CN114878624B - Method for assigning chitosan oligosaccharide NMR spectrum and method for measuring deacetylation degree - Google Patents
Method for assigning chitosan oligosaccharide NMR spectrum and method for measuring 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 174
- 238000000034 method Methods 0.000 title claims abstract description 73
- 230000006196 deacetylation Effects 0.000 title claims abstract description 40
- 238000003381 deacetylation reaction Methods 0.000 title claims abstract description 40
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 title claims abstract description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 111
- 238000005481 NMR spectroscopy Methods 0.000 claims abstract description 51
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 36
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 29
- 239000000126 substance Substances 0.000 claims abstract description 26
- HEMHJVSKTPXQMS-DYCDLGHISA-M Sodium hydroxide-d Chemical compound [Na+].[2H][O-] HEMHJVSKTPXQMS-DYCDLGHISA-M 0.000 claims abstract description 16
- 125000003047 N-acetyl group Chemical group 0.000 claims abstract description 9
- 239000011259 mixed solution Substances 0.000 claims abstract 3
- 239000000523 sample Substances 0.000 claims description 64
- 239000012488 sample solution Substances 0.000 claims description 21
- 238000012360 testing method Methods 0.000 claims description 18
- 238000001228 spectrum Methods 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 13
- 238000004364 calculation method Methods 0.000 claims description 12
- 230000002378 acidificating effect Effects 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 230000010354 integration Effects 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 4
- 230000008859 change Effects 0.000 abstract description 6
- -1 acetyl methyl proton Chemical group 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 2
- 229920001661 Chitosan Polymers 0.000 description 20
- 239000000243 solution Substances 0.000 description 20
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 11
- 239000002253 acid Substances 0.000 description 11
- 238000005259 measurement Methods 0.000 description 9
- 238000002156 mixing Methods 0.000 description 9
- 229910052739 hydrogen Inorganic materials 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
- 125000003277 amino group Chemical group 0.000 description 6
- 238000006073 displacement reaction Methods 0.000 description 6
- 239000001257 hydrogen Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 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 description 5
- 229920001542 oligosaccharide Polymers 0.000 description 5
- 150000002482 oligosaccharides Chemical class 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000003918 potentiometric titration Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 208000024827 Alzheimer disease Diseases 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
- 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 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
- 239000012670 alkaline solution Substances 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
- 230000003115 biocidal effect Effects 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
- 238000005119 centrifugation Methods 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
- 229940079593 drug Drugs 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 125000001976 hemiacetal group Chemical group 0.000 description 1
- 150000002373 hemiacetals Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000691 measurement method Methods 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
- 238000001225 nuclear magnetic resonance method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007789 sealing Methods 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
- 238000012546 transfer 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
- G01N24/088—Assessment or manipulation of a chemical or biochemical reaction, e.g. verification whether a chemical reaction occurred or whether a ligand binds to a receptor in drug screening or assessing reaction kinetics
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
- Y02A90/30—Assessment of water resources
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- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention relates to the technical field of nuclear magnetic analysis of chitosan oligosaccharide. Aiming at the problems that the chemical shift of a methyl proton peak of acetic acid added in a chitosan oligosaccharide product is similar to that of an acetyl methyl proton peak of chitosan oligosaccharide and the chitosan oligosaccharide is difficult to attribute in the technology of measuring the chitosan oligosaccharide deacetylation degree by a nuclear magnetic method, the method for attribute of the chitosan oligosaccharide NMR spectrum and the method for measuring the chitosan oligosaccharide deacetylation degree are provided: completely dissolving dry chitosan oligosaccharide sample in two deuteration reagents respectively, wherein deuteration reagent 1 is D 2 O or D 2 O and NaOD mixed solution, deuterated reagent 2 is D 2 Mixed solution of O and DCl; comparing nuclear magnetic resonance spectrograms of chitosan oligosaccharide samples dissolved by two deuterated reagents, and determining a methyl proton peak with small change of chemical shift value as a methyl proton peak H of N-acetyl of chitosan oligosaccharide AC The methyl proton peak with large change of chemical shift value is determined as methyl proton peak H of 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 attributing an NMR spectrum of chitosan oligosaccharide and a method for measuring deacetylation degree.
Background
The chitosan oligosaccharide is formed by two sugar 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, the chitosan oligosaccharide has good water solubility, and the biological activity of the chitosan oligosaccharide such as antibiosis, anti-tumor, immunity enhancing and regulating functions and the like is widely applied in the fields of medicine, cosmetics, food, textile, paper making and the like. Chitosan oligosaccharide, also known as low-polymerization degree water-soluble chitosan, is soluble in acidic, neutral and alkaline solutions. The Degree of Deacetylation (DD) can be defined as the average mole percent of glucosamine units in the chitosan oligosaccharide molecule. Glucosamine units are critical structures that determine the biological activity of chitosan oligosaccharides, and therefore DD is an important parameter for quality control of chitosan oligosaccharides.
Although many methods for measuring the degree of deacetylation of chitosan have been reported, few methods for measuring the degree of deacetylation of chitosan oligosaccharide have been reported, and for this reason, chitosan oligosaccharide products often contain acetic acid and/or hydrochloric acid to affect the accurate measurement of DD. The reason for acid contained in the chitosan oligosaccharide product is mainly as follows: 1) The molecular weight of the chitosan oligosaccharide is smaller, the unit mass of the chitosan oligosaccharide contains more reducing hemiacetal groups than chitosan, the hemiacetal base groups are easy to generate Maillard reaction with amino groups in glucosamine units in the chitosan oligosaccharide molecule, and acid is often added into a chitosan oligosaccharide product to protect the amino groups so as to inhibit the reaction; 2) In view of green, safety and environmental protection, most of chitosan oligosaccharide is derived from enzymatic degradation of chitosan, and acetic acid is often added to maintain the optimal pH of the chitosan; 3) The chitosan is insoluble in water, and is required to be dissolved in an acid solution before degradation, and acid residues exist in the degraded chitosan; 4) Chitosan can only be dissolved in a limited few acidic dilute solutions, dilute hydrochloric acid is the best, but hydrochloric acid is strong acid, so that chitosan oligosaccharide is easy to be degraded continuously to reduce the bioactivity of chitosan oligosaccharide, and acetic acid with the same low cost is often added into chitosan oligosaccharide products.
The addition of acetic acid brings great trouble to the accurate measurement of the deacetylation degree of chitosan oligosaccharide, and the methods commonly used for measuring the deacetylation degree of chitosan, such as an elemental analysis method, a potentiometric titration method, an infrared method and an ultraviolet method, cannot be applied to the measurement of the deacetylation degree of chitosan oligosaccharide. Element(s)The assay is based on the carbon to nitrogen content ratio (C/N) of the chitosan oligosaccharide molecule, whereas carbon-containing acetic acid interferes with its determination. The infrared method is based on quantitative determination of vibration peaks related to methyl and carbonyl of acetyl, and due to the existence of acid, the-NH in chitosan oligosaccharide molecule 2 Part or all of them being-NH 3 + The N-H bending vibration of the two groups is completely different, so that the C=O stretching vibration peak of the acetyl is influenced, and the deacetylation degree of chitosan oligosaccharide cannot be measured by using the infrared measurement method of chitosan. The potentiometric titration method is based on determination of amino groups in chitosan oligosaccharide molecules, and because acetic acid is weak acid and amino groups are weak base, determination of amino groups content is difficult to observe at titration sites, and furthermore, chitosan oligosaccharide often exists in the form of partial salts, possibly including acetate and hydrochloride, and the content of the salts is unknown, so that great trouble is brought to calculation of DD value, and moreover, the method is long in time consumption, low in detection efficiency and inconvenient for mass measurement. The ultraviolet method is based on acetyl in chitosan oligosaccharide molecule, and acetic acid added in chitosan oligosaccharide product directly affects the measurement result, and is more suitable for measuring chitosan DD without acetic acid. Therefore, the industry can only express the deacetylation degree of chitosan oligosaccharide by the mass ratio of acetyl in a chitosan oligosaccharide sample, but not the molar ratio of glucosamine units of a chitosan oligosaccharide molecular structure, and the structural characteristics of chitosan oligosaccharide molecules can be more reflected by measuring the deacetylation degree by the molar ratio.
The nuclear magnetic method in the prior art is a method for determining the deacetylation degree of chitosan oligosaccharide most accurately, and the DD value of the chitosan oligosaccharide is calculated by comparing the integral area of hydrogen protons of a test sample with the integral area of hydrogen protons of a selected reference hydrogen proton according to the difference of chemical displacement values of different hydrogen protons in chitosan oligosaccharide molecules. However, the process is not limited to the above-described process, 1 the H-NMR method also has disadvantages: the methyl proton peak of acetic acid added in the chitosan oligosaccharide product is similar to the chemical displacement of the methyl proton peak of acetyl, and is difficult to distinguish. Some scholars believe that the methyl proton peak at 1.92ppm is attributed to N-acetyl (Jiang, y., fu, c.h., wu, s.h., liu, g.h., guo, j.,&su, Z.Q. (2017), determination of the Deacetylation Degree of Chitooligosacharides. Marine drugs,15 (11)), are also knownThe inventors considered that they all belonged to N-acetyl (Jiang, z., liu, g, yang, y., shao, k., wang, y., liu, w.,&han, B. (2019). N-Acetyl chitooligosaccharides attenuate amyloid. Beta. -induced damage in animal and cell models of Alzheimer's disease. Process Biochemistry,84, 161-171.). If the methyl proton peak of acetyl is not clear, the detection of deacetylation degree is greatly deviated, which is extremely unfavorable for quality control and application of chitosan oligosaccharide. Therefore, accurate assignment of these two methyl proton peaks is a current urgent need to solve the problem that is a necessary premise for accurate determination of the deacetylation degree of chitosan oligosaccharide and the addition amount of acetic acid.
Disclosure of Invention
The invention aims to provide a spectrum attribution method of a nuclear magnetic resonance spectrogram of chitosan oligosaccharide and a method for measuring the deacetylation degree of the chitosan oligosaccharide, which aim to solve the problems that the chemical displacement of a methyl proton peak of acetic acid added in a chitosan oligosaccharide product is similar to that of an acetyl methyl proton peak of the chitosan oligosaccharide and is difficult to distinguish, and different attributions can directly lead to the occurrence of great deviation of a measurement result, and can accurately judge the methyl proton peak H of an N-acetyl group by comparing displacement change conditions of the methyl proton peak before and after the pH of a chitosan oligosaccharide nuclear magnetic sample is reduced AC Methyl proton peak H with acetic acid A The spectrum belonging to the chitosan oligosaccharide is adopted, and the accurate determination of the deacetylation degree of the chitosan oligosaccharide is further realized.
The invention is realized by the following technical scheme:
in one aspect, 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 magnetic sample solution
Completely dissolving a dried chitosan oligosaccharide sample in a deuteration reagent 1 and a deuteration reagent 2 respectively to obtain a sample solution 1 and a sample solution 2, wherein the deuteration reagent 1 is D 2 O or D 2 The final concentration of the NaOD is 0-0.02mol/L, the deuteration reagent 2 is an acidic deuteration reagent, and the composition of the deuteration reagent is D 2 O and DCl in a final concentration of 0.02-0.5mol/L；
(2) Sample testing
Respectively placing the sample solution 1 and the sample solution 2 prepared in the step (1) in a nuclear magnetic resonance instrument to obtain a nuclear magnetic resonance spectrogram 1 and a nuclear magnetic resonance spectrogram 2 of chitosan oligosaccharide;
(3) Spectrum assignment
Comparing the nuclear magnetic resonance spectrogram 2 in the step (2) with the nuclear magnetic resonance spectrogram 1, and determining a proton peak of 2.070+/-0.015 ppm, which does not change the chemical shift value or moves to the high field with smaller amplitude, as a methyl proton peak H of N-acetyl of chitosan oligosaccharide in a high field region AC The proton peak of 2.000.+ -. 0.085ppm, whose chemical shift value was greatly shifted to the lower field, was determined as methyl proton peak H due to addition of acetic acid in the chitosan oligosaccharide sample A The method comprises the steps of carrying out a first treatment on the surface of the Proton peak group with chemical shift value of 4.18-3.11ppm is H of chitosan oligosaccharide 2 -H 6 Sum H of 6 proton peaks of (C) 2-6 。
D in deuterated reagent 1 described above 2 O aims at making N-acetylmethyl proton peak H AC And methyl proton peak H generated by adding acetic acid in chitosan oligosaccharide sample A Independent of each other without interfering with the measurement, naOD is added to D 2 O is more beneficial to H AC And H is A Independent of each other without interfering with the assay.
The purpose of the above-mentioned acid deuteration reagent to dissolve the sample is to accurately assign methyl proton peak of acetic acid which moves to low field greatly due to acid deuteration reagent and acetyl methyl proton peak of chitosan oligosaccharide which moves to high field hardly or in small amplitude; if the concentration of DCl is too low, it is difficult to observe whether the methyl proton peak of acetic acid moves, and the methyl proton peak of acetic acid is shifted to a low field to coincide with the acetyl methyl proton peak of chitosan oligosaccharide, but if the concentration of DCl is too high, the chitosan oligosaccharide is degraded, and the detection cost is increased.
Further, the drying method of the chitosan oligosaccharide sample in the step (1) comprises the following steps: drying at 60deg.C for 4-10 hr, heating to 80deg.C for 2-2.5 hr, further heating to 105deg.C for 0.5-1 hr, and drying to constant weight.
The purpose of drying the chitosan oligosaccharide sample is to remove moisture so as to avoid interference of residual moisture in the chitosan oligosaccharide sample with measurement; 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 moisture is removed after the moisture is almost removed at high temperature (80 ℃ and 105 ℃) for a short time.
Further, the concentration of the chitosan oligosaccharide sample solution in the step (1) is 5-20mg/mL. The chitosan oligosaccharide concentration is enough to meet the requirement of nuclear magnetic resonance hydrogen spectrum on the concentration of a sample, the concentration is too low, the signal to noise ratio is too small, and the accurate measurement is affected; too high a concentration increases the solution viscosity, affecting signal acquisition.
Further, the concentrations of the chitosan oligosaccharide in the sample solution 1 and the sample solution 2 are the same.
Further, the concentration of the chitosan oligosaccharide in the sample solution 1 and the sample solution 2 is 8-12mg/mL, preferably 10mg/mL.
Further, the final concentration of NaOD of the deuterated reagent 1 in the step (1) is 0-0.01mol/L.
Further, the final concentration of NaOD of the deuterated reagent 1 in the step (1) is preferably 0.001 to 0.01mol/L, more preferably 0.005 to 0.01mol/L.
Further, the final concentration of DCl of deuterated reagent 2 in step (1) is 0.02-0.05mol/L.
Further, the test temperature in step (2) is 20-25 ℃, preferably 25 ℃.
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 AC Is integrated into the area of I AC The method comprises the steps of carrying out a first treatment on the surface of the Methyl proton peak H of acetic acid A Is integrated as I A The method comprises the steps of carrying out a first treatment on the surface of the 6 proton peaks H of Chitosan oligosaccharide 2-6 Is the integral sum of I 2-6 The method comprises the steps of carrying out a first treatment on the surface of the Degree of deacetylation DD and amount of acetic acid added D A The calculation formula of (2) is as follows:
compared with the prior art, the invention has the following advantages:
(1) By the displacement condition of two methyl proton peaks in the nuclear magnetic resonance hydrogen spectrum of chitosan oligosaccharide and the displacement condition of acetic acid and acetate under the condition of different solvent pH values, the proton peak which is positioned at about 2.07ppm can be accurately attributed to the methyl proton peak H of acetyl in chitosan oligosaccharide molecule AC The peak does not shift greatly due to the change of the pH of the solution; the proton peak at about 1.92-2.08ppm is the proton peak H of acetic acid added in chitosan oligosaccharide sample A The peak can be greatly shifted due to the difference of the pH values of the solutions;
(2) The deacetylation degree of the chitosan oligosaccharide can be 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 addition amount of acetic acid contained in the unit sugar unit in the chitosan oligosaccharide molecule can be measured;
(4) The operation method is simple, convenient and efficient, and the result accuracy is high;
(5) The detection process has no chemical change, so the reproducibility of the detection result is good.
Drawings
FIG. 1 shows three chitosan oligosaccharide samples measured by the infrared method of the prior art: stacked images of infrared spectra of COS-A, COS-B and COS-C;
FIG. 2 is a hydrogen spectrum of three chitosan oligosaccharide samples measured by the prior art nuclear magnetic resonance method (the solvent used for the test is D 2 O, testing at room temperature of 25 ℃, wherein the chitosan oligosaccharide sample COS-A, COS-B, COS-C in the figure is respectively from Qingdao Bozhi Hui biological technology Co., shanghai source leaf biological technology Co., shandong Lai City sea biological product Co., ltd.);
FIG. 3 is a hydrogen spectrum of a chitosan oligosaccharide sample of example 1 (test conditions are a.D, respectively 2 O/25℃、b.0.02M DCl/D 2 O/25℃、c.0.01M NaOD/D 2 O/25℃)；
FIG. 4 is a hydrogen spectrum of a chitosan oligosaccharide sample of example 2 (test conditions are a.D, respectively 2 O/25℃、b.0.05M DCl/D 2 O/25℃)；
FIG. 5 is a hydrogen spectrum of a chitosan oligosaccharide sample of example 3 (test conditions are a.D, respectively 2 O/25℃、b.0.01M NaOD/D 2 O/25℃)；
Detailed Description
The present invention is described in further detail below with reference to examples, but the implementation of the present invention is not limited to the examples, and all changes or equivalent substitutions without departing from the concept of the present invention are within the scope of the present invention.
The chitosan oligosaccharide samples used in the following examples: COS-A, COS-B and COS-C are from Qingdao Bozhi Hui biosciences, shanghai Yuan Ye biosciences, and Shandong Lai Hi-Chemicals, inc., respectively.
D 2 O (TMSP with internal standard) was purchased from sigma aldrich (Shanghai) trade company.
DCl (20%) solutions were purchased from Shanghai Alasdine Biochemical technologies Co.
Instrument: DD2 500Mhz Nuclear magnetic resonance apparatus Agilent technologies Co.
EXAMPLE 1 Spectrum assignment method of Nuclear magnetic resonance Spectrometry for Chitosan oligosaccharide
The method comprises the following steps:
(1) Preparation of Chitosan oligosaccharide sample (COS-A) solution
a. Drying
The specific operation is as follows: 10mg of chitosan oligosaccharide sample was weighed into three centrifuge tubes (centrifuge tube 1, centrifuge tube 2 and centrifuge tube 3), dried at 60℃for 2 hours, warmed to 80℃for 2 hours, and then warmed to 105℃for 1 hour until the weight was constant. And after the drying is finished, opening the oven, immediately sealing, and cooling in a dryer for standby.
b. Sample addition
500 microliters of D 2 O is added to the centrifugation of the chitosan oligosaccharide sample in step aTube 1, centrifuge tube 2 and centrifuge tube 3 were thoroughly mixed to dissolve the mixture to prepare a chitosan oligosaccharide solution having a concentration of 20mg/mL. Into centrifuge tube 1, 500. Mu.l of D was added 2 O, after thoroughly mixing, is added into the nuclear magnetic tube 1 by a pipette. 500 microliters of 0.04mol/L DCl solution was added to centrifuge tube 2, thoroughly mixed to a final DCl concentration of 0.02mol/L and a chitosan oligosaccharide concentration of 10mg/mL, and added to nuclear magnetic resonance tube 2 with a pipette. 500 microliters of 0.02mol/L NaOD solution was added to centrifuge tube 3, and the mixture was thoroughly mixed to give a final NaOD concentration of 0.01mol/L and a chitosan oligosaccharide concentration of 10mg/mL, and the mixture was added to nuclear magnetic resonance tube 3 by a pipette.
(2) Sample testing
And (3) positioning the nuclear magnetic tube filled with the sample in a nuclear magnetic resonance spectrometer, and obtaining a proton nuclear magnetic resonance spectrogram through the steps of field locking, shimming, probe tuning, parameter setting, data acquisition, data processing and the like. In terms of parameter setting, the test temperature is room temperature (25 ℃), the scanning times are 32 times, and the acquired data are stored for standby use
(3) Data processing
Obtaining nuclear magnetic resonance spectrogram of chitosan oligosaccharide from step (2) by using application software of nuclear magnetic resonance spectrometer, as shown in figure 3, comparing figure 3a (or figure 3 c) with figures 3b and D 2 O (or NaOD/D) 2 O) the proton peak with a chemical shift value of about 1.94ppm (or 1.92 ppm) in the dissolved chitosan oligosaccharide sample was shifted to 2.08ppm with the addition of DCl, thus belonging to D 2 Proton peak H with a chemical shift value of about 2.07ppm of acetic acid in O-dissolved chitosan oligosaccharide sample A ；D 2 Proton peak with chemical shift value of about 2.07ppm in O-dissolved chitosan oligosaccharide sample, which is attributed to methyl proton peak H of acetyl in chitosan oligosaccharide molecule, is not changed by adding acidic deuteration reagent into chitosan oligosaccharide sample AC . About 4.18-3.11ppm of proton peak group is H of chitosan oligosaccharide 2 -H 6 Sum H of 6 proton peaks of (C) 2-6 。
Example 2 method for determining the degree of deacetylation of Chitosan oligosaccharides
The method comprises the following steps:
(1) Preparation of Chitosan oligosaccharide sample (COS-A) solution
a. Drying
For specific operation, reference is made to example 1.
b. Sample addition
500 microliters of D 2 And (c) adding O into the centrifuge tube 1, the centrifuge tube 2 and the centrifuge tube 3 added with the chitosan oligosaccharide sample in the step a, and fully and uniformly mixing to dissolve the chitosan oligosaccharide sample to prepare a chitosan oligosaccharide solution. Into centrifuge tube 1, 500. Mu.l of D was added 2 O, after thoroughly mixing, is added into the nuclear magnetic tube 1 by a pipette. Into the centrifuge tube 2, 500. Mu.l of a 0.04mol/L DCl solution was added to give a final DCl concentration of 0.02mol/L, and after thoroughly mixing, the mixture was added to the nuclear magnetic resonance tube 2 by a pipette. 500. Mu.l of a 0.02mol/L NaOD solution was added to the centrifuge tube 3 to give a final NaOD concentration of 0.01mol/L, and the mixture was thoroughly mixed and then added to the nuclear magnetic resonance tube 3 by a pipette.
(2) Sample testing
Reference example 1
(3) Data processing
By utilizing application software of a nuclear magnetic resonance analyzer, a nuclear magnetic resonance spectrogram of the chitosan oligosaccharide is obtained from the step (2), and as shown in an attached figure 3 of the specification, a proton peak with a chemical shift value of about 2.07ppm is a methyl proton peak H of acetyl in chitosan oligosaccharide molecules by comparing the two nuclear magnetic resonance spectrograms obtained in the step (2) AC The peak was not changed in chemical shift value by the addition of acidic deuterating reagent to the chitosan oligosaccharide sample, and its integral was I AC The method comprises the steps of carrying out a first treatment on the surface of the A proton peak having a chemical shift value of about 1.94ppm was shifted to 2.08ppm by adding an acidic deuterating agent to the chitosan oligosaccharide sample (FIG. 3 b), and to 1.92ppm by adding a basic deuterating agent to the chitosan oligosaccharide sample, and thus the peak was methyl proton peak H generated by adding acetic acid to the chitosan oligosaccharide sample A Integral of which is I A . About 4.18-3.11ppm of proton peak group is H of chitosan oligosaccharide 2 -H 6 Sum H of 6 proton peaks of (C) 2-6 Integral of which is I 2-6 . The above values are shown in Table 1.
TABLE 1 Chitosan oligosaccharide samples in 1 Proton attribution and area integration (25 ℃ test) on HNMR spectrogram
| Sample number | Proton peak integration | H AC | H 2-6 | H A |
| 1 | Nuclear magnetic resonance tube 1 (D) 2 O) | 1 | 51.30 | 4.00 |
| 2 | Nuclear magnetic resonance tube 2 (acid) | 1 | 53.32 | 3.80 |
| 3 | Nuclear magnetic resonance tube 2 (alkali) | 1 | 50.54 | 3.97 |
According to a calculation formula of the deacetylation degree of chitosan oligosaccharide, the calculation result is as follows:
deacetylation Degree (DD) and acetic acid addition amount (D) of chitosan oligosaccharide sample in nuclear magnetic resonance tube 1 A ) The method comprises the following steps:
deacetylation Degree (DD) and acetic acid addition amount (D) of chitosan oligosaccharide sample in nuclear magnetic resonance tube 2 A ) The method comprises the following steps:
deacetylation Degree (DD) and acetic acid addition amount (D) of chitosan oligosaccharide sample in nuclear magnetic resonance tube 3 A ) The method comprises the following steps:
example 3 method for determining the degree of deacetylation of Chitosan oligosaccharides
The method comprises the following steps:
(1) Preparation of Chitosan oligosaccharide sample (COS-B) solution
a. Drying
For specific operation, reference is made to example 1.
b. Sample addition
500 microliters of D 2 And (c) adding O into the centrifuge tube 1 and the centrifuge tube 2 added with the chitosan oligosaccharide sample in the step a, and fully and uniformly mixing to dissolve the chitosan oligosaccharide sample to prepare a chitosan oligosaccharide solution. Into centrifuge tube 1, 500. Mu.l of D was added 2 O, after fully and evenly mixing, adding the mixture into a nuclear magnetic tube by a liquid transfer device1. Into the centrifuge tube 2, 500. Mu.l of a 0.10mol/L DCl solution was added to give a final DCl concentration of 0.05mol/L, and after thoroughly mixing, the mixture was added to the nuclear magnetic resonance tube 2 by a pipette.
(2) Sample testing
Reference example 1
(3) Data processing
By utilizing application software of a nuclear magnetic resonance analyzer, a nuclear magnetic resonance spectrogram of the chitosan oligosaccharide is obtained from the step (2), and as shown in an attached figure 4 of the specification, a proton peak with a chemical shift value of about 2.07ppm is a methyl proton peak H of acetyl in chitosan oligosaccharide molecules by comparing the two nuclear magnetic resonance spectrograms obtained in the step (2) AC The peak was not changed in chemical shift value by the addition of acidic deuterating reagent to the chitosan oligosaccharide sample, and its integral was I AC The method comprises the steps of carrying out a first treatment on the surface of the The proton peak having a chemical shift value of about 1.94ppm was shifted to 2.08ppm (b..in FIG. 4) by adding an acidic deuterating agent to the chitosan oligosaccharide sample, and therefore, this peak was methyl proton peak H generated by adding acetic acid to the chitosan oligosaccharide sample A Integral of which is I A . About 4.18-3.11ppm of proton peak group is H of chitosan oligosaccharide 2 -H 6 Sum H of 6 proton peaks of (C) 2-6 Integral of which is I 2-6 . The above values are shown in Table 2.
TABLE 2 Chitosan oligosaccharide samples in 1 Proton attribution and area integration (25 ℃ test) on HNMR spectrogram
| Sample number | Proton peak integration | H AC | H 2-6 | H A |
| 1 | Nuclear magnetic resonance tube 1 (D) 2 O) | 1 | 25.61 | 1.87 |
| 2 | Nuclear magnetic resonance tube 2 (acid) | 1 | 26.35 | 1.75 |
According to a calculation formula of the deacetylation degree of chitosan oligosaccharide, the calculation result is as follows:
deacetylation Degree (DD) and acetic acid addition amount (D) of chitosan oligosaccharide in Nuclear magnetic resonance tube 1 A ) The method comprises the following steps:
deacetylation Degree (DD) and acetic acid addition amount (D) of chitosan oligosaccharide in nuclear magnetic resonance tube 2 A ) The method comprises the following steps:
example 4 method for determining the degree of deacetylation of Chitosan oligosaccharides
The method comprises the following steps:
(1) Preparation of Chitosan oligosaccharide sample (COS-C) solution
a. Drying
For specific operation reference is made to example 1.
b. Sample addition
500 microliters of D 2 And (c) adding O into the centrifuge tube 1 and the centrifuge tube 2 added with the chitosan oligosaccharide sample in the step a, and fully and uniformly mixing to dissolve the chitosan oligosaccharide sample to prepare a chitosan oligosaccharide solution. Into centrifuge tube 1, 500. Mu.l of D was added 2 O, after thoroughly mixing, is added into the nuclear magnetic tube 1 by a pipette. 500. Mu.l of a 0.02mol/L NaOD solution was added to the centrifuge tube 2 to give a final NaOD concentration of 0.01mol/L, and the mixture was thoroughly mixed and then added to the nuclear magnetic resonance tube 2 by a pipette.
(2) Sample testing
Reference example 1
(3) Data processing
By utilizing application software of a nuclear magnetic resonance analyzer, a nuclear magnetic resonance spectrogram of the chitosan oligosaccharide is obtained from the step (2), and as shown in an attached figure 5 of the specification, a proton peak with a chemical shift value of about 2.07ppm is a methyl proton peak H of acetyl in chitosan oligosaccharide molecules by comparing the two nuclear magnetic resonance spectrograms obtained in the step (2) AC The peak was not changed in chemical shift value by the addition of acidic deuterating reagent to the chitosan oligosaccharide sample, and its integral was I AC The method comprises the steps of carrying out a first treatment on the surface of the A proton peak having a chemical shift value of about 1.95ppm was shifted to 1.92ppm (b..in FIG. 5) by adding an alkaline deuterating agent to the chitosan oligosaccharide sample, and therefore, this peak was methyl proton peak H generated by adding acetic acid to the chitosan oligosaccharide sample A Integral of which is I A . About 4.18-3.11ppm of proton peak group is H of chitosan oligosaccharide 2 -H 6 Sum H of 6 proton peaks of (C) 2-6 Integral of which is I 2-6 . The above values are shown in Table 3.
TABLE 3 Chitosan oligosaccharide samples in 1 Proton attribution and area integration (25 ℃ test) on HNMR spectrogram
| Sample number | Proton peak integration | H AC | H 2-6 | H A |
| 1 | Nuclear magnetic resonance tube 1 (D) 2 O) | 1 | 18.15 | 0.31 |
| 2 | Nuclear magnetic resonance tube 2 (alkali) | 1 | 18.74 | 0.30 |
According to a calculation formula of the deacetylation degree of chitosan oligosaccharide, the calculation result is as follows:
deacetylation Degree (DD) and acetic acid addition amount (D) of chitosan oligosaccharide in Nuclear magnetic resonance tube 1 A ) The method comprises the following steps:
deacetylation Degree (DD) and acetic acid addition amount (D) of chitosan oligosaccharide in nuclear magnetic resonance tube 2 A ) The method comprises the following steps:
the above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. The spectrum attribution method of the nuclear magnetic resonance spectrogram of the chitosan oligosaccharide is characterized by comprising the following steps of:
(1) Preparation of chitosan oligosaccharide nuclear magnetic sample solution
Completely dissolving a dried chitosan oligosaccharide sample in a deuteration reagent 1 and a deuteration reagent 2 respectively to obtain a sample solution 1 and a sample solution 2, wherein the deuteration reagent 1 is D 2 O or D 2 The final concentration of the NaOD is 0-0.02mol/L, the deuteration reagent 2 is an acidic deuteration reagent, and the composition of the deuteration reagent is D 2 A mixed solution of O and DCl, wherein the final concentration of the DCl is 0.02-0.05mol/L;
(2) Sample testing
Respectively placing the sample solution 1 and the sample solution 2 prepared in the step (1) in a nuclear magnetic resonance instrument to obtain a nuclear magnetic resonance spectrogram 1 and a nuclear magnetic resonance spectrogram 2 of chitosan oligosaccharide;
(3) Spectrum assignment
Comparing the nuclear magnetic resonance spectrum 2 in the step (2) with the nuclear magnetic resonance spectrum 1, and determining a proton peak of 2.07+/-0.02 ppm, which is unchanged in chemical shift value or moves to a high field with a smaller amplitude, as a methyl proton peak H of N-acetyl of chitosan oligosaccharide in a high field region AC The proton peak of 2.00.+ -. 0.09ppm, in which the chemical shift value was greatly changed and shifted to the low field, was determined as the proton peak of the chitosan oligosaccharide sample due to the addition of acetic acidThe resulting methyl proton peak H A The method comprises the steps of carrying out a first treatment on the surface of the Proton peak group with chemical shift value of 4.18-3.11ppm is H of chitosan oligosaccharide 2 -H 6 Sum H of 6 proton peaks of (C) 2-6 。
2. The method for assigning a nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 1, wherein the drying method of the chitosan oligosaccharide sample in the step (1) is as follows: drying at 60deg.C for 1-2 hr, heating to 80deg.C for 1-2 hr, further heating to 105deg.C for 0.5-1 hr, and drying to constant weight.
3. The method for assigning a nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 1, wherein the concentration of chitosan oligosaccharide in the sample solution 1 and the concentration of chitosan oligosaccharide in the sample solution 2 in the step (1) are the same.
4. The method for assigning a nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 1, wherein the concentration of chitosan oligosaccharide in the sample solution 1 and the sample solution 2 is 5-20mg/mL.
5. The method for assigning a nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 4, wherein the concentration of chitosan oligosaccharide in the sample solution 1 and the sample solution 2 in the step (1) is 10mg/mL.
6. The method for assigning a nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 1, wherein the final concentration of NaOD of deuterated reagent 1 in step (1) is 0-0.01mol/L.
7. The method of assigning a nuclear magnetic resonance spectrum to a chitosan oligosaccharide according to claim 6, wherein the final concentration of NaOD of the deuterated reagent 1 in the step (1) is 0.005-0.01mol/L.
8. The method for assigning a nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 1, wherein the test temperature in the step (2) is 20-25 ℃.
9. A method for determining the deacetylation degree of chitosan oligosaccharide, which is characterized by further comprising the following steps based on the method for assigning a spectrum to a nuclear magnetic resonance spectrum of chitosan oligosaccharide according to claim 1:
(4) Area integration and calculation of proton peaks
Calculating methyl proton peak H of N-acetyl by adopting nuclear magnetic resonance spectrogram of dissolved chitosan oligosaccharide sample of deuteration reagent 1 AC Is integrated into the area of I AC The method comprises the steps of carrying out a first treatment on the surface of the Methyl proton peak H of acetic acid A Is integrated as I A The method comprises the steps of carrying out a first treatment on the surface of the 6 proton peaks H of Chitosan oligosaccharide 2-6 Is the integral sum of I 2-6 The method comprises the steps of carrying out a first treatment on the surface of the Degree of deacetylation DD and amount of acetic acid added D A The calculation formula of (2) is as follows:
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911081A (en) * | 2016-04-18 | 2016-08-31 | 成都奥邦药业有限公司 | Method of identifying mannatide |
| CN109211957A (en) * | 2018-08-28 | 2019-01-15 | 青岛科技大学 | A method of measurement hydroxypropyl chitosan N- degree of substitution, total degree of substitution and degree of acetylation |
| CN110806421A (en) * | 2019-10-30 | 2020-02-18 | 中国科学院山西煤炭化学研究所 | NMR detection method for PAMAM and guest small molecule interaction mode |
| CN111337529A (en) * | 2020-03-12 | 2020-06-26 | 青岛科技大学 | Preparation and test method of polysaccharide sample with reduced viscosity and shifted water peak |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911081A (en) * | 2016-04-18 | 2016-08-31 | 成都奥邦药业有限公司 | Method of identifying mannatide |
| CN109211957A (en) * | 2018-08-28 | 2019-01-15 | 青岛科技大学 | A method of measurement hydroxypropyl chitosan N- degree of substitution, total degree of substitution and degree of acetylation |
| CN110806421A (en) * | 2019-10-30 | 2020-02-18 | 中国科学院山西煤炭化学研究所 | NMR detection method for PAMAM and guest small molecule interaction mode |
| CN111337529A (en) * | 2020-03-12 | 2020-06-26 | 青岛科技大学 | Preparation and test method of polysaccharide sample with reduced viscosity and shifted water peak |
Non-Patent Citations (2)
| Title |
|---|
| Determination of the Deacetylation Degree of Chitooligosaccharides;Yao Jiang et al.;《Mar. Drugs》;第332卷(第15期);1-14 * |
| Simultaneous determination of molar degree of substitution and its distribution fraction, degree of acetylation in hydroxypropyl chitosan by 1H NMR spectroscopy;Xingshuang Lv et al.;《Carbohydrate Polymers》;第263卷;117950 * |
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