WO2018040821A1 - Method for measuring degree of deacetylation of chitosan oligosaccharide by using first-order derivative ultraviolet spectrophotometry - Google Patents

Abstract

A method for measuring the degree of deacetylation of a chitosan oligosaccharide by using first-order derivative ultraviolet spectrophotometry, comprising the steps of 1) with a hydrochloric acid solution having a concentration of 0.3-1.0 mol/L as a blank solvent, preparing standard solutions of N-acetyl-D-glucosamine at a series of concentrations, measuring the ultraviolet absorbance values A of the standard solutions of N-acetyl-D-glucosamine within a wavelength range of 200-206 nm, and plotting a standard curve according to the concentrations of N-acetyl-D-glucosamine and ΔA/Δλ; and 2) dissolving a chitosan oligosaccharide sample with the blank solvent, with the blank solvent as a reference, measuring the absorbance value of the chitosan oligosaccharide sample at a wavelength of 200-206 nm, and calculating the degree of deacetylation of the chitosan oligosaccharide sample according to the standard curve.

Description

Method for determining chitosan oligosaccharide deacetylation degree by first derivative ultraviolet spectrophotometry Technical field

The invention belongs to the field of chemistry, and in particular relates to a method for determining the degree of deacetylation of chitosan oligosaccharides by first derivative ultraviolet spectrophotometry.

Background technique

Chitin is the second largest bio-polysaccharide on the earth after cellulose. It is mainly found in shrimp, crab, insect carapace, fungus, plant cell wall, animal joints and other hard parts, and muscle and bone joints. Chitosan oligosaccharide (COS) is a homopolymer or heteropolymer of glucosamine and N-acetylglucosamine linked by β-1,4 glycosidic bonds, which is degraded by chitin. The removal of more than 55% of the N-acetyl group of chitin is chitosan (CTS), and the CTS continues to degrade to 2-10 degrees of polymerization, which is COS. In actual production, 20 molecules with a degree of polymerization (about 3 kDa) are also called COS.

Compared with chitosan, chitosan oligosaccharides have the advantages of better solubility, lower viscosity and easier absorption by the human body, and have wide applications in medicine, agriculture, fine chemicals and other fields. Chitosan oligosaccharide not only has anti-tumor, antibacterial, anti-oxidant and other functions, but also has biological functions such as weight loss, lipid regulation and immunity enhancement. The research and development of chitosan oligosaccharides is currently a research hotspot.

The degree of deacetylation of chitosan oligosaccharides refers to the percentage of the number of saccharide residues in the total number of sugar residues in chitosan oligosaccharides, which can affect the biological, physical and chemical functions and activities of chitooligosaccharides. It is a manifestation of various functions of chitooligosaccharides and is one of the important indicators for measuring the quality of chitosan oligosaccharides.

At present, there are many mature methods for determining the degree of deacetylation of chitosan. For example, the 2015 edition of the Pharmacopoeia of the People's Republic of China, Part 4, 511-512, discloses the determination of shells by methyl orange acid base indicator method. Method for deacetylation degree of glycans; European Pharmacopeia 8.0 [M]. EDQM, 2014: 1841-1842) discloses the use of purified water as a solvent, using a first derivative ultraviolet A method for determining the degree of deacetylation of chitosan by photometric method. Chinese Patent Application No. 201310587162.5 discloses a method for determining the degree of deacetylation of a mixture of chitosan and chitooligosaccharide, comprising the steps of: dissolving a sample to be detected with dilute hydrochloric acid while using fully deacetylated chitosan oligosaccharide as an internal standard The absorbance at 199 nm of violet light was measured, and the degree of deacetylation of the sample was obtained according to a standard curve. A 0.001 mol/L hydrochloric acid solution was used as a solvent during the test.

Chitosan and chitosan oligosaccharides, because of the large difference in the nature of the two, the determination method of chitosan deacetylation degree is not necessarily suitable for the determination of chitosan oligosaccharide deacetylation degree. According to the determination method of chitosan deacetylation degree in the fourth edition of the 2015 edition of the Pharmacopoeia of the People's Republic of China, the pH of the indicator methyl orange is in the range of 3.1-4.4, and the acid color is red. The basic color is yellow. On the one hand, the aqueous solution of the chitosan oligosaccharide sample itself is pale yellow, and methyl orange is used as an indicator, so that the end point of the titration cannot be accurately judged; on the other hand, the isoelectric value of chitosan oligosaccharide is about 4.80. Exceeding the pH range of methyl orange, therefore, according to the 2015 edition of the Chinese Pharmacopoeia, the degree of deacetylation of chitosan oligosaccharides determined by the methyl orange acid base indicator method is not obvious, the reproducibility is poor, and the error is large. Disadvantages. Determination of chitosan deacetylation degree by reference to the European Pharmacopoeia When chitosan oligosaccharide deacetylation degree is determined by using water or 0.001 mol/L hydrochloric acid solution as a solvent, chitosan oligosaccharide has no maximum absorption wavelength, and therefore, the first derivative of the European Pharmacopoeia is disclosed. Ultraviolet spectrophotometry cannot be applied to the determination of chitosan oligodeacetylation degree.

Nuclear magnetic resonance spectroscopy ( ¹ H-NMR) has been included in the US Pharmacopoeia as the gold standard for determining the degree of deacetylation of chitosan. At the same time, the papers published by Kim et al., "Oligosaccharides and Their Derivatives" and Wang Shixin, are different. The mass analysis of the chitosan oligosaccharide product of the source discloses that the degree of deacetylation of chitosan oligosaccharide can be determined by nuclear magnetic resonance spectroscopy, and the error of the measurement result is small. However, the ¹ H-NMR method cannot be widely used because of its high instrument cost and the need for professional technicians.

At present, standard methods for determining the degree of deacetylation of chitooligosaccharides have not been established at home and abroad. Therefore, it is necessary to establish a rapid, convenient and accurate method for determining the degree of deacetylation of chitooligosaccharides.

Summary of the invention

In order to solve the problems existing in the prior art, the inventors conducted a large number of experiments and studies, and unexpectedly found that when the first derivative UV spectrophotometric method is used to determine the degree of deacetylation of chitooligosaccharides, the concentration of hydrochloric acid is 0.30 mol/mL. And above dilute hydrochloric acid as a solvent, chitosan oligosaccharide has the largest wavelength. Based on the above findings, the present invention has been completed.

The object of the present invention will be further embodied and illustrated by the following detailed description.

The invention provides a method for determining the degree of deacetylation of chitosan oligosaccharides by first derivative ultraviolet spectrophotometry, comprising the following steps:

1) A series of concentrations of N-acetyl-D-glucosamine standard solution were prepared with a concentration of 0.3-1.0 mol/L hydrochloric acid solution as a blank solvent, and different concentrations of N-acetyl-D were determined by using a blank solvent as a reference. - UV absorbance value A of the glucosamine standard solution in the wavelength range of 200-206 nm, a standard curve is drawn according to the concentration of N-acetyl-D-glucosamine and ΔA/Δλ, where ΔA=A _λ+1 —A _λ-1 , λ is 203-205 nm, Δλ=2 nm;

2) Taking the chitosan oligosaccharide sample, dissolving it with a blank solvent, taking the blank solvent as a reference, measuring the UV absorbance at a wavelength of 200-206 nm, and calculating the N-acetyl-D-glucosamine in the chitosan oligosaccharide sample according to the standard curve. Concentration, and calculate the degree of deacetylation of chitosan oligosaccharides according to the following formula:

Where DD% is the degree of deacetylation, %; C ₁ is the concentration of chitooligosaccharide sample, μg/mL; C ₂ is the concentration of N-acetyl-D-glucosamine in the chitosan oligosaccharide sample, μg/mL; M ₁ Is the molecular weight of 203, N-acetyl-D-glucosamine; 42 is the difference between the molecular weight of N-acetyl-D-glucosamine and the molecular weight of D-glucosamine.

Preferably, the concentration of the blank solvent is 0.3 mol/L.

Preferably, in step 1), the concentration of the N-acetyl-D-glucosamine standard solution is 1.60, respectively. Μg/mL, 20.0 μg/mL, 32.0 μg/mL, 40.0 μg/mL, 64.0 μg/mL, and 80.0 μg/mL.

Preferably, in step 1), λ is 204 nm.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The method for determining the degree of deacetylation of chitosan oligosaccharide by the first derivative ultraviolet spectrophotometry provided by the present invention, using a hydrochloric acid solution having a concentration of 0.3-1.0 mol/L as a solvent, using a first derivative ultraviolet spectrophotometry The content of N-acetyl-D-glucosamine in the sample was measured, and the degree of deacetylation of chitooligosaccharide was directly determined. The measurement result showed good linearity in the range of 0.0016-0.08 mg/mL (R ² =0.9992), relative standard deviation. 0.19%-0.27%, the recovery rate is 99.5%-100.3%; at the same time, compared with the measurement result of ¹ H-NMR method, the relative error is controlled within 0.08%, indicating the determination of chitosan oligosaccharide deacetylation degree provided by the present invention The method has high accuracy and practicality.

(2) The method for determining the degree of deacetylation of chitosan oligosaccharide by the first derivative ultraviolet spectrophotometry provided by the invention eliminates the interference of the coexisting D-glucosamine, turbidity and chromaticity, and improves the accuracy of the determination result. And sensitivity, the sample does not require special handling, and the operation is simple and easy.

(3) The method for determining the degree of deacetylation of chitosan oligosaccharide by the first derivative ultraviolet spectrophotometry provided by the present invention, wherein the relative standard deviation (RSD) of the same sample is controlled within 0.04%, indicating the chitosan oligosaccharide provided by the present invention The method of measuring the degree of deacetylation has good reproducibility and good precision.

DRAWINGS

Figure 1 shows the UV absorption spectra of N-acetyl-D-glucosamine, D-glucosamine, COS _MW1000 and COS _MW3000 in the range of 200 nm to 400 nm with purified water as solvent.

Figure 2 _shows the UV absorption spectra of COS _MW1000 in the range of 200 nm to 400 nm with different concentrations of hydrochloric acid solution as solvent.

Figure 3 _shows the UV absorption spectra of COS _MW3000 in the range of 200 nm to 400 nm with different concentrations of hydrochloric acid solution as solvent.

Figure 4 shows the ultraviolet absorption spectrum of N-acetyl-D-glucosamine, D-glucosamine, COS _MW1000 and COS _MW3000 in the range of 200 nm to 400 nm using a 0.30 mol/L hydrochloric acid solution as a solvent.

Figure 5 UV first-order derivative spectra of N-acetyl-D-glucosamine, D-glucosamine, COS _MW1000 and COS _MW3000 in the range of 200-210 nm.

Figure 6. First-order derivative ultraviolet spectrophotometric standard curve.

Figure 7 is a nuclear magnetic resonance spectrum (500 MHz) of COS _MW1000 , wherein AE represents the C2-C6 hydrogen signal on the sugar ring.

Figure 8 is a nuclear magnetic resonance spectrum (500 MHz) of COS _MW3000 , wherein AE represents the C2-C6 hydrogen signal on the sugar ring.

detailed description

The invention will be further described in detail below by way of specific examples.

The raw materials used in the examples of the present invention are all commercially available products, and some of the types and sources of equipment involved are as follows:

name	manufacturer
T6 New Century UV-Vis Spectrophotometer	Beijing Pu Analysis General Instrument Co., Ltd.
DK-8D type one thousandth electronic analytical balance	German Sartorius Group
Bruker 500MHz NMR	Bruker

Example 1 Determination of Solvent Selection

The inventors of the present invention found in the course of the determination of chitosan oligosaccharide according to the first derivative ultraviolet spectrophotometric method in the European Pharmacopoeia, using purified water as a solvent and N-acetyl-D-glucose. The amine is an internal standard, and is N-acetyl-D-glucosamine, D-glucosamine, COS _MW1000 (chitosan oligosaccharide sample with deacetylation degree ≥90%, average molecular weight _{≤1000) in the} wavelength range of 200 nm to 400 nm. COS _MW3000 (chitosan oligosaccharide sample with deacetylation degree ≥90% and average molecular weight ≤3000) was scanned. The scanning results are shown in Fig. 1. Curve 1 is the UV scan curve of D-glucosamine, and curve 2 is the UV of COS _MW3000 . Scan curve, curve 3 is the UV scan curve of COS _MW1000 , and curve 4 is the UV scan curve of N-acetyl-D-glucosamine. It can be seen from the results of Fig. 1 that according to the first derivative ultraviolet spectrophotometry in the European Pharmacopoeia, when purified water is used as a solvent, the chitosan oligosaccharide has no maximum absorption wavelength, and the first derivative ultraviolet spectrophotometry disclosed in the European Pharmacopoeia cannot be applied to chitooligosaccharides. Determination of the degree of deacetylation.

The inventors conducted a large number of experiments and studies, and unexpectedly found that the first derivative UV spectrophotometric method was used to determine the degree of deacetylation of chitosan oligosaccharides, using dilute hydrochloric acid with a hydrochloric acid concentration of 0.30 mol/mL or more as a solvent, chitosan oligosaccharide. Has the maximum absorption wavelength.

COS _MW1000 and COS _MW3000 were scanned in the wavelength range of 200 nm to 400 nm with different concentrations of dilute hydrochloric acid solution as a solvent. The scanning results are shown in Fig. 2 and Fig. 3. In FIG. 2, curves 1-5 are ultraviolet scanning curves of COS _MW1000 at a concentration of 0.05 mol/mL, 0.10 mol/mL, 0.15 mol/mL, 0.30 mol/mL, and 0.50 mol/mL hydrochloric acid solution respectively as a solvent; In Fig. 3, curves 1-5 are ultraviolet scanning curves of COS _MW3000 at a concentration of 0.05 mol/mL, 0.10 mol/mL, 0.15 mol/mL, 0.30 mol/mL, and 0.50 mol/mL hydrochloric acid solution as a solvent, _respectively .

2 and 3, the chitosan oligosaccharide has a maximum absorption wavelength when the concentration of hydrochloric acid reaches 0.30 mol/mL or more. Therefore, the degree of deacetylation of chitosan oligosaccharides can be determined by first-order derivative ultraviolet spectrophotometry using dilute hydrochloric acid having a hydrochloric acid concentration of 0.30 mol/mL or more as a solvent.

Example 2 Determination of wavelength

Chinese Patent Application No. 201310587162.5 discloses a method for determining the degree of deacetylation of chitosan and chitosan oligosaccharide mixture by measuring the absorbance at 199 nm violet light, but it was found that N-acetyl-D-glucosamine and chitooligosaccharide were There is terminal absorption at 199 nm, which causes interference to the measurement, and the error is large. Therefore, the present invention selects a wavelength of 200 nm to 400 nm as a research object.

40μg/mL N-acetyl-D-glucosamine, 40μg/mL D-glucosamine, 0.4mg/mL COS _MW1000 , 0.2mg/mL COS _{MW3000 were prepared with 0.30mol} /mL hydrochloric acid solution as solvent. Scanning in the wavelength range of 200nm-400nm, the results are shown in Figure 4, where curve 1 is the UV scan curve of D-glucosamine, curve 2 is the UV scan curve of N-acetyl-D-glucosamine, curve 3 is COS The UV scan curve of _MW3000 and curve 4 are the UV scan curves of COS _MW1000 . As can be seen from the results in Fig. 4, N-acetyl-D-glucosamine has the maximum absorption at 204 nm, but at the wavelength of 204 nm, the coexisting D-glucosamine also absorbs, which interferes with the determination of N-acetyl-D-glucosamine. Therefore, first-order derivative ultraviolet spectrophotometry is used to eliminate interference.

The ultraviolet absorption spectra of N-acetyl-D-glucosamine, D-glucosamine, COS _MW1000 and COS _MW3000 in the range of 200 nm to 210 nm were converted into first derivative spectra, and the results are shown in Fig. 5, wherein curve 1 is D- Glucosamine, curve 2 is COS _MW3000 , curve 3 is COS _MW1000 , and curve 4 is N-acetyl-D-glucosamine. It can be seen from the results of FIG. 5 that N-acetyl-D-glucosamine has absorption in the range of 203 nm to 206 nm, and has maximum absorption at 204 nm, while D-glucosamine has no absorption in the range of 200 nm to 210 nm. Therefore, one is used. Determination of the degree of deacetylation of chitosan oligosaccharides by the derivative derivative ultraviolet spectrophotometry can eliminate the interference of the coexisting D-glucosamine.

Example 3 Determination of Chitosan Oligosaccharide Deacetylation Degree by First Derivative Ultraviolet Spectrophotometry

1) Establishment of the standard curve

A hydrochloric acid solution having a concentration of 0.3 mol/L was used as a blank solvent, and a standard solution of 2.0 mg/mL N-acetyl-D-glucosamine was placed, and then diluted with a 0.3 mol/L hydrochloric acid solution to prepare a solution of 1.60 μg/mL and 20.0 μg. /mL, 32.0μg/mL, 40.0μg/mL, 64.0μg/mL, 80.0μg/mL N-acetyl-D-glucosamine standard solution, with 0.3mol/L hydrochloric acid solution as reference, with 1cm quartz For the cuvette, the UV absorbance value A of different concentrations of the N-acetyl-D-glucosamine standard solution in the wavelength range of 200-206 nm was determined, and a standard curve was drawn according to the concentration of N-acetyl-D-glucosamine and ΔA/Δλ. _{where, ΔA = A 205nm -A 203nm,} Δλ = (205-203) nm = 2nm; standard curve shown in Figure 6, wherein the regression equation ^{Y = 0.0021X-0.003, R 2} = 0.9992, N- acetylamino Glucosamine is linear in the range of 0.0016-0.08 mg/mL.

2) Determination of the degree of deacetylation of chitosan oligosaccharides

1.0 g of COS _MW1000 and COS _MW3000 were accurately weighed and _dissolved in a 0.3 mol/L hydrochloric acid solution, and then made up to 250 mL as a stock solution. Using a 0.3 mol/L hydrochloric acid solution as a reference, the UV absorbance value was measured at a wavelength of 200-206 nm, and the concentration of N-acetyl-D-glucosamine in the chitosan oligosaccharide sample was calculated according to a standard curve, and the shell was calculated according to the following formula. Degree of deacetylation of oligosaccharide samples:

Where DD% is the degree of deacetylation, %; C ₁ is the concentration of chitooligosaccharide sample, μg/mL; C ₂ is the concentration of N-acetyl-D-glucosamine in the chitosan oligosaccharide sample, μg/mL; M ₁ Is the molecular weight of 203, N-acetyl-D-glucosamine; 42 is the difference between the molecular weight of N-acetyl-D-glucosamine and the molecular weight of D-glucosamine.

3) Measurement results: the degree of deacetylation of COS _MW1000 is 93.45±0.04%; the degree of deacetylation of COS _MW3000 is 92.88±0.03%. The method for determining the degree of deacetylation of chitosan oligosaccharide by the first derivative ultraviolet spectrophotometry is provided by the invention, and the degree of deacetylation of the same batch of chitosan oligosaccharide samples is determined, and the relative standard deviation of the determination results is within 0.04%, which indicates that the present invention provides The method is precise.

Example 4 Investigation of recovery rate

Since chitosan oligosaccharides have no standard, the shells representing different degrees of deacetylation are mixed in different proportions of the two basic constituent N-acetyl-D-glucosamine and D-glucosamine in the chitosan oligosaccharide polymer. Oligosaccharides. 0.50 mL of N-acetyl-D-glucosamine standard solution 0.50 mL, 0.75 mL, and 1.00 mL were accurately weighed into a 100 mL volumetric flask, and D-glucosamine was added to the above volumetric flask to 0.0063 g and 0.0063 g, respectively. 0.0051 g was formulated into a chitosan oligosaccharide solution equivalent to a degree of deacetylation of 92.2%, 88.7% and 82.7%. The degree of deacetylation of the chitosan oligosaccharide solution was determined by the method described in the step 2) of Example 3, and the recovery was calculated. The results are shown in Table 1.

Table 1 Experimental results of determination of chitosan oligosaccharide deacetylation recovery by first derivative ultraviolet spectrophotometry

It can be seen from the results of Table 1 that the method for determining the degree of deacetylation of chitosan oligosaccharide by the first derivative ultraviolet spectrophotometry provided by the present invention has good recovery rate, and the determination result of the chitosan oligosaccharide deacetylation degree is high.

Comparative Example 1 ¹ H-NMR method for determination of degree of deacetylation

1) Determination of degree of deacetylation by ¹ H-NMR

Separately weigh 20mg COS _MW1000 and COS _MW3000 dissolved in 5mL of D ₂ O (99.96%) to obtain a sample solution with a concentration of 4mg/mL. Transfer the obtained sample solution to 8mm nuclear magnetic tube for measurement. The resonance frequency is 500MHz. The measured temperature is 297K, and finally the target signal is integrated. The hydrogen signal (Acetyl-H) of the acetyl group in the C2 acetamido group on the sugar ring is 1.9-2.1 ppm, and the C2-C6 hydrogen signal on the sugar ring is 2.6- 6.0ppm. The NMR spectra of COS _MW1000 and COS _MW3000 are shown in Figure 7, Figure 8. According to Table 2, the corresponding peaks of the nuclear magnetic resonance spectrum are integrated, and the calculation formula of the degree of deacetylation is:

DD(%)={1-[(7*A ₂ )/(3*A ₁ )]}*100

Wherein A ₂ represents an integral value of three hydrogen signals of an acetyl group at the C2 acetylamino group on the sugar ring; and A ₁ represents an integral value of a hydrogen signal at the C2-C6 position on the sugar ring.

Table 2 Hydrogen proton chemical shift in aqueous solution of chitosan oligosaccharide at 25 °C

The determination results of the deacetylation degree of COS _MW1000 and COS _MW3000 by using ¹ H-NMR method were 93.52±0.13 and 92.81±0.07, respectively.

2) Comparison of ¹ H-NMR method with the results of the acid-base indicator method of the present invention

^The results of the comparison between the ¹ H-NMR method and the acid-base indicator method of the present invention are shown in Table 3.

Table 3 ¹ H-NMR comparison with the acid-base indicator method of the present invention (n=6)

sample	1 H-NMR (%)	First derivative ultraviolet spectrophotometry (%)	Relative error
COS MW1000	93.52±0.13	93.45±0.04	0.07±0.69
COS MW3000	92.81±0.07	92.88±0.03	0.08±0.57

It can be seen from the results in Table 3 that the determination results of the chitosan oligosaccharide deacetylation degree by the first derivative ultraviolet spectrophotometry of the present invention are consistent with the results of the ¹ H-NMR method, and the relative error is controlled within 0.08%, indicating that the present invention provides The first derivative ultraviolet spectrophotometry is used to determine the degree of chitosan oligosaccharide deacetylation.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (4)

1. A method for determining the degree of deacetylation of chitosan oligosaccharides by first derivative ultraviolet spectrophotometry, comprising the steps of:

   1) A series of concentrations of N-acetyl-D-glucosamine standard solution were prepared with a concentration of 0.3-1.0 mol/L hydrochloric acid solution as a blank solvent, and different concentrations of N-acetyl-D were determined by using a blank solvent as a reference. - UV absorbance value A of the glucosamine standard solution in the wavelength range of 200-206 nm, a standard curve is drawn according to the concentration of N-acetyl-D-glucosamine and ΔA/Δλ, where ΔA=A _λ+1 —A _λ-1 , λ is 203-205 nm, Δλ=2 nm;

   2) Taking the chitosan oligosaccharide sample, dissolving it with a blank solvent, taking the blank solvent as a reference, measuring the UV absorbance at a wavelength of 200-206 nm, and calculating the N-acetyl-D-glucosamine in the chitosan oligosaccharide sample according to the standard curve. Concentration, and calculate the degree of deacetylation of chitosan oligosaccharides according to the following formula:

   

   Where DD% is the degree of deacetylation, %; C ₁ is the concentration of chitooligosaccharide sample, μg/mL; C ₂ is the concentration of N-acetyl-D-glucosamine in the chitosan oligosaccharide sample, μg/mL; M ₁ Is the molecular weight of 203, N-acetyl-D-glucosamine; 42 is the difference between the molecular weight of N-acetyl-D-glucosamine and the molecular weight of D-glucosamine.
2. The method for determining the degree of deacetylation of chitosan oligosaccharides by a first derivative ultraviolet spectrophotometry according to claim 1, wherein the concentration of the blank solvent is 0.3 mol/L.
3. The method for determining the degree of deacetylation of chitosan oligosaccharide by first derivative ultraviolet spectrophotometry according to claim 1, wherein the concentration of the N-acetyl-D-glucosamine standard solution in the step 1) They were 1.60 μg/mL, 20.0 μg/mL, 32.0 μg/mL, 40.0 μg/mL, 64.0 μg/mL, and 80.0 μg/mL, respectively.
4. The method for determining the degree of deacetylation of chitosan oligosaccharide by first derivative ultraviolet spectrophotometry according to claim 1, wherein in step 1), λ is 204 nm.

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