CN114965543A - Method for determining attribution of chitosan oligosaccharide NMR spectrum and related index of deacetylation degree - Google Patents
Method for determining attribution of chitosan oligosaccharide NMR spectrum and related index of deacetylation degree Download PDFInfo
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- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 title claims abstract description 186
- 238000000034 method Methods 0.000 title claims abstract description 52
- 230000006196 deacetylation Effects 0.000 title claims abstract description 38
- 238000003381 deacetylation reaction Methods 0.000 title claims abstract description 38
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 title claims abstract description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 114
- 239000000126 substance Substances 0.000 claims abstract description 42
- 238000012360 testing method Methods 0.000 claims abstract description 34
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 30
- 238000005481 NMR spectroscopy Methods 0.000 claims abstract description 28
- 238000001228 spectrum Methods 0.000 claims abstract description 17
- 125000003047 N-acetyl group Chemical group 0.000 claims abstract description 10
- HEMHJVSKTPXQMS-DYCDLGHISA-M Sodium hydroxide-d Chemical compound [Na+].[2H][O-] HEMHJVSKTPXQMS-DYCDLGHISA-M 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 54
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 26
- 229920001661 Chitosan Polymers 0.000 claims description 22
- 238000001035 drying Methods 0.000 claims description 18
- 238000004364 calculation method Methods 0.000 claims description 17
- MSWZFWKMSRAUBD-IVMDWMLBSA-N glucosamine group Chemical group OC1[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 13
- 238000006116 polymerization reaction Methods 0.000 claims description 12
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims description 11
- 238000010438 heat treatment Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 230000010354 integration Effects 0.000 claims description 4
- 230000005311 nuclear magnetism Effects 0.000 claims description 2
- 230000003595 spectral effect Effects 0.000 claims description 2
- QTBSBXVTEAMEQO-DYCDLGHISA-N deuterio acetate Chemical compound [2H]OC(C)=O QTBSBXVTEAMEQO-DYCDLGHISA-N 0.000 claims 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 abstract description 7
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 7
- 239000001257 hydrogen Substances 0.000 abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 abstract description 2
- -1 acetyl methyl Chemical group 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 20
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 10
- 125000003277 amino group Chemical group 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000012545 processing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000005259 measurement Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 3
- 150000001720 carbohydrates Chemical group 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 125000001976 hemiacetal group Chemical group 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000003918 potentiometric titration Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 239000003390 Chinese drug Substances 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Chemical group CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Chemical group CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000002542 deteriorative effect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 150000002373 hemiacetals Chemical class 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- OIIWPAYIXDCDNL-UHFFFAOYSA-M sodium 3-(trimethylsilyl)propionate Chemical compound [Na+].C[Si](C)(C)CCC([O-])=O OIIWPAYIXDCDNL-UHFFFAOYSA-M 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
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Abstract
The invention belongs to the technical field of nuclear magnetic analysis of chitosan oligosaccharide. The invention provides a method for measuring the NMR spectrum attribution and deacetylation degree related indexes of chitosan oligosaccharide, aiming at the problems that the chemical shifts of the proton peaks of the acetyl methyl of acetic acid and acetate and the methyl of N-acetyl in the structure of chitosan oligosaccharide in nuclear magnetic hydrogen spectrum are similar and difficult to be attributed: respectively completely dissolving the dried chitosan oligosaccharide samples in a deuterated reagent D 2 O, then regulating the pH value to 5.4-6.2 by using NaOD solution; testing at two different temperatures respectively, wherein the temperature difference of the two temperatures is enough to cause a distinguishable chemical shift of a high field region on a nuclear magnetic resonance spectrogram; the proton peak with unchanged chemical shift value or slightly shifted to high field is determined as the methyl proton peak H of the N-acetyl group of the chitosan oligosaccharide AC And determining the proton peak with the chemical shift value moving to the low field as the methyl proton peak H of the acetic acid A . The attribution method and the deacetylation degree measuring method are accurate, simple and convenient.
Description
Technical Field
The invention belongs to the technical field of nuclear magnetic analysis of chitosan oligosaccharide, and particularly relates to a method for determining the attribution of an NMR spectrum and related indexes of deacetylation degree of chitosan oligosaccharide.
Background
The chitosan oligosaccharide is composed of two saccharide units of glucosamine and N-acetylglucosamine, and is a degradation product of chitosan, so the molecular structure of the chitosan oligosaccharide is similar to that of chitosan, but the average polymerization degree of the chitosan oligosaccharide is greatly reduced. Compared with chitosan, chitosan oligosaccharide has good water solubility, and its biological activities such as antibacterial, antitumor, and immunoregulatory enhancement have been widely used in the fields of medicine, cosmetics, food, textile, paper making, etc. Chitosan oligosaccharide is also known as a low degree of polymerization water-soluble chitosan, which is soluble in acidic, neutral and alkaline solutions. The Degree of Deacetylation (DD) can be defined as the molar percentage of glucosamine units in the chitosan oligosaccharide molecule. Glucosamine units are the key structure determining the biological activity of chitosan oligosaccharide, therefore, DD is an important parameter for quality control of chitosan oligosaccharide.
Many reports are made about methods for measuring the deacetylation degree of chitosan, but the methods for measuring the deacetylation degree of chitosan oligosaccharide are only rarely reported, and the accurate measurement of DD is influenced because acetic acid and/or hydrochloric acid are often contained in a chitosan oligosaccharide product. The reasons for the acid content in the chitosan oligosaccharide product are as follows: (1) the molecular weight of the chitosan oligosaccharide is smaller, the reducing hemiacetal groups contained in unit mass are obviously more than that of the chitosan, the hemiacetal groups are easy to generate Maillard reaction with amino groups in glucosamine units in the chitosan oligosaccharide molecules, and in order to prevent the chitosan oligosaccharide from deteriorating, acid is often added into the chitosan oligosaccharide product to protect the amino groups so as to inhibit the reaction from going on; (2) due to the consideration of green, safety and environmental protection, most of the chitosan oligosaccharide is derived from enzymatic degradation of chitosan, and the optimal pH value of the enzymolysis of the chitosan oligosaccharide is always maintained by adding acetic acid; (3) chitosan is insoluble in water, and needs to be dissolved in an acidic solution before degradation, and acid residues exist in the degraded chitosan; (4) chitosan can only be dissolved in a limited number of acidic dilute solutions, and in view of low cost, dilute hydrochloric acid is the best, but hydrochloric acid is strong acid, so that chitosan oligosaccharide can be easily degraded continuously to reduce the biological activity of chitosan oligosaccharide, and therefore, acetic acid with the same low cost is often added into a chitosan oligosaccharide product.
The addition of acetic acid brings great trouble to the accurate determination of the deacetylation degree of chitosan oligosaccharide, and methods commonly used for determining the deacetylation degree of chitosan, such as an elemental analysis method, a potentiometric titration method, an infrared method and an ultraviolet first derivative method, cannot be applied to the determination of the deacetylation degree of chitosan oligosaccharide. Elemental analysis is based on the carbon to nitrogen ratio in the chitosan oligosaccharide molecule, and carbon-containing acetic acid interferes with its determination. The infrared method is based on the quantitative determination of the vibration peak related to methyl and carbonyl of acetyl, and due to the existence of acid, -NH in the chitosan oligosaccharide molecule 2 Partially or wholly converted to-NH 3 + On the other hand, the two groups have completely different N-H bending vibration and affect the C ═ O stretching vibration peak of the acetyl group, so that the deacetylation degree of the chitosan oligosaccharide cannot be measured by the infrared measurement method of chitosan. The potentiometric titration method is based on the determination of amino groups in chitosan oligosaccharide molecules, because acetic acid is weak acid and amino groups are weak base, the titration sites are difficult to observe to influence the determination of the amino group content, furthermore, chitosan oligosaccharide often exists in the form of partial salt, which may have both acetate and hydrochloride, which brings trouble to the calculation of DD value, in addition, the method is time-consuming, low in detection efficiency and inconvenient for large-scale determination. The determination of the ultraviolet first derivative method is based on acetyl in chitosan oligosaccharide molecules, and the acetic acid added in the chitosan oligosaccharide product can directly influence the determination result, so the method is more suitable for the determination of the chitosan oligosaccharide DD without the acetic acid. The deacetylation degree of the chitosan oligosaccharide is indirectly expressed by the mass ratio of a chitosan oligosaccharide sample in the industry for a long time, but the molar ratio of glucosamine units in the molecular structure of the chitosan oligosaccharide is not, and the structural characteristics of the chitosan oligosaccharide molecule can be better represented by measuring the deacetylation degree by the molar ratio.
The nuclear magnetic method in the prior art is a recognized method for determining the deacetylation degree of chitosan oligosaccharide most accurately,the method is characterized in that according to the difference of chemical displacement values of different hydrogen protons in chitosan oligosaccharide molecules, the DD value of the chitosan oligosaccharide is calculated by comparing the integral area of the hydrogen protons of a test sample with the integral area of a selected reference hydrogen proton. However, in the course of the above-mentioned operation, 1 H-NMR methods also have disadvantages: the chemical shift of the methyl proton peak of the acetic acid added in the chitosan oligosaccharide product is similar to that of the methyl proton peak of the acetyl group, and the methyl proton peak is difficult to distinguish. Some scholars believe that the methyl proton peak at 1.92ppm is attributable to the N-acetyl group (Jiang, y., Fu, c.h., Wu, s.h., Liu, g.h., Guo, j.,&su, Z.Q. (2017), Determination of the Determination Degree of the methods of Chinese drugs, Marine drugs,15(11), also by scholars, they all belong to the N-acetyl group (Jiang, Z., Liu, G., Yang, Y., Shao, K., Wang, Y., Liu, W., U, K., U., T.,&han, B. (2019). N-Acetyl chitin oligosaccharides expressed beta-induced damagein and cell models of Alzheimer's disease. Process Biochemistry,84, 161. su-171.). If the methyl proton peak of the acetyl group is not clearly assigned, the determination of the deacetylation degree is greatly deviated, which is extremely unfavorable for the quality control and application of the chitosan oligosaccharide. Therefore, the accurate assignment of the two methyl proton peaks is a problem which needs to be solved at present, and the solution of the problem is a necessary prerequisite for accurately determining the deacetylation degree and the acetic acid addition amount of the chitosan oligosaccharide.
Disclosure of Invention
The invention aims at providing a method for measuring spectrum attribution and deacetylation degree related indexes of chitosan oligosaccharide NMR (nuclear magnetic resonance), and by comparing the shift change conditions of methyl proton peaks on spectrograms of chitosan oligosaccharide nuclear magnetic samples at two temperatures, the methyl proton peak H of N-acetyl can be accurately judged AC Methyl proton peak with acetic acid H A The spectrum attribution of the chitosan oligosaccharide can further realize the accurate determination of the deacetylation degree of the chitosan oligosaccharide.
The invention is realized by the following technical scheme:
on one hand, the invention provides a spectrum attribution method of a nuclear magnetic resonance spectrogram of chitosan oligosaccharide, which specifically comprises the following steps:
(1) preparation of Chitosan oligosaccharide nuclear magnetism sample solution
Respectively completely dissolving the dried chitosan oligosaccharide samples in a deuterated reagent D 2 O, then adjusting the pH value to 5.4-6.2 by using a NaOD solution to obtain a sample solution;
(2) sample testing
Respectively placing the sample solutions prepared in the step (1) in a nuclear magnetic resonance instrument, and respectively testing at two different temperatures to obtain a nuclear magnetic resonance spectrogram of the chitosan oligosaccharide; the temperature difference between the two different temperatures is sufficient to cause a distinguishable change in chemical shift value in the low field region of the NMR spectrum;
(3) spectral attribution
Comparing the nuclear magnetic resonance spectrogram measured at higher temperature with the nuclear magnetic resonance spectrogram measured at lower temperature, and determining the proton peak at 2.07 +/-0.01 ppm with the chemical shift value unchanged or slightly shifted to high field due to the rise of the test temperature in the high-field region as the proton peak H of the N-acetyl group of the chitosan oligosaccharide AC The proton peak at 1.93. + -. 0.02ppm at which the chemical shift value shifts to a low field was determined as the methyl proton peak H generated by the addition of acetic acid in the chitooligosaccharide sample A (ii) a The proton peak group with the chemical shift value of 4.18-3.11ppm is H of chitosan oligosaccharide 2 -H 6 Sum of 6 proton peaks H 2-6 。
The purpose of using NaOD in step (1) is to make the N-acetyl methyl proton peak and the methyl proton peak generated by adding acetic acid in the chitosan oligosaccharide sample independent from each other without interfering the measurement.
Measuring the pH value of the solution at room temperature after the chitosan oligosaccharide nuclear magnetic sample is dissolved, and if the pH value is within the range of 5.4-6.2, directly measuring; if the pH is less than 5.4, NaOD solution is added for adjustment. The pH of the chitosan oligosaccharide solution is too low, which is not favorable for obtaining mutually independent methyl proton peaks; if the pH is too high, the amino groups in the chitosan oligosaccharide molecules are liable to react with hemiacetal, so that the chitosan oligosaccharide is deteriorated.
Further, the drying method of the chitosan oligosaccharide sample in the step (1) comprises the following steps: drying at 60 deg.C for 1-2 hr, heating to 80 deg.C for 1-2 hr, heating to 105 deg.C, drying for 0.5-1 hr, and drying to constant weight.
Drying the chitosan oligosaccharide sample for the purpose of removing water so as to prevent residual water in the chitosan oligosaccharide sample from interfering with the determination; the initial drying temperature is controlled at 60 ℃ to ensure that amino groups in chitosan oligosaccharide molecules do not react in the presence of moisture, and the temperature is raised for a short time (80 ℃, 105 ℃) to remove the moisture when the moisture is nearly removed.
Further, the concentration of the chitosan oligosaccharide sample in the step (1) is 5-20 mg/mL. The concentration of the chitosan oligosaccharide is enough to meet the requirement of a nuclear magnetic resonance hydrogen spectrum on the concentration of a sample, and the concentration is too low, the signal to noise ratio is too low, so that accurate determination is influenced; too high a concentration increases the solution viscosity and affects signal acquisition. Preferably, the concentration of the chitosan oligosaccharide sample in the step (1) is 8-12mg/mL, and more preferably 10 mg/mL.
Further, the range of one test temperature in the step (2) is 20-30 ℃, and the range of the other test temperature in the step (2) is 60-80 ℃; preferably, one test temperature is 25 deg.C and the other test temperature is 70 deg.C.
On the other hand, the invention provides a method for measuring the deacetylation degree of chitosan oligosaccharide, which is based on the spectrum attribution method of the nuclear magnetic resonance spectrogram of chitosan oligosaccharide, and further comprises the following steps:
(4) area integration and calculation of proton peaks
Calculation of the methyl proton Peak H of N-acetyl group AC Has an area integral of I AC (ii) a Methyl proton peak of acetic acid H A Has an integral of I A (ii) a 6 proton peaks H of Chitosan oligosaccharide 2-6 Is the integral sum of 2-6 (ii) a Degree of deacetylation DD and amount of acetic acid added D A The calculation formula of (a) is as follows:
further, the proton peak with a chemical shift value of 5.45ppm is the anomeric carbon proton of the terminal glucosamine unit of the chitosan oligosaccharidePeak H 1D-E The integral of which is I 1D-E (ii) a The proton peak with the chemical shift value of 5.22ppm is the anomeric carbon proton peak H of the terminal acetylglucosamine unit of the chitosan oligosaccharide 1A-E The integral of which is I 1A-E (ii) a The proton peak with a chemical shift value of 4.84ppm is the anomeric carbon proton peak H of the glucosamine unit in the chitooligosaccharide chain 1D-I The integral of which is I 1D-I (ii) a The proton peak at 4.62ppm was assigned as the anomeric carbon proton peak H of the acetylglucosamine unit in the chitooligosaccharide chain 1A-I The integral of which is I 1A-I (ii) a Anomeric carbon proton peak H of chitosan oligosaccharide 1 Integral I of 1 Is H 1D-E 、H 1A-E 、H 1D-I 、H 1A-I The sum of the integrals of (a); i is 2-6 6 equivalent replacement by I 1 ，I 1 Replacement by I 1D-E +I 1A-E +I 1D-I +I 1A-I ，I AC Replacement of/3 equivalents by I 1A-E +H 1A-I Then, the calculation formula of the deacetylation degree is as follows:
in a third aspect, the invention provides a method for determining the amount of acetic acid added to a chitosan oligosaccharide, wherein the amount of acetic acid added D is based on the determination of the degree of deacetylation A The calculation formula of (a) is as follows:
in a fourth aspect, the present invention provides a method for measuring the average degree of polymerization of chitosan oligosaccharide, wherein the calculation formula of the average degree of polymerization DP of chitosan oligosaccharide based on the measurement of the degree of deacetylation is as follows:
in a fifth aspect, the present invention provides a method for determining the number average molecular weight MW of chitosan oligosaccharide, wherein the calculation formula of the number average molecular weight MW of chitosan oligosaccharide is as follows, based on the determination of the average polymerization degree:
MW＝DP×(203.2-42DD)+18。
compared with the prior art, the invention has the following advantages:
(1) the pH value of the chitosan oligosaccharide sample solution is 5.4-6.2, and the methyl proton peak H of acetyl in the chitosan oligosaccharide molecule AC And proton peak H of acetic acid added to the Chitosan oligosaccharide sample A Do not interfere with each other, and H moving to a low field can be seen by raising the test temperature A And H that hardly moves or moves slightly to a high field AC ；
(2) The deacetylation degree of the chitosan oligosaccharide can be accurately measured, and the deacetylation degree measured by the method is measured according to the molar ratio, so that the structural characteristics of chitosan oligosaccharide molecules can be reflected;
(3) the content of acetic acid added in a chitosan oligosaccharide sample, the average polymerization degree and the average molecular weight of chitosan oligosaccharide can be accurately measured;
(4) the test at the high temperature of 70 ℃ is beneficial to improving the sensitivity of signal acquisition;
(5) the HOD peak can not overlap with the peak to be measured and interfere the measurement;
(6) the operation method is simple and efficient, the result accuracy is high, and the reproducibility is good.
Drawings
FIG. 1 shows the prior art infrared determination of three chitosan oligosaccharide samples: a stack of the infrared spectra of COS-A, COS-B and COS-C;
FIG. 2 is a chart showing the hydrogen spectra of three chitosan oligosaccharide samples measured by conventional nuclear magnetic method (the test conditions are a.D respectively) 2 O/25℃、b.D 2 The chitosan oligosaccharide samples COS-A, COS-B, COS-C in the O/70 ℃ are from QINGDAOBIZHIHUIJUHI Biotech, Shanghai-sourced leaf Biotech, Shandong Riizhou, Haili Bioproduct, Inc., respectively);
FIG. 3Is the hydrogen spectrum of the chitosan oligosaccharide COS-A sample of example 1 (the test conditions are a.D respectively) 2 O/25℃、 b.D 2 O/70℃)；
FIG. 4 is A hydrogen spectrum of COS-A sample of chitosan oligosaccharide of example 2 (test conditions D, respectively) 2 O/70 ℃, pH of the sample solution measured at room temperature is 5.42);
FIG. 5 is a hydrogen spectrum of COS-B sample of chitosan oligosaccharide from example 3 (test condition D) 2 O/70 ℃, pH of the sample solution measured at room temperature is 5.41);
FIG. 6 is a hydrogen spectrum of a sample of Chitosan oligosaccharide COS-C of example 4 (test condition 0.01M NaOD/70 ℃, pH of the sample solution measured at room temperature is 6.13).
Detailed Description
The present invention is described in further detail with reference to the following examples, but the embodiments of the present invention are not limited to these examples, and all changes or equivalent substitutions that do not depart from the spirit of the present invention are intended to be included within the scope of the present invention.
The following examples used chitosan oligosaccharide samples: COS-A, COS-B and COS-C from Bozhihui Biotech, Inc., Shanghai, and Haili Bioproducts, Inc., of Lyzhou, Shandong, respectively.
D 2 O (with internal standard TMSP) was purchased from sigma aldrich trade ltd.
DCl (20%) solution was purchased from Shanghai Aladdin Biotechnology Ltd.
The instrument comprises the following steps: agilent technologies, Inc. of DD 2500 Mhz NMR spectrometer.
Example 1 method for assigning spectrum of nuclear magnetic resonance spectrum of Chitosan oligosaccharide
(1) Preparation of Chitosan oligosaccharide sample (COS-A) solution
a. Drying
The specific operation is as follows: weighing 10mg of chitosan oligosaccharide sample in a centrifuge tube respectively, drying at 60 ℃ for 2 hours, heating to 80 ℃ for drying for 2 hours, heating to 105 ℃ for drying for 1 hour until the weight is constant. And after drying, opening the oven, immediately sealing, and placing in a dryer for cooling for later use.
b. Sample application
1 ml of D 2 And adding O into the dried chitosan oligosaccharide sample, fully and uniformly mixing to dissolve the chitosan oligosaccharide, measuring the pH value of the chitosan oligosaccharide solution at the concentration of 10mg/mL, measuring the pH value of the solution at room temperature to be 5.42, and then adding the solution into a nuclear magnetic tube by using a pipette.
(2) Sample testing
The nuclear magnetic resonance spectrometer comprises a nuclear magnetic resonance instrument, a proton nuclear magnetic resonance spectrogram, a probe, a detector, a data acquisition unit, a data processing unit and the like. In the aspect of parameter setting, the test temperatures are respectively 25 ℃ and 70 ℃ (data acquisition is carried out after the thermal equilibrium time is 10 minutes), the scanning times are 32 times, and the acquired data are stored for later use.
(3) Data processing
And (3) obtaining a nuclear magnetic resonance spectrum of the chitosan oligosaccharide from the step (2) by using application software of a nuclear magnetic resonance instrument, as shown in the attached figure 3 of the specification. Comparing FIG. 3a with FIG. 3b, D measured at 25 ℃ and 70 ℃ 2 The proton peak having a chemical shift value of about 1.94ppm in the O-solubilized chitosan oligosaccharide sample shifted to 1.99ppm with increasing temperature, and was thus assigned D 2 The proton peak with chemical shift value of about 2.07ppm in the O-dissolved chitosan oligosaccharide sample is the proton peak H of acetic acid A ；D 2 The proton peak with chemical shift value of about 2.07ppm in the chitosan oligosaccharide sample dissolved by O does not change with the change of nuclear magnetic test temperature of the chitosan oligosaccharide sample, and is assigned as the methyl proton peak H of acetyl in the chitosan oligosaccharide molecule AC . 4.18-3.11ppm proton peak group is H of chitosan oligosaccharide 2 -H 6 Sum of 6 proton peaks H 2-6 . The water peak measured at 70 deg.C will shift to low field to expose the anomeric carbon proton peak, and the proton peak with chemical shift value of about 5.45ppm is the anomeric carbon proton peak H of terminal glucosamine unit of chitosan oligosaccharide 1D-E B, carrying out the following steps of; the proton peak with chemical shift value of about 5.22ppm is the anomeric carbon proton peak H of the terminal acetylglucosamine unit of the chitosan oligosaccharide 1A-E B, carrying out the following steps of; the proton peak at a chemical shift value of about 4.84ppm is in the chitooligosaccharide chain(ii) anomeric carbon proton peak H of glucosamine unit 1D-I B, carrying out the following steps of; the proton peak with a chemical shift value of about 4.62ppm is the anomeric carbon proton peak H of the acetylglucosamine unit in the chitooligosaccharide chain 1A-I 。
Example 2 method for measuring degree of deacetylation of Chitosan oligosaccharide and amount of acetic acid added
The method comprises the following steps:
(1) preparation of Chitosan oligosaccharide sample (COS-A) solution
a. Drying
For specific reference example 1.
b. Sample application
1 ml of D 2 And adding O into the dried chitosan oligosaccharide sample, fully and uniformly mixing to dissolve the chitosan oligosaccharide sample, wherein the concentration of the chitosan oligosaccharide solution is 10mg/mL, and adding the chitosan oligosaccharide solution into a nuclear magnetic tube by using a pipette (the pH value of the chitosan oligosaccharide sample solution is 5.42 at room temperature).
(2) Sample testing
Reference example 1
(3) Data processing
Obtaining the nuclear magnetic resonance spectrogram of the chitosan oligosaccharide from the step (2) by using the application software of the nuclear magnetic resonance instrument, and comparing the two nuclear magnetic resonance spectrograms obtained in the step (2) as shown in the attached figure 3 of the specification, wherein the proton peak with the chemical shift value of about 2.06ppm is the methyl proton peak H of the acetyl in the chitosan oligosaccharide molecule AC The peak did not undergo a change in chemical shift value by raising the test temperature to 70 ℃ and its integral was I AC ；D 2 The proton peak of O-dissolved sample with chemical shift value of about 1.94ppm was shifted to 1.99ppm by raising the test temperature to 70 deg.C (FIG. 4), and thus, this peak was the methyl proton peak H generated by the addition of acetic acid in the chitosan oligosaccharide sample A The integral of which is I A . The proton peak group of about 4.18-3.11ppm is H of chitooligosaccharide 2 -H 6 Sum of 6 proton peaks H 2-6 The integral of which is I 2-6 . The above values are shown in table 1.
TABLE 1 Chitosan oligosaccharide samples in 1 Proton assignment on HNMR spectra and area integral thereof
| Sample number | Peak integral of proton | H AC | H 2-6 | H A |
| 1 | D 2 O (25 ℃ C.) test | 1 | 44.08 | 3.47 |
| 2 | D 2 O (70 ℃ C.) test | 1 | 45.38 | 3.50 |
According to the calculation formula of the deacetylation degree of the chitosan oligosaccharide, the calculation result is as follows:
degree of Deacetylation (DD) and amount of acetic acid addition (D) of Chitosan oligosaccharide No. 1 (test at 25 ℃ C.) A ) Comprises the following steps:
degree of Deacetylation (DD) and amount of acetic acid addition (D) of Chitosan oligosaccharide No. 2 (test at 70 ℃ C.) (see A ) Comprises the following steps:
example 3 method for measuring deacetylation degree, average polymerization degree of acetic acid added amount, and number average molecular weight of Chitosan oligosaccharide
The method comprises the following steps:
(1) preparation of Chitosan oligosaccharide sample (COS-B) solution
a. Drying
For specific reference example 1.
b. Sample application
1 ml of D 2 And adding O into the dried chitosan oligosaccharide sample, fully and uniformly mixing to dissolve the chitosan oligosaccharide sample, wherein the concentration of the chitosan oligosaccharide solution is 10mg/mL, and adding the chitosan oligosaccharide solution into a nuclear magnetic tube by using a pipette (the pH value of the chitosan oligosaccharide sample solution is 5.41 at room temperature).
(2) Sample testing
Reference example 1
(3) Data processing
Obtaining a nuclear magnetic resonance spectrogram of the chitosan oligosaccharide from the step (2) by using application software of a nuclear magnetic resonance instrument, and determining a proton peak with a chemical shift value of about 2.06ppm as a methyl proton peak H of an acetyl group in a chitosan oligosaccharide molecule by comparing the two nuclear magnetic resonance spectrograms obtained from the step (2) AC The integral of which is I AC (ii) a The proton peak (FIG. 5) with a chemical shift value of about 1.97ppm is the methyl proton peak H generated by adding acetic acid in the chitosan oligosaccharide sample A The integral of which is I A . 4.18-3.11ppm proton peak group is H of chitosan oligosaccharide 2 -H 6 Sum of 6 proton peaks H 2-6 The integral of which is I 2-6 . Chemical shift value is about 5.45ppmThe proton peak of (A) is the anomeric carbon proton peak H of the terminal glucosamine unit of the chitosan oligosaccharide 1D-E The integral of which is I 1D-E (ii) a The proton peak with chemical shift value of about 5.22ppm is the anomeric carbon proton peak H of the terminal acetylglucosamine unit of the chitosan oligosaccharide 1A-E The integral of which is I 1A-E (ii) a The proton peak with a chemical shift value of about 4.84ppm is the anomeric carbon proton peak H of the glucosamine unit in the chitooligosaccharide chain 1D-I The integral of which is I 1D-I (ii) a The proton peak with a chemical shift value of about 4.62ppm is the anomeric carbon proton peak H of the acetylglucosamine unit in the chitosan oligosaccharide chain 1A-I The integral of which is I 1A-I (ii) a Anomeric carbon proton peak H of chitosan oligosaccharide 1 Integral I of 1 Is H 1D-E 、H 1A-E 、H 1D-I 、H 1A-I The sum of the integrals of (a). The above values are shown in Table 2
TABLE 2 Chitosan oligosaccharide samples in 1 Proton assignment on HNMR spectrum and area integral thereof (70 ℃ C.)
| Peak of proton | H AC | H 2-6 | H 1D-E | H 1A-E | H 1D-I | H 1A-I | H 1 | H A |
| Integration | 1 | 23.75 | 0.41 | 0.07 | 3.25 | 0.65 | 4.38 | 1.77 |
1. The degree of deacetylation of a chitosan oligosaccharide sample can be calculated by the following formula:
①
②
③
2. the amount of acetic acid added to chitosan oligosaccharide (amount of acetic acid added per saccharide unit) can be calculated by the following formula:
①
②
3. the average polymerization degree of the chitosan oligosaccharide is as follows:
4. the number average molecular weight of the chitosan oligosaccharide is as follows:
MW＝DP×(203.2-42DD)+18＝9.13×(203.2-42×91.58％)+18＝1521
example 4 method for measuring deacetylation degree, average polymerization degree of acetic acid added amount, and number average molecular weight of Chitosan oligosaccharide
The method comprises the following steps:
(1) preparation of Chitosan oligosaccharide sample (COS-C) solution
a. Drying
For specific reference example 1.
b. Sample application
Will D 2 And adding O into the dried chitosan oligosaccharide sample, adding a NaOD solution, fully and uniformly mixing to ensure that the final concentration of NaOD is 0.01mol/L and the concentration of the chitosan oligosaccharide is 10mg/mL, and adding the mixture into a nuclear magnetic tube by using a pipette (the pH value of the chitosan oligosaccharide sample solution is 6.13 at room temperature).
(2) Sample testing
Reference example 1
(3) Data processing
Obtaining a nuclear magnetic resonance spectrogram of the chitosan oligosaccharide from the step (2) by using application software of a nuclear magnetic resonance instrument, and comparing the two nuclear magnetic resonance spectrograms obtained from the step (2), wherein a proton peak with a chemical shift value of about 2.06ppm is a methyl proton peak H of an acetyl group in a chitosan oligosaccharide molecule AC The integral of which is I AC (ii) a The proton peak (FIG. 6) with a chemical shift value of about 1.94ppm is the methyl proton peak H generated by adding acetic acid in the chitosan oligosaccharide sample A . 4.18-3.11ppm proton peak group is H of chitosan oligosaccharide 2 -H 6 Sum of 6 proton peaks of H 2-6 . The proton peak with chemical shift value of about 5.45ppm is the anomeric carbon proton peak H of the terminal glucosamine unit of the chitosan oligosaccharide 1D-E B, carrying out the following steps of; the proton peak with chemical shift value of about 5.22ppm is the anomeric carbon proton peak H of the terminal acetylglucosamine unit of the chitosan oligosaccharide 1A-E A step of,; the peak of the proton with a chemical shift value of about 4.84ppm is the anomeric carbon of the glucosamine unit in the chitosan oligosaccharide chainProton peak H 1D-I B, carrying out the following steps of; the proton peak with a chemical shift value of about 4.62ppm is the anomeric carbon proton peak H of the acetylglucosamine unit in the chitosan oligosaccharide chain 1A-I . The above values are shown in Table 3.
TABLE 3 Chitosan oligosaccharide samples in 1 Proton assignment on HNMR spectrum and area integral thereof (70 ℃ C.)
| Peak of proton | H AC | H 2-6 | H 1D-E | H 1A-E | H 1D-I | H 1A-I | H 1 | H A |
| Integration | 1 | 18.37 | 0.26 | 0.03 | 2.24 | 0.50 | 3.03 | 0.31 |
1. The deacetylation degree of the chitosan oligosaccharide sample can be calculated by the following formula:
①
②
③
2. the amount of acetic acid added to chitosan oligosaccharide (amount of acetic acid added per saccharide unit) can be calculated by the following formula:
①
②
3. the average polymerization degree of the chitosan oligosaccharide is as follows:
4. the number average molecular weight of the chitosan oligosaccharide is as follows:
MW＝DP×(203.2-42DD)+18＝10.45×(203.2-42×89.1％)+18＝1751
the above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention and are intended to be equivalent substitutions are included in the scope of the present invention.
Claims (10)
1. A spectrum attribution method of a nuclear magnetic resonance spectrogram of chitosan oligosaccharide is characterized by comprising the following steps:
(1) preparation of Chitosan oligosaccharide nuclear magnetism sample solution
Dissolving dried chitosan oligosaccharide in deuterated reagent D completely 2 O, then adjusting the pH value to 5.4-6.2 by using a NaOD solution to obtain a sample solution;
(2) sample testing
Respectively placing the sample solutions prepared in the step (1) in a nuclear magnetic resonance instrument, and respectively testing at two different temperatures to obtain a nuclear magnetic resonance spectrogram of the chitosan oligosaccharide; the temperature difference between the two different temperatures is sufficient to cause a change in the distinguishable chemical shift value of the high field region on the nuclear magnetic resonance spectrum;
(3) spectral attribution
Comparing the nuclear magnetic resonance spectrogram of the chitosan oligosaccharide sample measured at a higher temperature with the nuclear magnetic resonance spectrogram measured at a lower temperature, and determining the proton peak at 2.07 +/-0.01 ppm with unchanged chemical shift value or slightly shifted to a high field as the methyl proton peak H of the N-acetyl group of the chitosan oligosaccharide in the high field AC And the proton peak at 1.93 + -0.02 ppm with the chemical shift value shifted to the low field is determined as the methyl proton peak H generated by adding acetic acid in the chitosan oligosaccharide sample A (ii) a The proton peak group with the chemical shift value of 4.18-3.11ppm is H of the chitosan oligosaccharide 2 -H 6 Sum of 6 proton peaks H 2-6 。
2. The method for assigning spectrum of nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 1, wherein the drying method of the chitosan oligosaccharide sample in step (1) comprises: drying at 60 deg.C for 1-2 hr, heating to 80 deg.C for 1-2 hr, heating to 105 deg.C, drying for 0.5-1 hr, and drying to constant weight.
3. The method of claim 1, wherein the concentration of the sample of chitooligosaccharide in step (1) is 5-20 mg/mL.
4. The method of claim 1, wherein the temperature range of one test in step (2) is 20-30 ℃ and the temperature range of the other test in step (2) is 60-80 ℃.
5. The method of claim 4, wherein the temperature in step (2) is 25 ℃ for one test and 70 ℃ for the other test.
6. A method for determining the degree of deacetylation of a chitosan oligosaccharide, which comprises the following steps based on the method for assigning a nuclear magnetic resonance spectrum of a chitosan oligosaccharide of claim 5:
(4) area integration and calculation of proton peaks
Calculation of the methyl proton Peak H of N-acetyl group AC Has an area integral of I AC (ii) a Methyl proton peak of acetic acid H A Is an integral of I A (ii) a 6 proton peaks H of Chitosan oligosaccharide 2-6 Is the integral sum of 2-6 (ii) a Degree of deacetylation DD and amount of acetic acid added D A The calculation formula of (a) is as follows:
7. the method for determining the deacetylation degree of a chitosan oligosaccharide according to claim 6, wherein the proton peak having a chemical shift value of 5.45ppm is the anomeric carbon proton peak H of the terminal glucosamine unit of the chitosan oligosaccharide 1D-E The integral of which is I 1D-E (ii) a The proton peak with the chemical shift value of 5.22ppm is the anomeric carbon proton peak H of the terminal acetylglucosamine unit of the chitosan oligosaccharide 1A-E The integral of which is I 1A-E (ii) a The proton peak with a chemical shift value of 4.84ppm is the anomeric carbon proton peak H of the glucosamine unit in the chitosan oligosaccharide chain 1D-I Product of itIs divided into 1D-I (ii) a The proton peak at 4.62ppm was assigned as the anomeric carbon proton peak H of the acetylglucosamine unit in the chitooligosaccharide chain 1A-I The integral of which is I 1A-I (ii) a Anomeric carbon proton peak H of chitosan oligosaccharide 1 Integral I of 1 Is H 1D-E 、H 1A-E 、H 1D-I 、H 1A-I The sum of the integrals of (a); i is 2-6 6 equivalent replacement by I 1 ，I 1 Replacement by I 1D-E +I 1A-E +I 1D-I +I 1A-I ，I AC Replacement of/3 equivalents by I 1A-E +H 1A-I Then, the calculation formula of the degree of deacetylation is as follows:
8. a method for measuring the addition amount of acetic acid in chitosan oligosaccharide, which is characterized in that, on the basis of claim 7, the addition amount of acetic acid D A The calculation formula of (a) is as follows:
9. a method for measuring the average degree of polymerization of chitosan oligosaccharide, characterized in that, on the basis of claim 7, the calculation formula of the average degree of polymerization DP of chitosan oligosaccharide is as follows:
10. a method for determining the number average molecular weight of a chitosan oligosaccharide, characterized in that, on the basis of claim 7, the calculation formula of the number average molecular weight MW of the chitosan oligosaccharide is as follows:
MW＝DP×(203.2-42DD)+18。
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