CN108254328A - A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis - Google Patents
A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis Download PDFInfo
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- CN108254328A CN108254328A CN201711500093.4A CN201711500093A CN108254328A CN 108254328 A CN108254328 A CN 108254328A CN 201711500093 A CN201711500093 A CN 201711500093A CN 108254328 A CN108254328 A CN 108254328A
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 59
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 47
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 36
- 239000002253 acid Substances 0.000 title claims abstract description 32
- 238000002798 spectrophotometry method Methods 0.000 title claims abstract description 22
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims abstract description 41
- 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 abstract description 39
- 229960002442 glucosamine Drugs 0.000 claims abstract description 39
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 24
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 50
- 239000000523 sample Substances 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 27
- 238000002835 absorbance Methods 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000012224 working solution Substances 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- JCZPMGDSEAFWDY-SQOUGZDYSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanamide Chemical compound NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO JCZPMGDSEAFWDY-SQOUGZDYSA-N 0.000 claims description 8
- 230000031700 light absorption Effects 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- CHVZQMAANSUXJU-JJKGCWMISA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanamide;hydrochloride Chemical compound Cl.NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO CHVZQMAANSUXJU-JJKGCWMISA-N 0.000 claims description 6
- 150000007522 mineralic acids Chemical class 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 238000012417 linear regression Methods 0.000 claims description 5
- 238000005086 pumping Methods 0.000 claims description 5
- FZRCKLPSHGTOAU-UHFFFAOYSA-N 6-amino-1,4-dimethylcyclohexa-2,4-diene-1-carbaldehyde Chemical compound CC1=CC(N)C(C)(C=O)C=C1 FZRCKLPSHGTOAU-UHFFFAOYSA-N 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000006467 substitution reaction Methods 0.000 claims description 3
- 230000006196 deacetylation Effects 0.000 claims description 2
- 238000003381 deacetylation reaction Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 4
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 9
- 238000005903 acid hydrolysis reaction Methods 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 229920002101 Chitin Polymers 0.000 description 4
- 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 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- VQLYBLABXAHUDN-UHFFFAOYSA-N bis(4-fluorophenyl)-methyl-(1,2,4-triazol-1-ylmethyl)silane;methyl n-(1h-benzimidazol-2-yl)carbamate Chemical compound C1=CC=C2NC(NC(=O)OC)=NC2=C1.C=1C=C(F)C=CC=1[Si](C=1C=CC(F)=CC=1)(C)CN1C=NC=N1 VQLYBLABXAHUDN-UHFFFAOYSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- QKPLRMLTKYXDST-NSEZLWDYSA-N (3r,4r,5s,6r)-3-amino-6-(hydroxymethyl)oxane-2,4,5-triol;hydrochloride Chemical compound Cl.N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O QKPLRMLTKYXDST-NSEZLWDYSA-N 0.000 description 1
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 150000004265 D-glucosamines Chemical class 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- -1 aminoglucose saccharide Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003402 anti-promotion Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000002473 artificial blood Substances 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 150000002302 glucosamines Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 208000018875 hypoxemia Diseases 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- FZUJWWOKDIGOKH-UHFFFAOYSA-N sulfuric acid hydrochloride Chemical compound Cl.OS(O)(=O)=O FZUJWWOKDIGOKH-UHFFFAOYSA-N 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
- 230000029663 wound healing Effects 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
Landscapes
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Saccharide Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention relates to a kind of methods using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis, belong to macromolecular technology for hydrolyzing field.The present invention is using the yield of GAH in chitosan hydrolyzate liquid as index; chitosan hydrolyzate condition is optimized in the environment of nitrogen protection; hydrolysising condition will be screened using acetylacetone method; it is preferred that optimum hydrolysising condition; and chitosan hydrolyzate generation product Glucosamine is verified, with the content of determined by ultraviolet spectrophotometry Glucosamine.The present invention has the characteristics that at low cost, simple for process, yield is high, has the prospect of industrialization.
Description
Technical field
The present invention relates to a kind of methods using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis, belong to macromolecular water
Solve technical field.
Background technology
Chitin (chitin) is the cell wall constituent of the mushrooms such as ectoskeleton and the mushroom of the shell-fish such as shrimp, crab, beetle etc.,
It is widely present in nature.Chitosan (chitosan) is the product of chitin deacetylate, is a kind of few in number, day
Right, commercialization alkaline polysaccharide, stock number are only second to cellulose.Since chitosan has good film forming, biology can
Degradability and certain antibacterial anti-inflammatory and promotion wound healing effect, it is all in weaving, printing and dyeing, papermaking, food, medicine, environmental protection etc.
It is multi-field to be used widely.In terms of medical treatment, surgical cable, artificial blood vessel and artificial skin can be made, styptic sponge and
Wound specialized dressing；In terms of pharmaceutical preparation, slow releasing pharmaceutical and anticancer drug etc. can be done.In addition chitosan, which also has, promotes human body
The growth of enteral Bifidobacterium, the accumulation for preventing body's cholesterol promote the functions such as humoral immunity and cellular immunity.
D-Glucosamine Hydrochloride be also known as glucosamine HCL (D-Glucosamine Hydrochloride,
GAH), it is the final product of chitosan hydrolyzate.GAH has important physiological significance in vivo：The removing toxic substances of liver kidney is participated in, is played anti-
The effect of inflammation, protect liver, anti-reactive, anti-hypoxemia；Stimulate hyperplasia of Bifidobacterium etc. in baby intestinal.The use of D- Glucosamines
It is very extensive on the way, there are huge scientific value, social benefit and economic interests, domestic and international demand is increasingly being.At present,
Most of GAH is prepared using chitin or chitosan as raw material, but due in open type hydrolytic process, between chitosan molecule and dividing
It can be destroyed by the oxygen in air after inside son hydrogen bond fracture and influence chromogenic reaction, measured value is relatively low, the rate of recovery 30%~
40%.Again due to being hydrolyzed in acid hydrolysis using single acid.The concentration of acid is not high, generally needs to hydrolyze ability for a long time
Higher hydrolysis yield is obtained, hydrolysis efficiency is low.GAH content assaying methods have very much, the common measure in chitosan measure
The method of reduced sugar mainly has high performance liquid chromatography, phend-sulphuric acid, Elson-Morgan methods, DNS methods etc..Wherein,
Elson-Morgan methods are the methods of classical measure Glucosamine, free Glucosamine under alkaline condition with acetyl
Condensation of acetone adds lustre to former 2- methyl -3- diacetyl azole derivatives, and original of adding lustre to is again with p- dimethylaminobenzaldehyde in dense salt
It develops the color in sour ethanol solution, there is good specificity to Glucosamine.
Invention content
The purpose of the present invention is be directed to the hydrolysate yield of existing chitosan is low, hydrolysis efficiency is low, it is ropy not
Foot part, the method that a kind of lower sour hydrolyzing chitosan of nitrogen protection efficient, environmentally protective, that yield is high is provided.The present invention is with shell
The yield of GAH is index in glycan hydrolyzate, and chitosan hydrolyzate condition is optimized in the environment of nitrogen protection, will be used
Acetylacetone method screens hydrolysising condition, preferably optimum hydrolysising condition.
A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis includes the following steps：
1) 300mg chitosans are weighed and add in flask, 40ml inorganic acid solutions is measured and adds in flask, sealing device uses vacuum
Pumping uses up air, then is passed through nitrogen, is repeated 3 times.Start constant temperature blender with magnetic force, 100 DEG C of controlling reaction temperature, rotating speed 200r/
Min, reaction time are 12h~48h, and take out appropriate hydrolyzate in different time respectively with sampling probe is sealed respectively, as
It is spare to place refrigerator for sample；
2) content of determined by ultraviolet spectrophotometry Glucosamine：
A) standard curve of the Glucosamine titer of A and various concentration is drawn：1.5mL a certain concentrations ladder is drawn respectively
The Glucosamine titer of degree is settled to 2mL in 10mL colorimetric cylinders with water, is separately added into acetylacetone,2,4-pentanedione solution 1.5mL, shakes
It is even, 80 DEG C of water-bath 30min.It removes and is cooled to room temperature, add paradime thylaminobenzaldehyde solution 5.0mL respectively, add absolute ethyl alcohol extremely
10mL, 60 DEG C of water-bath 60min, is removed and is cooled to room temperature, compared with blank tube, wavelength is measured on ultraviolet specrophotometer
Light absorption value during 534nm.Three groups of parallel tests are done respectively, survey each pipe absorbance.To measure absorbance A as ordinate, aminoglucose
Sugared content is abscissa, draws standard curve；
B) preparation of sample working solution：Hydrolyzate sample 1.0mL in step 1) is taken in 50mL volumetric flasks, to be settled to water
It is fully shaken up to get sample working solution after 50mL；
C) content of Glucosamine determines in sample：Sample working solution 1.5mL in step b) is taken, by step a) detection sides
Method is measured, its absorbance value A is measured at 534nm, and A substitution equations of linear regression are acquired Glucosamine in sample
Content；
3) calculation formula of aminoglucose sugar yield is in chitosan hydrolyzate liquid：
In formula：m1It is the content that aminoglucose hydrochloride is calculated by standard curve, unit is μ g/mL；
m2It is the content of chitosan sample, unit is μ g/mL；
0.8309 is coefficient of the aminoglucose hydrochloride conversion for Glucosamine.
A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis as described above, the chitosan
Deacetylation is more than 80%；The molecular weight ranges of chitosan are 20,000~300,000.
A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis as described above, the inorganic acid are
Hydrochloric acid, one or more of phosphoric acid and sulfuric acid.
A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis as described above, the inorganic acid are
The hydrochloric acid of a concentration of 6mol/L.
A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis as described above, amino in step a)
The content range of glucose is the μ g/mL of 3 μ g/mL~24.
Test example：
1. the verification of hydrolysis generation Glucosamine
It weighs 300mg chitosans and adds in flask, measure 40.00ml 6mol/L HCl solutions and add in flask, sealing device,
Air is use up, then be passed through nitrogen with vacuum pumping, be repeated 3 times.Start constant temperature blender with magnetic force, arrange parameter (t=100 DEG C, n=
200r/min), start to hydrolyze.Hydrolyzate was extracted at the 12nd, 24,36,48 hour and Glucosamine standard solution exists respectively
Point sample is carried out on thin layer silica gel plate, point sample is placed on the chromatography cylinder equipped with chromatographic solution, ascending method expansion.After the completion of expansion, electricity consumption
Blowing drying silica gel plate, is coloured, then silica gel plate is made to develop the color with hair dryer hot wind with 0.1% ninhydrin solution.As a result such as Fig. 1
It is shown, from left to right hydrolysis time be followed successively by 12,24,36,48h, hydrolyzate develops the color gradually apparent (left on silica gel plate：Amino
Dextrose standard sample is right：Chitosan hydrolyzate liquid), show the content of Glucosamine in hydrolyzate with the increase of hydrolysis time and
Increase.And the apparent elongation that develops the color after 36 hours, it is because the Glucosamine of chitosan hydrolyzate generation starts gradually to be degraded brokenly
It is bad, therefore the hydrolyzate of subsequent experimental can take 24-36h to be detected.
2. the drafting of standard curve
120 μ g/mL Glucosamines titers 0,0.25,0.5,0.75,1.00,1.25,1.50,2.00mL are drawn respectively
2mL is added to water in 10mL colorimetric cylinders, acetylacetone,2,4-pentanedione solution 1.5mL is separately added into, shakes up, 80 DEG C of water-bath 30min.It removes cold
But to room temperature, respectively plus paradime thylaminobenzaldehyde solution 5.0mL, absolute ethyl alcohol is added to remove to 10mL, 60 DEG C of water-bath 60min
It is cooled to room temperature, is compared with blank tube, the light absorption value when measuring wavelength 534nm on ultraviolet specrophotometer.Three groups are done respectively
Each pipe absorbance is surveyed in parallel test.To measure absorbance (A) as ordinate, GAH contents (μ g/mL) are abscissa (standard curve
Concentration is followed successively by 3,9,15,18,24 μ g/mL to middle taken point from left to right), draw standard curve.Obtain regression equation：Y=
0.0113x-0.0012, R2=0.9943, show to measure GAH contents between 3~24 μ g/mL, the extinction when wavelength is 534nm
Value is good with the amount linear relationship of Glucosamine.
3. the comparison of yield is hydrolyzed under differential responses time conditions
300mg chitosans are weighed in flask, measure 40mL6mol/LHCl solution in flask, it is closed, it vacuumizes, fills out
Inflated with nitrogen is repeated 3 times.T=100 DEG C of constant temperature blender with magnetic force arrange parameter continues constant temperature hydrolysis.Take the appropriate 24th respectively, 30,
36th, 42,48 hours hydrolyzates, it is spare.
The the 24th, 30,36,42,48 hour hydrolyzate 1.0mL is taken to be settled to 50mL with water, respectively in 50mL volumetric flasks respectively
1.5mL dilutions are drawn in 10mL colorimetric cylinders, same standard solution is handled, is compared with blank tube, on ultraviolet specrophotometer
Light absorption value when measuring wavelength 534nm.Three groups of parallel tests are done respectively.
The 1 differential responses time of table hydrolyzes the comparison result (n=3) of yield
The result shows that as hydrolysis time is longer, the yield of GAH is slowly increased, and hydrolysis time reaches 48 hours, GAH productions
Rate is still rising, and whether 48 hours be that optimum reacting time needs further to be verified.
4. the comparison of yield is hydrolyzed under the conditions of hydrochloric acid and mixed acid (middle strong acid) each ratio
It weighs 300mg chitosans and measures 40.00ml 6mol/L HCl solutions and add in flask, sealing device takes out sky to the greatest extent
Gas, then nitrogen is passed through, it is repeated 3 times.Constant temperature blender with magnetic force parameter (t=100 DEG C, n=200r/min) is set, continues thermostatted water
Solution.Hydrolyzate was extracted at the 24th, 30,36 hour respectively, it is spare.
300mg chitosans are weighed in flask, measure 20mL 9.0mol/L HCl solutions and 20mL3.0mol/L respectively
H3PO4(3: 1), 20mL 8.0mol/L HCl solutions and 20mL 4.0mol/L H3PO4(2: 1), 20mL 6.0mol/L HCl are molten
Liquid and 20mL 6.0mol/L H3PO4(1: 1) solution is in flask, constant temperature blender with magnetic force arrange parameter (t=100 DEG C, n=
200r/min).Continue constant temperature hydrolysis.Hydrolyzate was extracted at the 24th, 30,36 hour respectively, it is spare.
The the 24th, 30,36 hour 6mol/L HCl, mixed-acid hydrolysis chitosan hydrolyzate 1.0mL are taken respectively in 50mL capacity
Bottle, remaining handles same standard solution.It is compared with blank tube, the light absorption value when measuring wavelength 534nm on ultraviolet specrophotometer.
Three groups of parallel tests are done respectively.
The comparison result (n=3) of 2 hydrochloric acid of table and mixed-acid hydrolysis yield
The result shows that hydrolysis same time, when phosphoric acid ratio is smaller, hydrolysis effect is not so good as the rate of recovery of independent HCl/water solution
Height, and HCl-H3PO4Ratio be 1: 1 when hydrolysis effect become worse, it may be possible to because mixing acidacidity it is inadequate, chitosan this
When hydrolysis it is incomplete, therefore the yield of the GAH of HCl/water solution is used all to be higher than and uses HCl-H3PO4The yield of hydrolysis, the hydrolysis of hydrochloric acid
Effect is preferable.
5. the comparison of yield is hydrolyzed under the conditions of hydrochloric acid and mixed acid (strong acid)
300mg chitosans are weighed in flask, measure 20mL 9.6mol/L HCl solutions and 20mL2.4mol/L respectively
H3SO4(4: 1), 20mL 9.0mol/L HCl solutions and 20mL 3.0mol/L H2SO4(3: 1), 20mL 8.0mol/L HCl are molten
Liquid and 20mL 4.0mol/L H2SO4(2: 1), 20mL 6.0mol/L HCl solutions and 20mL 6.0mol/L H2SO4(1: 1) is molten
Liquid is in flask, constant temperature blender with magnetic force arrange parameter (t=100 DEG C, n=200r/min).Continue constant temperature hydrolysis.Respectively
24th, hydrolyzate is extracted within 30,36 hours, it is spare.
The the 24th, 30,36 hour 6mol/L HCl, mixed-acid hydrolysis chitosan hydrolyzate 1.0mL are taken respectively in 50mL capacity
Bottle, the remaining same standard solution of processing.It is compared with blank tube, the light absorption value when measuring wavelength 534nm on ultraviolet specrophotometer.Point
Three groups of parallel tests are not done.
The comparison result (n=3) of 3 hydrochloric acid of table and mixed-acid hydrolysis yield
The result shows that hydrolysis same time, (such as HCl-H when strong acid ratio is smaller2SO4(4: 1)), the rate of recovery approaches
The hydrolysis rate of recovery of hydrochloric acid is used alone, but hydrolyzes yield to be still hydrochloric acid higher, and (such as HCl- when strong acid large percentage
H25O4(1: 1)), hydrolysis effect is significantly not as good as hydrochloric acid, it may be possible to because sulfuric acid concentration is excessively high, and make most of chitosan by carbon
Change destruction or the destruction because the Glucosamine of most of hydrolysis production is degraded under strong acid.As it can be seen that use HCl/water solution
GAH yield generally all be higher than HCl-H2SO4The yield of hydrolysis, the hydrolysis effect of hydrochloric acid are preferable.
6. hydrochloric acid and mixed acid (strong acid) hydrolyze the comparison of yield for 24 hours
By the above results as it can be seen that in the short time i.e. for 24 hours under hydrolysising condition, hydrochloric acid is 3: 1,4: 1 with strong acid mixed proportion
When hydrolysis yield it is higher.
4 hydrochloric acid of table and mixed acid hydrolyze the comparison result (n=3) of yield for 24 hours
The result shows that when hydrochloric acid-strong acid ratio 3: 1, the rate of recovery of short time is higher than returning when hydrochloric acid is used alone
Yield hydrolyzes 24 hours, and hydrolysis yield is up to 72.8%.Thus, from hydrolysis time is shortened, reduce and examined in hydrolysis cost angle
Consider, adjust the proportioning of mixed acid, when hydrochloric acid-sulfuric acid ratio 3: 1, hydrolysis larger can improve hydrolysis yield in 24 hours.
The present invention has following technical advantage compared with prior art：
1) this experiment is use up air, and be passed through nitrogen with vacuum pumping, is repeated 3 times, in nitrogen in hydrolysis stage, sealing device
Chitosan is hydrolyzed under gas shielded, chitosan hydrolyzate chain breaking process is avoided to react with oxygen, to Glucosamine
Molecular structure is preferably protected.
2) experiment discussion chitosan hydrolyzate method, respectively it has been investigated the selecting of acid, the concentration of acid, the reaction time,
Influence of the reaction temperature to hydrolysis effect measures aminoglucose sugar yield using ultraviolet spectrophotometry and evaluates different hydrolysising conditions
Effect, finally show that chitosan hydrolyzes optimised process under nitrogen protection.
3) the content good linearity of determined by ultraviolet spectrophotometry Glucosamine, specificity are good：Under optimum experimental condition,
According to the corresponding A of the Glucosamine of determination of experimental method various concentration, standard curve is drawn, the results showed that, Glucosamine
There are good linear relationships with A in the range of the μ g/mL of 3 μ g/mL of concentration~24；And ultraviolet spectrophotometry (acetylacetone,2,4-pentanedione
Method) Glucosamine is measured to free Glucosamine with preferable specificity.
Description of the drawings
Fig. 1 chitosan hydrolyzates liquid and aminoglucose saccharide comparison TLC figures.
The standard curve of the Glucosamine titer of Fig. 2 absorbances and various concentration.
Specific embodiment
The present invention is further described, but the invention does not limit the present invention in any way below by way of specific embodiment
The range of patent protection.
Embodiment 1
A kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis includes the following steps：
1) 300mg chitosans are weighed and add in flask, the hydrochloric acid solution for measuring 40ml 6mol/L adds in flask, sealing device,
Air is use up, then be passed through nitrogen with vacuum pumping, be repeated 3 times.Start constant temperature blender with magnetic force, 100 DEG C of controlling reaction temperature, rotating speed
200r/min, reaction time 48h hydrolyze liquid-tight envelope with sampling probe taking-up and preserve, as sample, it is spare to place refrigerator in right amount；
2) content of determined by ultraviolet spectrophotometry Glucosamine：
A) standard curve of the Glucosamine titer of A and various concentration is drawn：1.5mL a certain concentrations ladder is drawn respectively
The Glucosamine titer of degree is settled to 2mL in 10mL colorimetric cylinders with water, is separately added into acetylacetone,2,4-pentanedione solution 1.5mL, shakes
It is even, 80 DEG C of water-bath 30min.It removes and is cooled to room temperature, add paradime thylaminobenzaldehyde solution 5.0mL respectively, add absolute ethyl alcohol extremely
10mL, 60 DEG C of water-bath 60min, is removed and is cooled to room temperature, compared with blank tube, wavelength is measured on ultraviolet specrophotometer
Light absorption value during 534nm.Three groups of parallel tests are done respectively, survey each pipe absorbance.To measure absorbance A as ordinate, aminoglucose
Sugared content is abscissa, draws standard curve；
With titer, empirically method draws sample curves, and result is A=for the equation of linear regression of standard items
0.0113c-0.0012 shows to measure GAH contents between 3~24 μ g/mL, light absorption value and amino Portugal when wavelength is 534nm
The amount linear relationship of grape sugar is good；
B) preparation of sample working solution：The hydrolyzate sample 1.0mL of reaction 48 hours in step 1) is taken in 50mL volumetric flasks,
It is fully shaken up to get sample working solution after being settled to 50mL with water；
C) content of Glucosamine determines in sample：Sample working solution 1.5mL in step b) is taken, by step a) detection sides
Method is measured, its absorbance value A is measured at 534nm, and A substitution equations of linear regression are acquired Glucosamine in sample
Content；
Sample working solution 1mL is taken, empirically method is measured, its absorbance value A is measured at 534nm, A is substituted into line
Property regression equation acquires the content of Glucosamine in sample, the results are shown in Table 5；
3) calculation formula of aminoglucose sugar yield is in chitosan hydrolyzate liquid：
By m1、m2The calculation formula for substituting into aminoglucose sugar yield in chitosan hydrolyzate liquid acquires amino in sample
The yield of glucose, the results are shown in Table 5；
In formula：m1It is the content that aminoglucose hydrochloride is calculated by standard curve, unit is μ g/mL；m2It is chitosan
The content of sample, unit are μ g/mL；0.8309 is coefficient of the aminoglucose hydrochloride conversion for Glucosamine.
Table 5 reacts the comparison result (n=3) of 48 hours hydrolysis yields
The range of linearity and detection limit
Under optimum experimental condition, according to the corresponding A of the Glucosamine of determination of experimental method various concentration, standard is drawn
Curve, the results showed that, there are good linear relationship, equations of linear regression with A in the range of 3 μ g/mL-24 μ g/mL for GAH contents
For A=0.0113c-0.0012, related coefficient 0.9943, the method range of linearity is wide, 0.9206 μ g/mL of detection limit.
Claims (5)
1. a kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis includes the following steps：
1) 300mg chitosans are weighed and add in flask, 40ml inorganic acid solutions is measured and adds in flask, sealing device, with vacuum pumping
Air to the greatest extent, then nitrogen is passed through, it is repeated 3 times；Startup constant temperature blender with magnetic force, 100 DEG C of controlling reaction temperature, rotating speed 200r/min,
Reaction time is for 24 hours~48h, and take out appropriate hydrolyzate in different time respectively with sampling probe is sealed respectively, as sample,
It is spare to place refrigerator；
2) content of determined by ultraviolet spectrophotometry Glucosamine：
A) standard curve of the Glucosamine titer of A and various concentration is drawn：1.5mL a certain concentration gradients are drawn respectively
Glucosamine titer is settled to 2mL in 10mL colorimetric cylinders with water, is separately added into acetylacetone,2,4-pentanedione solution 1.5mL, shakes up, 80
DEG C water-bath 30min；It removes and is cooled to room temperature, respectively plus paradime thylaminobenzaldehyde solution 5.0mL, add absolute ethyl alcohol to 10mL,
60 DEG C of water-bath 60min, are removed and are cooled to room temperature, compared with blank tube, when measuring wavelength 534nm on ultraviolet specrophotometer
Light absorption value；Three groups of parallel tests are done respectively, survey each pipe absorbance；To measure absorbance A as ordinate, aminoglucose sugared content is
Abscissa draws standard curve；
B) preparation of sample working solution：Hydrolyzate sample 1.0mL is taken in step 1) in 50mL volumetric flasks, after being settled to 50mL with water
It fully shakes up to get sample working solution；
C) content of Glucosamine determines in sample：Sample working solution 1mL in step b) is taken, is carried out by step a) detection methods
It measures, its absorbance value A is measured at 534nm, A substitution equations of linear regression are acquired to the content of Glucosamine in sample；
3) calculation formula of aminoglucose sugar yield is in chitosan hydrolyzate liquid：
In formula：m1It is the content that aminoglucose hydrochloride is calculated by standard curve, unit is μ g/mL；m2It is chitosan sample
Content, unit is μ g/mL；0.8309 is coefficient of the aminoglucose hydrochloride conversion for Glucosamine.
2. a kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis according to claim 1, special
Sign is that the deacetylation of the chitosan is more than 80%；The molecular weight ranges of chitosan are 20,000~300,000.
3. a kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis according to claim 1, special
Sign is, the inorganic acid is hydrochloric acid, one or more of phosphoric acid and sulfuric acid.
4. a kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis according to claim 1, special
Sign is that the inorganic acid is the hydrochloric acid of a concentration of 6mol/L.
5. a kind of method using determined by ultraviolet spectrophotometry chitosan acid percent hydrolysis according to claim 1, special
Sign is, the content range of Glucosamine is the μ g/mL of 3 μ g/mL~24 in step a).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112557552A (en) * | 2020-12-29 | 2021-03-26 | 中国科学院烟台海岸带研究所 | Method for measuring chitosan content |
| CN116698776A (en) * | 2023-06-28 | 2023-09-05 | 湖北工程学院 | Color development-free spectrophotometry for quantitatively analyzing chitosan |
| 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 |
-
2017
- 2017-12-30 CN CN201711500093.4A patent/CN108254328A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112557552A (en) * | 2020-12-29 | 2021-03-26 | 中国科学院烟台海岸带研究所 | Method for measuring chitosan content |
| CN116698776A (en) * | 2023-06-28 | 2023-09-05 | 湖北工程学院 | Color development-free spectrophotometry for quantitatively analyzing chitosan |
| 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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