CN106248607A - Utilize the method that first derivative ultraviolet spectro-photometry measures oligochitosan deacetylation - Google Patents
Utilize the method that first derivative ultraviolet spectro-photometry measures oligochitosan deacetylation Download PDFInfo
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- CN106248607A CN106248607A CN201610765268.3A CN201610765268A CN106248607A CN 106248607 A CN106248607 A CN 106248607A CN 201610765268 A CN201610765268 A CN 201610765268A CN 106248607 A CN106248607 A CN 106248607A
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- oligochitosan
- deacetylation
- acetyl
- glucosamine
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 96
- 230000006196 deacetylation Effects 0.000 title claims abstract description 58
- 238000003381 deacetylation reaction Methods 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000002798 spectrophotometry method Methods 0.000 title claims abstract description 30
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 47
- 239000002904 solvent Substances 0.000 claims abstract description 32
- 239000000243 solution Substances 0.000 claims description 21
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound 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 19
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 19
- -1 D-glucosamine amine Chemical class 0.000 claims description 18
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 claims description 17
- 229950006780 n-acetylglucosamine Drugs 0.000 claims description 17
- 239000012086 standard solution Substances 0.000 claims description 10
- 230000031700 light absorption Effects 0.000 claims description 5
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims 1
- 241000219094 Vitaceae Species 0.000 claims 1
- 229960002442 glucosamine Drugs 0.000 claims 1
- 235000021021 grapes Nutrition 0.000 claims 1
- 238000003556 assay Methods 0.000 abstract description 4
- 238000002835 absorbance Methods 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract 1
- 238000003908 quality control method Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 17
- 238000005259 measurement Methods 0.000 description 13
- BNSTVBLCTRZUDD-CBQIKETKSA-N N-acetyl-D-glucoseamine Chemical compound CC(=O)N[C@@]1(O)O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O BNSTVBLCTRZUDD-CBQIKETKSA-N 0.000 description 11
- 238000010521 absorption reaction Methods 0.000 description 11
- 229910052739 hydrogen Inorganic materials 0.000 description 11
- 239000001257 hydrogen Substances 0.000 description 11
- 238000005160 1H NMR spectroscopy Methods 0.000 description 10
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 7
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 6
- 230000000850 deacetylating effect Effects 0.000 description 6
- 125000003047 N-acetyl group Chemical group 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 239000002696 acid base indicator Substances 0.000 description 5
- STZCRXQWRGQSJD-GEEYTBSJSA-M methyl orange Chemical compound [Na+].C1=CC(N(C)C)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 STZCRXQWRGQSJD-GEEYTBSJSA-M 0.000 description 5
- 229940012189 methyl orange Drugs 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 229920002101 Chitin Polymers 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920001542 oligosaccharide Polymers 0.000 description 3
- 150000002482 oligosaccharides Chemical class 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 235000009392 Vitis Nutrition 0.000 description 2
- 241000219095 Vitis Species 0.000 description 2
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000371997 Eriocheir sinensis Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229920000140 heteropolymer Polymers 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
Landscapes
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Saccharide Compounds (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention provides a kind of method utilizing first derivative ultraviolet spectro-photometry to measure oligochitosan deacetylation, comprises the steps: 1) using hydrochloric acid solution that concentration is 0.3 1.0mol/L as blank solvent, Criterion curve；2) take oligochitosan sample, after dissolving by blank solvent, with blank solvent as reference, at 200 206nm wavelength, measure its absorbance, calculate its deacetylation according to standard curve.The method utilizing first derivative ultraviolet spectro-photometry mensuration oligochitosan deacetylation of present invention offer, operation is simple, can eliminate the interference of concurrent, turbidity and colourity, accuracy and precision high, practical.The assay method of the oligochitosan deacetylation that the present invention provides is simple, quick, accurate, contributes to the quality control in oligochitosan preparation process.
Description
Technical field
The invention belongs to chemical field, particularly relate to one and utilize first derivative ultraviolet spectro-photometry mensuration oligochitosan to take off
The method of acetyl degree.
Background technology
Chitin is the second largest biological polyoses being only second to cellulose on the earth, is primarily present in the first of shrimp, Eriocheir sinensis, insecticide
At shell, fungus, the cell wall of plant, the tough parts such as the joint of animal and muscle and synosteosis etc..Oligochitosan (COS) is ammonia
Homopolymer that base glucose and 2-Acetamido-2-deoxy-D-glucose are formed by connecting with β-Isosorbide-5-Nitrae glycosidic bond or heteropolymer, by degradation of chitin
?.The N-acetyl group of chitin is sloughed more than 55% and is chitosan (CTS), and CTS continues degraded to 2-10 the degree of polymerization i.e.
For COS.In actual production, the molecule of 20 degree of polymerization (about 3kDa) is also referred to as COS.
Compared with chitosan, it is more preferable that oligochitosan has dissolubility, the lower advantage being more easy to be absorbed by the body of viscosity, doctor
The fields such as medicine, agricultural, fine chemistry industry have a wide range of applications.Oligochitosan not only has the merits such as antitumor, bacteriostasis antibiosis, antioxidation
Can, also there is fat-reducing, adjust the biological function such as fat, enhancing immunity.The research and development of oligochitosan is current study hotspot.
The deacetylation of oligochitosan refers to remove in saccharide residue number total in oligochitosan shared by the saccharide residue number of acetyl group
Percentage ratio, it is possible to affect the biology of oligochitosan, physics, chemical functional with activity, be the embodiment of the various function of oligochitosan, be weighing apparatus
One of important indicator of amount oligochitosan quality.
At present, the method for existing more maturation is applied to measure the deacetylation of chitosan, such as: version " middle Chinese in 2015
People republic pharmacopeia " the 4th the 511-512 page disclose methyl orange acid-base indicator method and measure the side of deacetylating degree of chitosan
Method；European Pharmacopoeia (European Pharmacopeia8.0 [M] .EDQM, 2014:1841-1842) discloses and with purified water is
Solvent, utilizes the method that first derivative ultraviolet spectro-photometry measures deacetylating degree of chitosan.Chinese patent application
201310587162.5 disclose a kind of method measuring chitosan and the deacetylation of oligochitosan mixture, including walking as follows
Rapid: by sample diluted hydrochloric acid dissolution to be detected, simultaneously with complete deacetylated oligochitosan as internal standard, measure respectively 199nm's
Absorbance under purple light, and according to standard curve, obtain sample deacetylation, with the hydrochloric acid solution of 0.001mol/L during detection
As solvent.
, there is bigger difference, the therefore survey of deacetylating degree of chitosan in nature due to both in chitosan and oligochitosan
The method of determining is not necessarily applied to the mensuration of oligochitosan deacetylation.According to the version Pharmacopoeia of the People's Republic of China the 4th in 2015
When in portion, the assay method of deacetylating degree of chitosan measures the deacetylation of oligochitosan, indicator methyl orange pH color change interval exists
3.1-4.4, acid color is red, and alkali formula color is yellow, on the one hand, oligochitosan sample aqueous solution itself presents faint yellow, uses
Methyl orange is as indicator so that titration end-point cannot accurately judge；On the other hand, oligochitosan isoelectric point, IP value about 4.80,
Beyond methyl orange pH color change interval, therefore, the shell of methyl orange acid-base indicator method mensuration is used according to 2015 editions " Chinese Pharmacopoeia "
It is inconspicuous to there is the change of titration end-point color in oligosaccharide deacetylation, poor reproducibility, the shortcoming that error is bigger.Survey with reference to European Pharmacopoeia
The method determining deacetylating degree of chitosan measures oligochitosan deacetylation using the hydrochloric acid solution of water or 0.001mol/L as solvent
Time, oligochitosan is without maximum absorption wavelength, and therefore, first derivative ultraviolet spectro-photometry disclosed in European Pharmacopoeia cannot be applicable to shell
The mensuration of oligosaccharide deacetylation.
Proton nmr spectra (1H-NMR) as measuring the goldstandard of deacetylating degree of chitosan, loaded American Pharmacopeia,
Meanwhile, Kim etc. deliver paper " Oligosaccharides and Their Derivatives " and Wang Shixin etc. are delivered
Paper " quality analysis of the oligochitosan product of separate sources " discloses available hydrogen nuclear magnetic resonance spectroscopy and measures the de-of oligochitosan
Acetyl degree, measurement result error is little.But1H-NMR method needs technical professional due to its instrument cost height and operation, because of
And use cannot be widely popularized.
The most not yet set up the standard method measuring oligochitosan deacetylation.Therefore, it is necessary to set up a kind of fast
Speed, convenient and mensuration oligochitosan deacetylation accurately method.
Summary of the invention
For solving problems of the prior art, inventor has carried out substantial amounts of test and research, it has unexpectedly been found that: adopt
With first derivative ultraviolet spectro-photometry measure oligochitosan deacetylation time, according to concentration of hydrochloric acid reach 0.30mol/mL and with
On dilute hydrochloric acid have maximum wavelength as solvent, oligochitosan.Based on above-mentioned discovery, thus complete the present invention.
The purpose of the present invention will be further described in detail below reflect and description.
The present invention provides a kind of method utilizing first derivative ultraviolet spectro-photometry to measure oligochitosan deacetylation, including
Following steps:
1) hydrochloric acid solution using concentration as 0.3-1.0mol/L is as blank solvent, configure the N-acetyl of a series of concentration-
D-Glucose amine standard solution, with blank solvent as reference, the N-ACETYL-D-GLUCOSAMINE standard solution measuring variable concentrations exists
Ultraviolet light absorption angle value A in 200-206nm wave-length coverage, concentration and Δ A/ Δ λ according to N-ACETYL-D-GLUCOSAMINE draw mark
Directrix curve, wherein, Δ A=Aλ+1—Aλ-1, λ is 203-205nm, Δ λ=2nm；
2) take oligochitosan sample, after dissolving by blank solvent, with blank solvent as reference, survey at 200-206nm wavelength
Its ultraviolet light absorption angle value fixed, calculates the concentration of N-ACETYL-D-GLUCOSAMINE in oligochitosan sample according to standard curve, and according to
The deacetylation of lower formula calculating oligochitosan sample:
In formula, D.D% is deacetylation, %；C1For oligochitosan sample concentration, μ g/mL；C2For N-second in oligochitosan sample
The concentration of acyl-D-Glucose amine, μ g/mL；M1It is 203, the molecular weight of N-ACETYL-D-GLUCOSAMINE；42 is N-acetyl-D-Fructus Vitis viniferae
The difference of the molecular weight of the molecular weight of osamine and D-glucosamine amine.
Preferably, the concentration of described blank solvent is 0.3mol/L.
Preferably, step 1) in, the concentration of N-ACETYL-D-GLUCOSAMINE standard solution is respectively 1.60 μ g/mL, 20.0 μ
g/mL、32.0μg/mL、40.0μg/mL、64.0μg/mL、80.0μg/mL。
Preferably, step 1) in, λ is 204nm.
Compared with prior art, the invention has the beneficial effects as follows:
(1) method utilizing first derivative ultraviolet spectro-photometry mensuration oligochitosan deacetylation of present invention offer, with
Concentration be the hydrochloric acid solution of 0.3-1.0mol/L as solvent, use first derivative ultraviolet spectro-photometry, by measuring sample
In the content of N-ACETYL-D-GLUCOSAMINE, directly measure the deacetylation of oligochitosan, measurement result is at 0.0016-0.08mg/
In good linear (R in the range of mL2=0.9992), relative standard deviation is 0.19%-0.27%, and the response rate is 99.5%-
100.3%；Meanwhile, with1The measurement result of H-NMR method is compared, and relative error controls within 0.08%, illustrates that the present invention provides
Oligochitosan deacetylation assay method accuracy high, practical.
(2) method utilizing first derivative ultraviolet spectro-photometry mensuration oligochitosan deacetylation of present invention offer, disappears
Except concurrent D-glucosamine amine, turbidity and the interference of colourity, improve accuracy and the sensitivity of measurement result, sample
Need not special process, operation is simple.
(3) method utilizing first derivative ultraviolet spectro-photometry mensuration oligochitosan deacetylation of present invention offer, with
The relative standard deviation (RSD) of one sample measurement result controls within 0.04%, illustrates that the oligochitosan that the present invention provides takes off second
Acyl degree assay method favorable reproducibility, precision is good.
Accompanying drawing explanation
Fig. 1 with purified water as solvent, N-acetyl-D-glucose amine, D-glucosamine amine, COSMW1000And COSMW3000
Ultra-violet absorption spectrum in the range of 200nm-400nm.
Fig. 2 with the hydrochloric acid solution of variable concentrations as solvent, COSMW1000Uv absorption light in the range of 200nm-400nm
Spectrum.
Fig. 3 with the hydrochloric acid solution of variable concentrations as solvent, COSMW3000Uv absorption light in the range of 200nm-400nm
Spectrum.
Fig. 4 with 0.30mol/L hydrochloric acid solution as solvent, N-acetyl-D-glucose amine, D-glucosamine amine,
COSMW1000And COSMW3000Ultra-violet absorption spectrum in the range of 200nm-400nm.
Fig. 5 N-acetyl-D-glucose amine, D-glucosamine amine, COSMW1000And COSMW3000At 200-210nm model
Enclose interior ultraviolet first derivative spectrum.
Fig. 6 first derivative ultraviolet spectro-photometry standard curve.
Fig. 7 COSMW1000Hydrogen nuclear magnetic resonance spectrogram (500MHz), wherein, A-E represent sugar ring on C2-C6 position hydrogen signal.
Fig. 8 COSMW3000Hydrogen nuclear magnetic resonance spectrogram (500MHz), wherein, A-E represent sugar ring on C2-C6 position hydrogen signal.
Detailed description of the invention
Below by specific embodiment, the present invention is described in further detail.
Raw materials used in the embodiment of the present invention being commercially available prod, wherein, the equipment component model related to and source are as follows:
| Title | Manufacturing enterprise |
| T6 new century ultraviolet-visible spectrophotometer | Beijing Puxi General Instrument Co., Ltd |
| DK-8D type one thousandth electronic analytical balance | Sai Duolisi group of Germany |
| Bruker500MHz nuclear magnetic resonance analyser | Brooker company of Germany |
Embodiment 1 measures the selection of solvent
Inventor finds in research process, surveys according to the first derivative ultraviolet spectro-photometry in European Pharmacopoeia
When determining chitosan acetyl degree method mensuration oligochitosan, using purified water as solvent, with N-acetyl-D-glucose amine as internal standard,
To N-acetyl-D-glucose amine, D-glucosamine amine, COS in 200nm-400nm wave-length coverageMW1000(deacetylation
>=90%, the oligochitosan sample of mean molecule quantity≤1000) and COSMW3000(deacetylation >=90%, mean molecule quantity≤3000
Oligochitosan sample) be scanned, scanning result is shown in Fig. 1, and wherein curve 1 is the UV scanning curve of D-glucosamine amine,
Curve 2 is COSMW3000UV scanning curve, curve 3 is COSMW1000UV scanning curve, curve 4 is N-acetyl group-D-
The UV scanning curve of glucamine.From Fig. 1 result, according to first derivative ultraviolet spectro-photometry in European Pharmacopoeia, with
When purified water is as solvent, oligochitosan does not has maximum absorption wavelength, first derivative ultraviolet spectro-photometry disclosed in European Pharmacopoeia
The mensuration of oligochitosan deacetylation cannot be applicable to.
Inventor has carried out substantial amounts of test and research, it has unexpectedly been found that: use first derivative ultraviolet spectro-photometry to survey
When determining oligochitosan deacetylation, use concentration of hydrochloric acid to reach 0.30mol/mL and above dilute hydrochloric acid has as solvent, oligochitosan
Maximum absorption wavelength.
Using the dilute hydrochloric acid solution of variable concentrations as solvent, to COS in 200nm-400nm wave-length coverageMW1000With
COSMW3000Being scanned, scanning result is shown in Fig. 2 and Fig. 3.In Fig. 2, curve 1-5 be respectively with concentration as 0.05mol/mL,
The hydrochloric acid solution of 0.10mol/mL, 0.15mol/mL, 0.30mol/mL, 0.50mol/mL is as COS during solventMW1000Ultraviolet
Scanning curve；In Fig. 3, curve 1-5 is respectively with concentration as 0.05mol/mL, 0.10mol/mL, 0.15mol/mL, 0.30mol/
The hydrochloric acid solution of mL, 0.50mol/mL is as COS during solventMW3000UV scanning curve.
From Fig. 2 and Fig. 3 result, when concentration of hydrochloric acid reach 0.30mol/mL and above time oligochitosan have maximum absorption wave
Long.Therefore, reach 0.30mol/mL and above dilute hydrochloric acid as solvent using concentration of hydrochloric acid, available first derivative ultraviolet spectrometry
Spectrphotometric method for measuring oligochitosan deacetylation.
Embodiment 2 measures the selection of wavelength
Chinese patent application 201310587162.5 discloses and measures shell and gather by measuring the absorbance under 199nm purple light
The method of the deacetylation of sugar and oligochitosan mixture, but research process finds, N-acetyl-D-glucose amine and oligochitosan
There is end absorption at 199nm, interfere mensuration, error is very big, and therefore, the present invention selects 200nm-400nm wavelength
As object of study.
Using the hydrochloric acid solution of 0.30mol/mL as solvent, prepare the N-acetyl-D-glucose amine of 40 μ g/mL, 40 μ g/
The D-glucosamine amine of mL, 0.4mg/mL COSMW1000、0.2mg/mL COSMW3000, in 200nm-400nm wave-length coverage
Being scanned, result is shown in Fig. 4, and wherein curve 1 is the UV scanning curve of D-glucosamine amine, and curve 2 is N-acetyl group-D-
The UV scanning curve of glucamine, curve 3 is COSMW3000UV scanning curve, curve 4 is COSMW1000UV scanning
Curve.From Fig. 4 result, N-acetyl-D-glucose amine has absorption maximum at 204nm, but at 204nm wavelength,
Concurrent D-glucosamine amine also has absorption, the mensuration of interference N-acetyl-D-glucose amine, therefore, uses first derivative
Ultraviolet spectrophotometry eliminates interference.
By N-acetyl-D-glucose amine, D-glucosamine amine, COSMW1000And COSMW3000At 200nm-210nm model
Enclosing interior ultra-violet absorption spectrum and be converted into first derivative spectrum, result is shown in Fig. 5, and wherein, curve 1 is D-glucosamine amine, bent
Line 2 is COSMW3000, curve 3 is COSMW1000, curve 4 is N-acetyl-D-glucose amine.From Fig. 5 result, N-acetyl
Base-D-Glucose amine all has absorption in the range of 203nm-206nm, has an absorption maximum at 204nm, and D-glucosamine amine
Without absorbing in the range of 200nm-210nm, therefore, the deacetylation measuring oligochitosan with first derivative ultraviolet spectro-photometry can
Eliminate the interference of concurrent D-glucosamine amine.
Embodiment 3 first derivative ultraviolet spectro-photometry measures oligochitosan deacetylation
1) foundation of standard curve
Hydrochloric acid solution using concentration as 0.3mol/L, as blank solvent, configures 2.0mg/mLN-acetyl-D-Glucose amine
Standard solution, then with the dilution of the hydrochloric acid solution of 0.3mol/L, be configured to 1.60 μ g/mL, 20.0 μ g/mL, 32.0 μ g/mL,
40.0 μ g/mL, 64.0 μ g/mL, the N-ACETYL-D-GLUCOSAMINE standard solution of 80.0 μ g/mL, with the hydrochloric acid solution of 0.3mol/L
Do reference, with the quartz colorimetric utensil of 1cm, measure the N-ACETYL-D-GLUCOSAMINE standard solution of variable concentrations at 200-206nm ripple
Ultraviolet light absorption angle value A in the range of length, according to concentration and the Δ A/ Δ λ drafting standard curve of N-ACETYL-D-GLUCOSAMINE, its
In, Δ A=A205nm—A203nm, Δ λ=(205-203) nm=2nm；As shown in Figure 6, wherein, regression equation is Y to standard curve
=0.0021X-0.003, R2=0.9992,2-Acetamido-2-deoxy-D-glucose amine in the range of 0.0016-0.08mg/mL in good
Linearly.
2) mensuration of oligochitosan deacetylation
Precision weighs 1.0g COS respectivelyMW1000And COSMW3000, after dissolving with the hydrochloric acid solution of 0.3mol/L, it is settled to
250mL is as storing solution.With the hydrochloric acid solution of 0.3mol/L as reference, at 200-206nm wavelength, measure its ultraviolet absorptivity
Value, calculates the concentration of N-ACETYL-D-GLUCOSAMINE in oligochitosan sample according to standard curve, and it is few to calculate shell according to below equation
The deacetylation of sugar-like product:
In formula, D.D% is deacetylation, %；C1For oligochitosan sample concentration, μ g/mL；C2For N-second in oligochitosan sample
The concentration of acyl-D-Glucose amine, μ g/mL；M1It is 203, the molecular weight of N-ACETYL-D-GLUCOSAMINE；42 is N-acetyl-D-Fructus Vitis viniferae
The difference of the molecular weight of the molecular weight of osamine and D-glucosamine amine.
3) measurement result: COSMW1000Corresponding deacetylation is 93.45 ± 0.04%；COSMW3000Corresponding deacetylation
It is 92.88 ± 0.03%.What the present invention provided utilizes first derivative ultraviolet spectro-photometry to measure the side of oligochitosan deacetylation
Method, measures same batch oligochitosan sample deacetylation, and the relative standard deviation of its measurement result, within 0.04%, illustrates this
The method precision that invention provides is good.
Embodiment 4 response rate is investigated
Owing to oligochitosan does not has standard substance, therefore, by the basic composition chain link N-second of two kinds in chitin polymer molecule
Acyl group-D-Glucose amine represents the oligochitosan of different deacetylations with the mixing of D-glucosamine amine different proportion.Precision respectively
Measure 2.0mg/mLN-acetyl-D-glucose amine standard solution 0.50mL, 0.75mL, 1.00mL in 100mL volumetric flask, point
In above-mentioned volumetric flask, do not add D-glucosamine amine 0.0063g, 0.0063g, 0.0051g be configured to be equivalent to deacetylation
It is the oligochitosan solution of 92.2%, 88.7% and 82.7%.By step 2 in embodiment 3) described method mensuration oligochitosan solution
Deacetylation also calculates the response rate, the results are shown in Table 1.
Table 1 first derivative ultraviolet spectro-photometry measures oligochitosan deacetylation response rate experimental result
As seen from the results in Table 1, it is deacetylated that what the present invention provided utilizes first derivative ultraviolet spectro-photometry to measure oligochitosan
The response rate of the method for degree is good, and oligochitosan deacetylation measurement result accuracy is high.
Comparative example 11H-NMR method measures deacetylation
1)1H-NMR method measures deacetylation
Precision weighs 20mg COS respectivelyMW1000And COSMW3000It is dissolved in the D of 5mL2In O (99.96%), obtaining concentration is
The sample solution of 4mg/mL, is transferred to gained sample solution in 8mm nuclear magnetic tube be measured, and resonant frequency is 500MHz, surveys
Fixed temperature is 297k, finally to echo signal integration, and the wherein hydrogen signal of acetyl group in the acetamido of C2 position on sugar ring
(Acetyl-H) at 1.9-2.1ppm, on sugar ring, C2-C6 position hydrogen signal is at 2.6-6.0ppm.COSMW1000, COSMW3000Nuclear-magnetism
Resonance hydrogen spectrogram is shown in Fig. 7, Fig. 8 respectively.According to table 2, being integrated hydrogen nuclear magnetic resonance spectrogram correspondence peak, deacetylation calculates public affairs
Formula is:
D.D (%)={ 1-[(7*A2)/(3*A1)]}*100
Wherein, A2Represent on sugar ring the integrated value of 3 hydrogen signals of acetyl group in the acetylamino of C2 position；A1Represent on sugar ring
The integrated value of C2-C6 position hydrogen signal.
Oligochitosan deuterium-oxide solution Hydrogen Proton chemical shift at 2 25 DEG C of table
Utilize1H-NMR method records COSMW1000, COSMW3000Corresponding deacetylation measurement result is respectively 93.52 ±
0.13,92.81 ± 0.07.
2)1H-NMR method and the contrast of acid-base indicator method measurement result of the present invention
1H-NMR method is shown in Table 3 with the comparing result of acid-base indicator method measurement result of the present invention.
Table 31H-NMR contrasts (n=6) with acid-base indicator method measurement result of the present invention
As can be seen from Table 3, first derivative ultraviolet spectro-photometry of the present invention measures the survey of oligochitosan deacetylation
Determine result with1The measurement result of H-NMR method is consistent, and relative error controls within 0.08%, and the single order that the present invention provides is described
It is high that derivative ultraviolet spectrophotometry is used for measuring oligochitosan deacetylation accuracy.
Above content is to combine concrete preferred implementation further description made for the present invention, it is impossible to assert
Being embodied as of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of present inventive concept, it is also possible to make some simple deduction or replace, all should be considered as belonging to the present invention's
Protection domain.
Claims (4)
1. one kind utilizes the method that first derivative ultraviolet spectro-photometry measures oligochitosan deacetylation, it is characterised in that: include
Following steps:
1) hydrochloric acid solution using concentration as 0.3-1.0mol/L is as blank solvent, configures the N-acetyl-D-Portugal of a series of concentration
Grapes glucosamine standard solution, with blank solvent as reference, measures the N-ACETYL-D-GLUCOSAMINE standard solution of variable concentrations at 200-
Ultraviolet light absorption angle value A in 206nm wave-length coverage, concentration and Δ A/ Δ λ according to N-ACETYL-D-GLUCOSAMINE draw standard song
Line, wherein, Δ A=Aλ+1—Aλ-1, λ is 203-205nm, Δ λ=2nm；
2) take oligochitosan sample, after dissolving by blank solvent, with blank solvent as reference, at 200-206nm wavelength, measure it
Ultraviolet light absorption angle value, calculates the concentration of N-ACETYL-D-GLUCOSAMINE in oligochitosan sample according to standard curve, and according to following public affairs
The deacetylation of formula calculating oligochitosan sample:
In formula, D.D% is deacetylation, %；C1For oligochitosan sample concentration, μ g/mL；C2For N-acetyl-D-in oligochitosan sample
The concentration of glucamine, μ g/mL；M1It is 203, the molecular weight of N-ACETYL-D-GLUCOSAMINE；42 is N-ACETYL-D-GLUCOSAMINE
The difference of molecular weight of molecular weight and D-glucosamine amine.
The method utilizing first derivative ultraviolet spectro-photometry to measure oligochitosan deacetylation the most according to claim 1,
It is characterized in that: the concentration of described blank solvent is 0.3mol/L.
The method utilizing first derivative ultraviolet spectro-photometry to measure oligochitosan deacetylation the most according to claim 1,
It is characterized in that: step 1) in, the concentration of N-ACETYL-D-GLUCOSAMINE standard solution be respectively 1.60 μ g/mL, 20.0 μ g/mL,
32.0μg/mL、40.0μg/mL、64.0μg/mL、80.0μg/mL。
The method utilizing first derivative ultraviolet spectro-photometry to measure oligochitosan deacetylation the most according to claim 1,
It is characterized in that: step 1) in, λ is 204nm.
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| PCT/CN2017/095114 WO2018040821A1 (en) | 2016-08-30 | 2017-07-31 | Method for measuring degree of deacetylation of chitosan oligosaccharide by using first-order derivative ultraviolet spectrophotometry |
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| WO2018040821A1 (en) * | 2016-08-30 | 2018-03-08 | 广东药科大学 | Method for measuring degree of deacetylation of chitosan oligosaccharide by using first-order derivative ultraviolet spectrophotometry |
| CN108535203A (en) * | 2018-04-03 | 2018-09-14 | 金川集团股份有限公司 | The derivative spectrophotometric detecting method of saccharin sodium in a kind of pure Ni solution |
| CN116698776A (en) * | 2023-06-28 | 2023-09-05 | 湖北工程学院 | Color development-free spectrophotometry for quantitatively analyzing chitosan |
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| CN116879210A (en) * | 2023-08-11 | 2023-10-13 | 青岛科技大学 | Method for determining deacetylation degree of chitosan oligosaccharide by using UV negative first derivative method |
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