T40173—2021
Pipette 500ml of sample solution and add 500ml.0.5mol/1. sodium hydroxide solution and 500l.M13TH reagent into a test tube and mix well: place in a water bath at 80℃ for 13min, quickly add 1000uL ammonium ferric sulfate reagent while hot. Mix well and let stand for 1h to develop color. Adjust the zero point with water and measure the absorbance value at a wavelength of 590nm: substitute the difference between the absorbance value of the sample to be tested and the absorbance value of the blank tube with zero mass concentration into the standard curve. Calculate the content of reducing end sugar in the sample, and calculate and express the results
Calculation of results
The content of reducing end sugar in the sample is calculated according to formula (1) X_Ar-An-C.
Wherein:
The content of reducing end sugar in the sample, in millimole per gram (mmol/g): Absorbance value of the sample to be tested;
Absorbance value of the blank tube;
Intercept of the standard curve;
Slope of the standard curve, in milliliter per milligram (mL/mg): Mass concentration of water-soluble chitosan, in milligram per milliliter (mg/mL): The mass of glucosamine hydrochloride.215.63g/mol. 9.2 Result expression
The calculated result is expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions. The result retains one significant figure. 10 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. -rKaeerkca-
GB/T 40173-2021
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People's Republic of China
National Standard
Determination of reducing end sugar in water-soluble chitosan by spectrophotometry
GB/T4C1732021
Published by China Standards Press
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First edition in May 2021
Book number: 155066: 1-67376
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