CN108051386B - Method for accurately measuring chitosan content by ultraviolet spectrophotometry - Google Patents
Method for accurately measuring chitosan content by ultraviolet spectrophotometry Download PDFInfo
- Publication number
- CN108051386B CN108051386B CN201711500040.2A CN201711500040A CN108051386B CN 108051386 B CN108051386 B CN 108051386B CN 201711500040 A CN201711500040 A CN 201711500040A CN 108051386 B CN108051386 B CN 108051386B
- Authority
- CN
- China
- Prior art keywords
- chitosan
- solution
- molecular
- concentration
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 120
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000002798 spectrophotometry method Methods 0.000 title claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 42
- 238000002835 absorbance Methods 0.000 claims abstract description 31
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 22
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 22
- QBZIEGUIYWGBMY-FUZXWUMZSA-N (5Z)-5-hydroxyimino-6-oxonaphthalene-2-sulfonic acid iron Chemical compound [Fe].O\N=C1/C(=O)C=Cc2cc(ccc12)S(O)(=O)=O.O\N=C1/C(=O)C=Cc2cc(ccc12)S(O)(=O)=O.O\N=C1/C(=O)C=Cc2cc(ccc12)S(O)(=O)=O QBZIEGUIYWGBMY-FUZXWUMZSA-N 0.000 claims abstract description 21
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 21
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims abstract description 14
- 239000007974 sodium acetate buffer Substances 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 239000012224 working solution Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000002775 capsule Substances 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 8
- 239000004094 surface-active agent Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- 239000011550 stock solution Substances 0.000 claims description 7
- 238000012417 linear regression Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000011084 recovery Methods 0.000 claims description 5
- 239000012086 standard solution Substances 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 4
- 229960000583 acetic acid Drugs 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 15
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 239000000523 sample Substances 0.000 description 33
- 239000007853 buffer solution Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 229920002101 Chitin Polymers 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 230000000171 quenching effect Effects 0.000 description 4
- 241000242587 Aurelia Species 0.000 description 3
- 238000005903 acid hydrolysis reaction Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 229910020820 NaAc-HAc Inorganic materials 0.000 description 1
- 235000010678 Paulownia tomentosa Nutrition 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 244000153888 Tung Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 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 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 108010025899 gelatin film Proteins 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000155 polyglutamine Polymers 0.000 description 1
- 108010040003 polyglutamine Proteins 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- HFQQZARZPUDIFP-UHFFFAOYSA-M sodium;2-dodecylbenzenesulfonate Chemical compound [Na+].CCCCCCCCCCCCC1=CC=CC=C1S([O-])(=O)=O HFQQZARZPUDIFP-UHFFFAOYSA-M 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
Images
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
Abstract
The invention relates to a method for accurately measuring chitosan content by an ultraviolet spectrophotometry, belonging to the field of macromolecular detection. The invention utilizes the reaction of chitosan in acetic acid-sodium acetate buffer solution with naphthol green B and Tween 20 solution to generate association, and the absorbance value of the association increases along with the increase of the chitosan content in a certain concentration range and presents a certain linear relationship. The method is influenced by the molecular weight of the chitosan, and has the advantages of cheap reagent, high sensitivity, good reproducibility, simple and convenient operation and the like.
Description
Technical Field
The invention relates to a method for accurately measuring chitosan content by an ultraviolet spectrophotometry, belonging to the field of macromolecule detection.
Background
Chitosan (chitosan) is a degradation product of chitin. The chemical name of the chitosan is polyglutamine (1, 4) -2-amino-2-deoxy-D-glucan, which is a main component of exoskeletons of crustaceans (shrimps and crabs) and insects, and chitosan is widely distributed in nature and belongs to a high-molecular green material. The understanding of chitosan in human beings has been from the beginning of the 19 th century, and the research on chitin/chitosan has become more active until the 60 th century. Internationally, since 1894, 6 conferences on chitin/chitosan have been held; asia also held the first chitin academic conference in 1994, now 2 times. However, only in recent years, China pays attention to the development and application of chitin/chitosan. The first chitin chemistry conference was held in the world in 1996 at 10 months. The chitosan and the derivatives thereof have wide application prospects in the industries of food, medicine, environmental protection, chemical industry and the like. The chitosan has good biocompatibility and bioactivity, is non-toxic, harmless and free of immunological antigenicity, and has five functions of strengthening immunity, inhibiting aging, preventing diseases, promoting disease recovery and regulating physiological functions. The chitosan products on the market are different in size and formula, and the content of chitosan directly influences the function of the chitosan. Therefore, the establishment of an accurate quantitative analysis method is very important to guarantee the stability and controllability of the product quality.
At present, methods for analyzing, researching and measuring chitosan mainly comprise spectrophotometry, fluorescence analysis, electrochemistry, infrared spectrometry, liquid chromatography, gas chromatography and the like. For example, Han Mei Na et al research establishes a fluorescence quenching method to determine the content of chitosan in a chitosan gel film, and establishes a fluorescence quenching method to determine the content of chitosan by utilizing the fact that chitosan has an obvious fluorescence quenching effect on Fluorescein Isothiocyanate (FITC) in a PBS (phosphate buffer solution) and the fluorescence quenching degree and the amount of chitosan are in a linear relationship within a certain range. The method is characterized in that the content of chitosan oligosaccharide in the raw materials and the compound premix is determined by high performance liquid chromatography established by two people, namely the Shangjun and the Wangbi. Tung school, MxBin et al established a new method for indirectly measuring non-electroactive chitosan (Chitosan) using Alizarin Red (AR) as an electroactive probe. Because chitosan is easy to degrade and ultraviolet absorption is weak, an acid hydrolysis indirect colorimetric method is often adopted for content analysis in experiments, concentrated sulfuric acid is often used in the method, operation has certain danger, and the method is not suitable for analyzing and detecting the content of trace chitosan. When the content of the chitosan is measured by the gas chromatography, the chitosan needs to be subjected to derivatization treatment before measurement, and the operation is complex. When the HPLC method is used for measuring the chitosan content, the chitosan is usually measured after being subjected to acid hydrolysis. The determination result of chitosan content is greatly influenced by the procedures of acid hydrolysis or derivatization, and some methods for directly determining chitosan content are gradually concerned.
Based on the method, the invention provides a method for accurately measuring the chitosan content by an ultraviolet spectrophotometry.
Disclosure of Invention
In order to overcome the technical defects that the content of chitosan is difficult to quantify and the operation technology is complicated in the prior art, the invention provides a method for accurately measuring the content of chitosan by an ultraviolet spectrophotometry. The method is influenced by the molecular weight of the chitosan, has the advantages of cheap reagent, high sensitivity, good reproducibility, simple and convenient operation and the like, and is suitable for popularization and application in practical application.
A method for accurately measuring the content of chitosan by an ultraviolet spectrophotometry comprises the following steps:
1) standard curves were drawn for Δ a and chitosan concentrations of different molecular weights: 1.0mL of low molecular chitosan standard solution with a certain concentration gradient is sequentially added into a 10mL colorimetric tube, and the concentration of 1.5mL is 2.0 multiplied by 10-4mol/L Naphthol Green B solution, 1.50ml 6.0X 10-2Adding 1.00ml of acetic acid-sodium acetate buffer solution with pH4.50 into tween 20 surfactant solution in g/L, adding distilled water to constant volume, shaking thoroughly, heating in 80 deg.C water bath for 8min, cooling to room temperature, measuring absorbance with water as reference at wavelength of 478nm on an ultraviolet spectrophotometer; wherein the reagent blank is marked as A0The chitosan-containing solution was designated as A, and Δ A ═ A-A was calculated0Establishing a standard curve C1 of the delta A and the concentration of the low molecular chitosan; replacing a low-molecular-weight chitosan solution with a high-molecular-weight chitosan solution to establish a standard curve C2 of the concentration of the medium-molecular-weight chitosan and the delta A; and replacing the low-molecular-weight chitosan solution with the high-molecular-weight chitosan solution to establish a standard curve C3 of the delta A and the concentration of the high-molecular-weight chitosan.
2) Preparation of 10. mu.g/mL sample working solution: weighing a certain amount of capsule to be detected, dissolving with glacial acetic acid, diluting to desired volume to obtain a sample stock solution, filtering with absorbent cotton, and filtering to obtain filtrate
6000r/min, centrifuging for 20min by a centrifuge, taking 2.5mL of supernatant into a 100mL volumetric flask, and performing constant volume to obtain a sample working solution with the concentration of 10 mug/mL.
3) Selecting a standard curve according to the molecular weight of chitosan: and (2) drawing a standard curve of the delta A and the sample working solution by using the detection method in the step 1), comparing the standard curve with chitosan standard curves with different molecular weights, and determining a regression equation used by the corresponding molecular weight of the chitosan.
4) Determination of chitosan content in the sample: taking 1mL of working solution, measuring according to the detection method in the step 1), measuring the absorbance value A at 478nm, and calculating that delta A is A-A0Substituting the delta A into a linear regression equation to obtain the content of chitosan in the sample, and simultaneously performing standard addition recoveryAnd (4) testing.
In the method for accurately measuring the chitosan content by the ultraviolet spectrophotometry, the pH value of the acetic acid-sodium acetate buffer solution is 4.50.
In the method for accurately measuring the chitosan content by the ultraviolet spectrophotometry, the solution in the step 1 is added in the order of firstly adding the chitosan standard solution, secondly adding naphthol green B, thirdly adding the Tween 20 surfactant solution and lastly adding the acetic acid-sodium acetate buffer solution.
The method for accurately measuring the chitosan content by ultraviolet spectrophotometry as described above, wherein the concentration is 6.0X 10-2The addition amount of the Tween 20 surfactant solution is 1.50 ml.
The method for accurately measuring the chitosan content by the ultraviolet spectrophotometry is characterized in that the time from cooling to room temperature to measuring is 8min-2 h.
The method for accurately determining the chitosan content by ultraviolet spectrophotometry as described above, step 1) standard curve C1, C2 and C3 has chitosan concentration range of 1.0-4.0 μ g/mL.
Sample pretreatment: removing the capsule shell of the capsule to be detected, weighing a certain amount of sample, dissolving with glacial acetic acid, and fixing the volume to obtain a sample stock solution. Filtering the stock solution with absorbent cotton in a funnel, centrifuging the filtrate for 20min at 6000r/min by a centrifuge, taking 2.5mL of supernatant in a 100mL volumetric flask, and performing constant volume to obtain a sample working solution with the concentration of 10 mug/mL.
And (3) sample determination: drawing a sample curve by using a sample working solution according to an experimental method, comparing the sample curve with chitosan standard curves with different molecular weights, determining the molecular weight of chitosan in the sample according to a regression equation of the sample, and determining the used delta A and a standard curve of chitosan concentration according to the molecular weight of the corresponding chitosan;
a sample of working solution (1 mL) was taken, and the absorbance A was measured at 478nm, and Δ A ═ A was calculated0Substituting the delta A into a linear regression equation to obtain the content of the chitosan in the sample, and simultaneously performing a standard addition recovery test
Experimental example:
1. ultraviolet spectral diagram of system
FIG. 1 is a graph showing the UV absorption of the system. The CTS concentration and the self-absorbance of the CTS, the Tween 20 and the naphthol green B are sequentially reduced from top to bottom. As can be seen from FIG. 1, the absorbance values of CTS, Tween 20 and naphthol green B are very low, but when CTS is combined with naphthol green B and Tween 20, the absorbance values are obviously increased, and the linear relationship at 478nm is the best, the scattering value of the blank group of the experiment is lower, and the scattering value of the system is increased along with the increase of the concentration of CTS in the experimental group, so that the linear relationship exists.
2. Buffer system and pH
The influence of three buffer solutions, namely glycine-hydrochloric acid, B-R buffer solution and acetic acid-sodium acetate HAc-NaAc, on the absorbance of the system is respectively inspected in the range of pH3.0-6.0. The result shows that the reaction of naphthol green B and chitosan has higher sensitivity in HAc-NaAc buffer solution. As shown in FIG. 2, the effect of the acidity of the HAc-NaAc buffer solution on the reaction system was observed, and the HAc-NaAc buffer solution was selected at pH4.50 because the acidity of the buffer solution significantly affects the reaction system and the sensitivity of the system was high and stable at pH 4.50.
3. Influence of the amount of buffer solution added
The buffer solution provides a proper acidity binding environment for an experimental system, the addition amount of the buffer solution has certain influence on the formation of the ionic associate, other experimental conditions are not changed, the addition amount of the acetic acid-sodium acetate buffer solution with the pH value of 3.0 is changed to 1.0, 1.5, 2.0, 3.0 and 4.0mL, and the absorbance value is measured. The results are shown in FIG. 3. The curve Δ A-V was plotted and the results showed that the optimum amount of buffer solution was 1.50 ml.
4. Effect of Naphthol Green B addition
Naphthol green B as a dye probe is combined with the target substance chitosan, the addition amount of the naphthol green B directly influences the formation of the ion association complex, other experimental conditions are not changed, and the concentration is changed to be 2.0 multiplied by 10-4The absorbance values of naphthol green B added in mol/L were measured at 1.00, 1.50,2.00,2.50,3.00,3.50, 4.00 mL. As a result, as shown in FIG. 4, the amount of naphthol green B added significantly affected the reaction system, and when the amount was more than 1.50mL, the absorbance tended to decrease, so that2.0×10-4The optimal addition amount of naphthol green B in mol/L is 1.50 mL.
5. Influence of the selection and amount of sensitizer
Some surfactants can play a role in sensitization, so the test investigates the influence of SDS, SLS, OP-10, polyvinyl alcohol, Tween 20, Tween 80 and SDBS on the absorbance of the system. As shown in FIG. 5, the reaction of naphthol green B with chitosan resulted in the best sensitization in Tween 20 solution.
The absorbance values were measured and the Δ A-V curves were plotted by adding Tween 20 to the system at 1.00, 1.50,2.00,2.50,3.00,3.50mL, with the other conditions being unchanged. As shown in FIG. 6, the influence of the amount of Tween 20 on the system shows that the sensitivity is highest when the optimal amount of Tween 20 is 1.50 ml. Therefore, the dosage of the Tween 20 is 1.50 ml.
6. Influence of the order of addition
Under experimental conditions, as shown in table 1, the effect of 12 different addition sequences of naphthol green B, acetate-sodium acetate buffer, CTS and tween 20 on the sensitivity of the system was examined. The result is shown in FIG. 7, and the optimal adding sequence is "CTS + Naphthol Green B + Tween 20+ acetic acid-sodium acetate buffer", at this time, the absorbance of the system is maximum, the sensitivity is highest, and the reproducibility is best.
TABLE 1 Effect of the order of addition
7. Influence of reaction temperature and heating time
Under the experimental conditions, the influence of different temperatures of 50-90 ℃ on the sensitivity of the system is examined, the result is shown in figure 8, and the temperature has influence on the absorbance value of the system. The absorbance value is in an increasing trend along with the increase of the temperature, the maximum sensitivity is reached at 80 ℃, the reproducibility is better at the reaction temperature, and the reaction temperature is in a decreasing trend after 80 ℃, so the reaction temperature is selected to be 80 ℃.
Under the optimized condition of the temperature, the influence of the heating time on the system is considered, and the experimental result shows that when the CTS-NGB system is heated at 80 ℃ for 8min, the sensitivity delta A of the system is maximum and most stable. Therefore, the heating condition of the system is selected to be 80 ℃ water bath for 8 min.
8. Influence of reaction time and stabilization time
After the reaction was complete, the reaction was cooled to room temperature according to the experimental conditions. The absorbance values were measured every 10min for the first half hour and every 20min after half an hour. The result shows that the absorbance of the system can be stable within 10min, basically does not change within 2h, and the stability is good.
9. Influence of the Ionic Strength
In the experiment, the influence of NaCl (0-0.30 mol/L) on the absorbance value of the reaction was examined. The results show (as in FIG. 9) that the general trend of Δ A as a function of NaCl concentration is first decreasing and then decreasing, with the reaction tending to plateau negatively at 0-0.05mol/L, probably because NaCl competes with NGB for CTS so that Δ A decreases.
10. Influence of coexisting substances
The interference of 20 coexisting substances on the system is examined, and the concentration of chitosan in the system is 1 mug/mL. The allowable amounts of the respective coexisting materials within. + -. 5% relative errors are shown in Table 2. Common amino acids, sugars, cations Na+，K+，NH4 +，Zn2+Etc. with a small interference and a large allowable amount; cu2+，Hg2+And Mn2+The allowable amount is relatively small.
TABLE 2 Effect of coexisting materials
11. Linear range and detection limit
Under the best experimental conditions, delta A corresponding to chitosan with different concentrations is measured according to an experimental method, a standard curve is drawn, and the result shows that the chitosan has a good linear relation with the delta A within the concentration range of 1.0-4.0 mu g/mL, the linear regression equation is that y is 0.0142x-0.0011, the correlation coefficient is 0.9974, and the method has a wide linear range. According to the formula: all the obtained DL-3S/K were substituted into 0.2470 μ g/mL.
12. Comparison of results for chitosans of different molecular weights
Under the optimal experimental conditions, the difference of the measurement results of the system on chitosan with different molecular weights is examined. Low, medium and high molecular weight chitosans were investigated separately. Through statistical analysis, the chitosan results with different molecular weights have obvious difference, and the method for determining the content of chitosan is influenced by different molecular weights.
TABLE 3 results for chitosans of different molecular weights
Compared with the prior art, the invention has the following technical advantages:
1) good linearity and low detection limit: under the best experimental condition, the corresponding delta A of chitosan with different concentrations is measured according to an experimental method, a standard curve is drawn, and the result shows that the chitosan has a good linear relation with the delta A within the concentration range of 1.0-4.0 mu g/mL, and the detection limit is 0.2470 mu g/mL.
2) The method is influenced by the molecular weight of chitosan, and in the actual sample measurement, a standard substance with similar molecular weight is taken as a quantitative standard, so that the method has the advantages of cheap reagent, high sensitivity, good reproducibility, simple and convenient operation and the like.
Drawings
FIG. 1 shows the absorption spectrum of CTS-naphthol green B-Tween 20 system.
FIG. 2 is a graph showing the effect of different pH values on absorbance of the buffer system.
FIG. 3 is a graph showing the effect of the amount of acetic acid-sodium acetate (NaAc-HAc) buffer added on the absorbance value.
FIG. 4 shows the effect of the amount of naphthol green B solution added on the absorbance of the system.
FIG. 5 Effect of different sensitizers on the absorbance of the system.
FIG. 6 influence of the amount of Tween 20 added on the absorbance of the system.
FIG. 7 Effect of the order of reagent addition on the absorbance of the system.
FIG. 8 influence of reaction temperature on system absorbance.
FIG. 9 Effect of ionic strength on the absorbance of the System
Detailed Description
The invention is further described below by means of specific examples, but said invention is not in any way intended to limit the scope of the patent protection of the invention.
Application example
A method for accurately measuring the content of chitosan by an ultraviolet spectrophotometry comprises the following steps:
1) drawing a standard curve of delta A and chitosan concentration with different molecular weights
1.0mL of low molecular chitosan standard solution with a certain concentration gradient is sequentially added into a 10mL colorimetric tube, and the concentration of 1.5mL is 2.0 multiplied by 10-4mol/L Naphthol Green B solution, 1.50ml 6.0X 10-2Adding 1.00ml of acetic acid-sodium acetate buffer solution with pH4.50 into tween 20 surfactant solution of g/L, adding distilled water to constant volume, shaking thoroughly, heating in 80 deg.C water bath for 8min, cooling to room temperature, measuring absorbance with water as reference at a wavelength of 478nm of the system; wherein the reagent blank is marked as A0The chitosan-containing solution was designated as A, and Δ A ═ A-A was calculated0Establishing a standard curve C1 of the delta A and the concentration of the low molecular chitosan; replacing a low-molecular-weight chitosan solution with a medium-molecular-weight chitosan solution to establish a standard curve C2 of delta A and medium-molecular-weight chitosan concentration; replacing a low-molecular-weight chitosan solution with a high-molecular-weight chitosan solution to establish a standard curve C3 of delta A and high-molecular-weight chitosan concentration;
2) preparation of 10. mu.g/mL sample working solution: removing capsule shell of the Aurelia chitin capsule, weighing 0.04g of the Aurelia chitin capsule into a 100mL volumetric flask, dissolving the Aurelia chitin capsule in 0.5mol/L glacial acetic acid, and fixing the volume to obtain a sample stock solution. Filtering the stock solution with absorbent cotton in a funnel, centrifuging the filtrate for 20min at 6000r/min by a centrifuge, taking 2.5mL of supernatant in a 100mL volumetric flask, and performing constant volume to obtain a sample working solution with the concentration of 10 mug/mL.
3) Selecting a standard curve according to the molecular weight of chitosan: drawing a standard curve of delta A and the sample working solution by using the detection method in the step 1), comparing the standard curve with chitosan standard curves with different molecular weights, and determining a regression equation used by the corresponding molecular weight of the chitosan;
and (3) drawing a sample curve by using the sample working solution according to an experimental method, comparing the sample curve with chitosan standard curves with different molecular weights, wherein the result is that the linear regression equation of the sample is delta A (0.0222 c-0.0001), the correlation coefficient is 0.9983, the result is close to the chitosan standard curve with the medium molecular weight, and the standard curve with the medium molecular weight chitosan is used as a quantitative standard curve.
1.00mL of sample working solution with a concentration of 10. mu.g/mL and 1.50mL of sample working solution with a concentration of 2.0X 10- 4mol/L Naphthol Green B solution, 1.50ml of 6.0X 10-2The Tween 20 solution of g/L and acetic acid-sodium acetate buffer solution of 1.00ml pH4.50 form stable ion association complex, and simultaneously, blank control is carried out. Heating in 80 deg.C water bath for 8 min. Cooled to room temperature and the absorbance a was measured with a uv spectrophotometer at λ 478nm, complete within 2 h. Calculating Delta A ═ A-A0Substituting the delta A into a linear regression equation to obtain the content of the chitosan in the sample, and simultaneously performing a standard addition recovery test. The results were: the content of chitosan in the chitin capsule is 1125.6mg/g, and the RSD is 1.57%. The recovery rates are shown in Table 4.
TABLE 4 results of sample analysis
Claims (1)
1. A method for accurately measuring the content of chitosan by an ultraviolet spectrophotometry comprises the following steps:
1) standard curves were drawn for Δ a and chitosan concentrations of different molecular weights: 1.0mL of low molecular chitosan standard solution with a certain concentration gradient is sequentially added into a 10mL colorimetric tube, and the concentration of 1.5mL is 2.0 multiplied by 10-4mol/L Naphthol Green B solution, 1.50ml 6.0X 10-2Adding 1.00ml of acetic acid-sodium acetate buffer solution with pH4.50 into tween 20 surfactant solution in g/L, adding distilled water to constant volume, shaking thoroughly, heating in 80 deg.C water bath for 8min, cooling to room temperature, measuring absorbance with water as reference at optimum wavelength of 478 nm; wherein the reagent blank is marked as A0The chitosan-containing solution was designated as A, and Δ A ═ A-A was calculated0Establishing a standard curve C1 of the concentration of the delta A and the low molecular weight chitosan; replacing a low-molecular-weight chitosan solution with a low-molecular-weight chitosan solution to establish a standard curve C2 of delta A and the concentration of the medium-molecular-weight chitosan; replacing a low-molecular-weight chitosan solution with a high-molecular-weight chitosan solution to establish a standard curve C3 of delta A and high-molecular-weight chitosan concentration;
2) preparation of 10. mu.g/mL sample working solution: weighing a certain amount of capsules of a sample to be detected, dissolving the capsules by using glacial acetic acid, fixing the volume to obtain a sample stock solution, filtering the stock solution by using a funnel absorbent cotton, centrifuging the filtrate for 20min at 6000r/min by using a centrifugal machine, taking 2.5mL of supernatant into a 100mL volumetric flask, and fixing the volume to obtain a sample working solution with the concentration of 10 mug/mL;
3) selecting a standard curve according to the molecular weight of chitosan: drawing a standard curve of delta A and the sample working solution by using the detection method in the step 1), comparing the standard curve with chitosan standard curves with different molecular weights, and determining a regression equation used by the corresponding molecular weight of the chitosan;
4) determination of chitosan content in the sample: taking 1mL of working solution, measuring according to the detection method in the step 1), measuring the absorbance value A at 478nm, and calculating that delta A is A-A0Substituting the delta A into a linear regression equation to obtain the content of the chitosan in the sample, and simultaneously performing a standard addition recovery test;
the pH value of the acetic acid-sodium acetate buffer solution is 4.50; the adding sequence of the solution in the step 1) is that firstly chitosan standard solution is added, secondly naphthol green B is added, thirdly Tween 20 surfactant solution is added, and finally acetic acid-sodium acetate buffer solution is added; the addition amount of the Tween 20 surfactant solution with the concentration of 6.0 multiplied by 10 < -2 > g/L is 1.50 ml; cooling to room temperature until the measuring time is 8min-2 h; step 1) the concentration range of chitosan in the standard curves C1, C2 and C3 is 1.0-4.0 mug/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711500040.2A CN108051386B (en) | 2017-12-30 | 2017-12-30 | Method for accurately measuring chitosan content by ultraviolet spectrophotometry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711500040.2A CN108051386B (en) | 2017-12-30 | 2017-12-30 | Method for accurately measuring chitosan content by ultraviolet spectrophotometry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108051386A CN108051386A (en) | 2018-05-18 |
| CN108051386B true CN108051386B (en) | 2021-06-29 |
Family
ID=62126072
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711500040.2A Active CN108051386B (en) | 2017-12-30 | 2017-12-30 | Method for accurately measuring chitosan content by ultraviolet spectrophotometry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108051386B (en) |
-
2017
- 2017-12-30 CN CN201711500040.2A patent/CN108051386B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108051386A (en) | 2018-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104833671B (en) | A kind of assay method of the absolute amylose content of rice | |
| CN103884676B (en) | A kind of rapid assay methods of Chinese crude drug multi-target ingredient content | |
| WO2018040821A1 (en) | Method for measuring degree of deacetylation of chitosan oligosaccharide by using first-order derivative ultraviolet spectrophotometry | |
| JP4659407B2 (en) | Analytical method for oligomeric proanthocyanidins (OPC) | |
| CN106124433B (en) | A method of it quickly detects pyrethroid pesticide remained | |
| Peng et al. | A ratiometric fluorescent sensor based on carbon dots for rapid determination of bisulfite in sugar | |
| CN108051386B (en) | Method for accurately measuring chitosan content by ultraviolet spectrophotometry | |
| CN102830114B (en) | Detection method for content of tannin in plant extract liquid | |
| CN107703080A (en) | A kind of assay method of dandelion extract general flavone content | |
| Shokrollahi et al. | Determination of erythrosine in food samples by CPE-scanometry as a new method and comparison with spectrophotometric results | |
| CN108254326B (en) | Method for accurately measuring chitosan content in capsule shell by fading spectrophotometry | |
| CN106053399A (en) | Method for measuring chitosan content by using water-soluble aniline blue as probe | |
| Su et al. | A colorimetric sensing strategy for detecting 10-hydroxy-2-decenoic acid in royal jelly based on Ag (I)-tetramethylbenzidine | |
| WO2018040820A1 (en) | Process for measuring degree of deacetylation of chitosan oligosaccharide using acid-base indicator method | |
| Heydari et al. | Simultaneous determination of Tropaeolin O and brilliant blue in food samples after cloud point extraction | |
| CN115326764A (en) | Preparation method of nitrogen-phosphorus-codoped red fluorescent carbon dots and detection method of nitrogen-phosphorus-codoped red fluorescent carbon dots on malachite green | |
| Janghel et al. | A new method for determination of ascorbic acid in fruit juices, pharmaceuticals and biological samples | |
| Delgado Andrade et al. | Optimised procedure to analyse maillard reaction-associated fluorescence in cereal-based products | |
| WO2020048269A1 (en) | Method for measuring content of chitosan oligosaccharide | |
| CN107796684B (en) | Method for detecting content of sodium copper chlorophyllin | |
| CN110927088A (en) | Method for rapidly detecting anthocyanin in lycium ruthenicum murr | |
| CN106645074B (en) | Direct fluorescence spectrum detection method for cystine content in cystine tablets | |
| Hewala et al. | Detection and determination of interfering 5–hydroxymethylfurfural in the analysis of caramel–coloured pharmaceutical syrups | |
| CN110487939A (en) | A method of measurement chitosan content | |
| CN103398960A (en) | Method for detecting nadroparin calcium product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |