WO2018040820A1 - Process for measuring degree of deacetylation of chitosan oligosaccharide using acid-base indicator method - Google Patents

Abstract

A process for measuring the degree of deacetylation of a chitosan oligosaccharide using an acid-base indicator method, comprising the steps of S1. measuring the content of water in a chitosan oligosaccharide sample, as a reduced weight under dry weight loss item of the chitosan oligosaccharide sample; S2. weighing a chitosan oligosaccharide sample, dissolving the sample with distilled water, adjusting the pH to 8.0, adding a dilute hydrochloric acid titrant while adding 1-2 drops of a 1% bromocresol green indicator, and after uniform mixing, titrating the solution with a sodium hydroxide titrant until the solution becomes green, i.e. reaching the titration end point; and S3. calculating the degree of deacetylation of the chitosan oligosaccharide sample. The method is an accurate, rapid method for measuring the degree of deacetylation of a chitosan oligosaccharide; moreover, the instruments used are simple, the operation is simple, no special sample pretreatment is required, the titration end point is obviously determined, the measurement error is small, and the method is suitable for the quality control in the process of the preparation of a chitosan oligosaccharide.

Description

Method for determining chitosan oligosaccharide deacetylation degree by acid-base indicator method Technical field

The invention belongs to the field of chemistry, and in particular relates to a method for determining the degree of deacetylation of chitooligosaccharides by using an acid-base indicator method.

Background technique

Chitooligosaccharides are homopolymers or heteropolymers of glucosamine and N-acetylglucosamine linked by β-1,4 glycosidic bonds. Chitosan oligosaccharide is a degradation product of chitosan, but compared with chitosan, chitosan oligosaccharide has the advantages of better solubility, lower viscosity and easier absorption by the human body. It has a wide range of fields in medicine, agriculture, fine chemicals and other fields. application. 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 degree of deacetylation of chitooligosaccharides can affect the biological, physical and chemical functions and activities of chitosan oligosaccharides. It is a manifestation of various functions of chitooligosaccharides and one of the important indicators for measuring the quality of chitosan oligosaccharides. The degree of deacetylation of chitooligosaccharides refers to the percentage of the number of sugar residues from which the acetyl group is removed in the total number of sugar residues in chitosan oligosaccharides, and the content of free amino groups is the basis of its various functions.

At present, there are many methods for determining the degree of deacetylation of chitosan reported at home and abroad: including alkali method (acid-base titration, potentiometric titration, double-bump titration), infrared spectroscopy, colloidal titration, refractive index method Destruction sample method (elemental analysis, acid hydrolysis liquid chromatography and thermal analysis). Determination of chitosan deacetylation by methyl orange acid base indicator method has been included in the 2015 edition of the Chinese Pharmacopoeia, and the amount of chitosan was determined by alkali method using methyl orange-aniline blue (1:2) as an indicator. The method of acetyl content is widely used and has become the standard method for the Chinese aquatic industry issued by the Ministry of Agriculture. However, the determination method using methyl orange or methyl orange-aniline blue as an indicator is used to determine the degree of deacetylation of chitosan oligosaccharide, which has the disadvantage that the color change of the endpoint is not obvious, the reproducibility is poor, and the error is large. According to the 2015 edition. The Chinese Pharmacopoeia uses the methyl orange acid base indicator method to determine the chitosan oligosaccharide deacetylation degree error of more than 60%. 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.

After searching, there is no report in the prior art for determining the degree of deacetylation of chitosan oligosaccharide by an acid-base indicator method favoring bromocresol green as an indicator, and a standard method for the degree of deacetylation of chitooligosaccharides has not been established.

Summary of the invention

In order to solve the problems in the prior art, the present invention provides a method for determining the degree of deacetylation of chitooligosaccharides using an acid-base indicator method. The method provided by the invention is a method for accurately and rapidly determining the degree of deacetylation of chitosan oligosaccharides. At the same time, the method provided by the invention is simple to use, simple and easy to operate, no special pretreatment is required for the sample, and the titration end point is judged obviously, and the measurement is obvious. The error is small and is suitable for quality control in the preparation of chitosan oligosaccharides.

The invention provides a method for determining the degree of deacetylation of chitooligosaccharides by using an acid-base indicator method, comprising the following steps:

S1, determining the moisture content in the chitosan oligosaccharide sample as a weight loss under the weight loss of the chitosan oligosaccharide sample;

S2, take the shell oligosaccharide sample, dissolve it in distilled water, adjust the pH to 8.0, add dilute hydrochloric acid titration solution, while adding 0.1-1% bromocresol green indicator 1-6 drops, mix and use with hydroxide The titration of the sodium titration until the solution turns green is the end point of the titration;

S3. Calculate the degree of deacetylation of the chitosan oligosaccharide sample, and the formula is as follows:

Where DD% is the degree of deacetylation, %; N _HCl is the concentration of hydrochloric acid titration solution, mol/L; V _HCl is the volume of hydrochloric acid titration solution, mL; N _NaOH is the concentration of sodium hydroxide titration solution, mol/L V _NaOH is the volume of sodium hydroxide titration solution, mL; G is the weight of the test sample, g; W is the weight loss under the weight loss of drying, %; 0.016 is the equivalent amount of amino acid of 1 mol / L hydrochloric acid, g; 9.94 % is the theoretical amino content.

Further, the chitosan oligosaccharide sample has a concentration of 6-10 mg/mL.

Further, the concentration of the diluted hydrochloric acid titration solution is 0.1-0.5 mol/L.

Further, the indicator is used in an amount of 1% of bromocresol green indicator 1-2 drops.

Further, the sodium hydroxide titration solution has a concentration of 0.1 to 0.5 mol/L.

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

(1) The method for determining the degree of deacetylation of chitosan oligosaccharides by the acid-base indicator method provided by the invention can accurately and quickly determine the degree of deacetylation of chitosan oligosaccharides, and the method provided by the invention is simple to use and easy to operate. The sample does not need special pre-treatment, the titration end point is judged obviously, the measurement error is small, suitable Used for quality control in the preparation of chitosan oligosaccharides.

(2) The method for determining the degree of deacetylation of chitosan oligosaccharide by the acid-base indicator method provided by the invention, using bromocresol green as an indicator, the color change of the titration end point is obvious, the titration end point can be accurately determined, and the measurement error is reduced.

(3) In the process of measuring the degree of deacetylation of chitosan oligosaccharide by the acid-base indicator method, the inventors of the present invention found that the pH value of the aqueous solution of chitosan oligosaccharide has a great influence on the determination of the degree of deacetylation of the sample. In the aqueous oligosaccharide solution, the acetylamino group just reached the state of complete liberation at pH 8.0. At this time, the degree of deacetylation of chitosan oligosaccharide was determined by the acid-base indicator method, and the accuracy was high, while the pH of the aqueous solution of the chitosan oligosaccharide was less than 8.0. Error, a pH greater than 8.0 causes a positive error.

DRAWINGS

Figure 1 is a nuclear magnetic resonance spectrum (500 MHz) of chitosan oligosaccharide (COS _MW1000 ) having an average molecular weight of ≤ 1000 Daltons, wherein AE represents a characteristic hydrogen signal at the C2-C6 position on the sugar ring.

Figure 2 is a nuclear magnetic resonance spectrum (500 MHz) of chitosan oligosaccharide (COS _MW3000 ) having an average molecular weight of ≤ 3000 Daltons, wherein AE represents a characteristic hydrogen signal at the C2-C6 position on the sugar ring.

detailed description

The invention will be further described in detail below by way of specific examples, and the scope of the invention is not limited to the following 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
S-25 potential pH meter	Shanghai Lei Magnetic Precision Scientific Instrument Co., Ltd.
DK-8D type one thousandth electronic analytical balance	German Sartorius Group
Sartourius MA150 Infrared Moisture Analyzer	German Sartorius Group
Bruker 500MHz NMR	Bruker

Example 1 Determination of Chitosan Oligosaccharide Deacetylation Degree by Acid-Base Indicator Method

S1, weigh 0.5g of chitosan oligosaccharide sample and determine its moisture content in infrared moisture analyzer, and record the result as weight loss under the weight loss of the batch of chitosan oligosaccharide sample;

S2, taking 0.5g of chitosan oligosaccharide sample, accurately weighed, precision added 50mL distilled water, at room temperature Stir it to dissolve completely, adjust the pH to 8.0, precisely add 0.3mL/L dilute hydrochloric acid titration solution 18mL, while adding 1% bromocresol green indicator 1-2 drops, mix and use 0.15mol/L The titration of the sodium hydroxide titration until the solution turns green is the end point of the titration;

S3. Calculate the degree of deacetylation of the chitosan oligosaccharide sample, and the formula is as follows:

Where DD% is the degree of deacetylation, %; N _HCl is the concentration of hydrochloric acid titration solution, mol/L; V _HCl is the volume of hydrochloric acid titration solution, mL; N _NaOH is the concentration of sodium hydroxide titration solution, mol/L V _NaOH is the volume of sodium hydroxide titration solution, mL; G is the weight of the test sample, g; W is the weight loss under the weight loss of drying, %; 0.016 is the equivalent amount of amino acid of 1 mol / L hydrochloric acid, g; 9.94 % is the theoretical amino content.

_{Determination of the} degree of deacetylation of COS _MW1000 (chitosan oligosaccharide sample with deacetylation degree ≥90%, average molecular weight ≤1000) _and COS _MW3000 (chitosan oligosaccharide sample with deacetylation degree ≥90% and average molecular weight ≤3000) See Table 1, Table 2.

Table 1 Determination of the degree of deacetylation of COS _MW1000

According to Table 1, it can be seen that COS _MW1000 has a deacetylation degree DD of 93.38% and an RSD of 0.28 (n=6) by the method provided by the present invention.

Table 2 Determination of the degree of deacetylation of COS _MW3000

According to Table 2, it was found that the chitosan oligosaccharide sample of COS _MW3000 was found to have a degree of deacetylation of 92.64% and an RSD of 0.53 (n = 6) by the method provided by the present invention.

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 1 and Figure 2, respectively. According to Table 3, 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 3 Hydrogen proton chemical shift in aqueous solution of chitosan oligosaccharide at 25 ° C

The deacetylation degree of COS _MW1000 and COS _MW3000 was determined by ¹ H-NMR method to be 93.52±0.13 (n=6) and 92.81±0.07 (n=6), 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 4.

Table 4 ¹ H-NMR comparison with the acid-base indicator method of the present invention (n=6)

sample	1 H-NMR (%)	Bromocresol green indicator method (%)	Relative error
COS MW1000	93.52±0.13	93.38±0.28	0.14±0.15
COS MW3000	92.81±0.07	92.64±0.53	0.17±0.46

From the results of Table 4, it can be seen that the relative error of the determination result of the chitosan oligosaccharide deacetylation degree by the acid-base indicator method of the present invention is less than 0.2%, which is consistent with the measurement result of the ¹ H-NMR method. The acid-base indicator method provided by the present invention is used for determining the degree of deacetylation degree of chitosan oligosaccharide.

Comparative Example 2 Determination of Chitosan Oligosaccharide Deacetylation by Methyl Orange Acid Base Indicator Method

The degree of deacetylation of COS _MW1000 and COS _{MW3000 was} determined by _{referring to the} method for determining the degree of deacetylation of chitosan in the Chinese Pharmacopoeia of 2015. The results are shown in Tables 5 and 6.

Table 5 COS _MW1000 deacetylation measurement results

Table 6 Determination of the degree of deacetylation of COS _MW3000

From the results of Tables 5 and 6, it can be seen that the deacetylation degree of COS _MW1000 and COS _MW3000 is 30.39±0.70 and 30.32±0.56, respectively, measured by the determination method of chitosan deacetylation degree in the 2015 edition of Chinese Pharmacopoeia. _Compared with the deacetylation degree of COS _MW1000 and COS _MW3000 measured by ¹ H-NMR method, the degree of deacetylation was 93.52±0.13 and 92.81±0.07, and the measurement error was 60% or more.

The aqueous solution of chitosan sample is a colorless solution with an isoelectric point of about 3.88. The pH of methyl orange is in the range of 3.1-4.4, and the acid color is red, and the basic color is yellow. Therefore, methyl orange is used as the acid. The alkali indicator is used to determine the degree of deacetylation of chitosan by acid-base titration. The titration end point is obvious, the measurement error is small, and the accuracy is high. For chitosan oligosaccharides, on the one hand, the aqueous solution of chitosan oligosaccharide sample itself is pale yellow, and methyl orange is used as an indicator, so that the titration end point 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, the degree of deacetylation of chitosan oligosaccharide measured by the methyl orange acid base indicator method according to the 2015 edition of the Chinese Pharmacopoeia is very large. The pH of bromocresol green is in the range of 3.8-5.4, the acid color is yellow, the basic color is green (blue-green), and bromocresol green is used as the indicator of the acid-base indicator method. The color change of the titration end point is obvious. Accurately determine the endpoint of the titration, which can significantly reduce the measurement error.

Comparative Example 3 Determination of Chitosan Oligosaccharide Deacetylation Degree by Phenolphthalic Acid Base Indicator Method

Refer to the method for determining the degree of deacetylation of chitosan in the 2015 Chinese Pharmacopoeia, and determine the degree of deacetylation of COS _MW1000 and COS _MW3000 using phenolphthalein as an indicator. The results are shown in Tables 7 and 8.

Table 7 COS _MW1000 deacetylation measurement results

Table 8 Determination of the degree of deacetylation of COS _MW3000

From the results of Tables 7 and 8, it can be seen that the deacetylation degree of COS _MW1000 and COS _MW3000 is 66.72±0.23 and 62.01±0.29, respectively, and COS _MW1000 measured by ¹ H-NMR method. _Compared with the degree of deacetylation of COS _MW3000 of 93.52±0.13 and 92.81±0.07, there is a measurement error of about 30%.

Comparative Example 4 pH investigation

The inventors of the present invention have found through extensive studies that the pH value of the chitosan oligosaccharide aqueous solution has a great influence on the determination of the degree of deacetylation of chitosan oligosaccharide samples.

The inventors of the present invention refer to the method for determining the degree of deacetylation of chitosan in the Chinese Pharmacopoeia of 2015, and determine the degree of deacetylation of COS _MW1000 and COS _MW3000 by using bromocresol green as an indicator of the acid-base indicator method, and titrating the end point color. The change was remarkable, and the measurement error was remarkably lowered. However, compared with the measurement method of the ¹ H-NMR method, the measurement error was still 10% or more.

During the research, it was found that the pH of the commercially available chitosan oligosaccharide solution was about 5.12 due to the preparation process of chitosan oligosaccharide, which showed weak acidity. Chitosan oligosaccharide is a basic aminopolysaccharide. If the aqueous solution is weakly acidic, the degree of deacetylation measured by the acid-base indicator method will definitely cause measurement error.

The effects of pH on the measurement results of the chitosan oligosaccharide deacetylation degree by the acid-base indicator method were examined under the same conditions as in Example 1 except that the pH was changed. The results are shown in Table 9.

Table 9 Effect of different pH values of chitosan oligosaccharide solution on the determination of chitosan oligodeacetylation degree (n=6)

pH	7.60	7.80	7.90	8.00	8.10	8.20
DD MW1000 (%)	87.45±0.18	90.94±0.21	91.64±0.31	93.44±0.13	95.62±0.20	97.65±0.25
DD MW3000 (%)	88.14±0.19	89.86±0.20	90.82±0.16	92.69±0.11	94.25±0.18	97.12±0.23

According to the results of Table 9, the pH value of the chitosan oligosaccharide aqueous solution has a great influence on the determination of the degree of deacetylation of chitosan oligosaccharide samples, which may be due to the combination of acetamido and hydrochloric acid to form chitooligosaccharide hydrochloride in an acidic environment. The acetylamino group cannot be completely released, resulting in a large measurement error. As the acidity and alkalinity of the solution changed, the acetylamino group slowly liberated, reaching a state of just completely free at pH=8.00, pH<8.00 caused a negative error, and pH>8.00 caused a positive error.

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 (5)

1. A method for determining the degree of deacetylation of chitosan oligosaccharides by using an acid-base indicator method, comprising: the following steps:

   S1, determining the moisture content in the chitosan oligosaccharide sample as a weight loss under the weight loss of the chitosan oligosaccharide sample;

   S2, take the shell oligosaccharide sample, dissolve it in distilled water, adjust the pH to 8.0, add dilute hydrochloric acid titration solution, while adding 0.1-1% bromocresol green indicator 1-6 drops, mix and use with hydroxide The titration of the sodium titration until the solution turns green is the end point of the titration;

   S3. Calculate the degree of deacetylation of the chitosan oligosaccharide sample, and the formula is as follows:

   

   Where DD% is the degree of deacetylation, %; N _HCl is the concentration of hydrochloric acid titration solution, mol/L; V _HCl is the volume of hydrochloric acid titration solution, mL; N _NaOH is the concentration of sodium hydroxide titration solution, mol/L V _NaOH is the volume of sodium hydroxide titration solution, mL; G is the weight of the test sample, g; W is the weight loss under the weight loss of drying, %; 0.016 is the equivalent amount of amino acid of 1 mol / L hydrochloric acid, g; 9.94 % is the theoretical amino content.
2. The method for determining the degree of deacetylation of chitosan oligosaccharides by an acid-base indicator method according to claim 1, wherein the chitosan oligosaccharide sample has a concentration of 6-10 mg/mL.
3. The method for determining the degree of deacetylation of chitosan oligosaccharides by an acid-base indicator method according to claim 1, wherein the concentration of the diluted hydrochloric acid titration solution is 0.1 to 0.5 mol/L.
4. The method for determining the degree of deacetylation of chitosan oligosaccharides by the acid-base indicator method according to claim 1, wherein the indicator is used in an amount of 1% of bromocresol green indicator 1-2 drops.
5. The method for determining the degree of deacetylation of chitosan oligosaccharides by an acid-base indicator method according to claim 1, wherein the sodium hydroxide titration solution has a concentration of 0.1 to 0.5 mol/L.

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