CN108279222B - Method for measuring chitosan content by dual-wavelength resonance Rayleigh scattering method - Google Patents

Method for measuring chitosan content by dual-wavelength resonance Rayleigh scattering method Download PDF

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CN108279222B
CN108279222B CN201711500100.0A CN201711500100A CN108279222B CN 108279222 B CN108279222 B CN 108279222B CN 201711500100 A CN201711500100 A CN 201711500100A CN 108279222 B CN108279222 B CN 108279222B
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苏政权
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Abstract

The invention relates to a method for determining chitosan content by a dual-wavelength resonance Rayleigh scattering method, belonging to the field of macromolecular detection. In order to overcome the technical defects that the content of chitosan in a finished product is difficult to quantify and the operation technology is complicated in the prior art, the invention provides a method for measuring the content of chitosan by using a dual-wavelength resonance Rayleigh scattering method. Under the selected conditions, the chitosan concentration is in the range of 0.005-1.8 mug/mL and has good linear relation with the resonance Rayleigh scattering intensity, and accordingly a dual-wavelength resonance Rayleigh scattering method for measuring the chitosan content is established. The method for measuring the molecular weight of the chitosan is influenced by the molecular weight of the chitosan, and has the advantages of cheap reagent, high sensitivity, good reproducibility, strong anti-interference capability, simple and convenient operation and the like.

Description

Method for measuring chitosan content by dual-wavelength resonance Rayleigh scattering method
Technical Field
The invention relates to a method for determining chitosan content by a dual-wavelength resonance Rayleigh scattering method, belonging to the field of macromolecular detection.
Background
Chitin (chitin), also known as chitin, is a polysaccharide of N-acetyl-2-amino-2-deoxy-D-glucose linked in the form of β -1, 4 glycosidic bonds. It is widely found in shells of crustacean such as shrimp, crab and insect, cells of lower plant fungi and algae in nature, and cell walls of higher plants. The amount of chitin biosynthesized per year is as much as 1000 million tons, and the stock is second only to cellulose in nature.
Chitosan (CTS) is a substance generated by linear amino polysaccharide chitin through deacetylation reaction, a plurality of amino groups, hydroxyl groups and deacetylation groups are distributed on a molecular chain, the molecular weight of the chitosan is different from hundreds of thousands to millions, various intra-molecular and intermolecular hydrogen bonds are formed in an aqueous solution, and the hydrogen bonds enable the chitosan to form hydrophobic micro-regions, so that a double-helix structure of the chitosan is formed. In an acidic solution, amino groups adsorb protons to form positively charged ions, and the positively charged ions and negatively charged anions can form associations through electrostatic binding and hydrophobic interaction. The excellent characteristics of chitosan are widely applied to various industries such as medicine and medicine, health food, water treatment, metal extraction and recovery and the like, and particularly the application in the aspect of health food is gradually wide, so that the accurate determination of the content of chitosan has important significance for researching and developing chitosan. At present, the chitosan content can be measured by four methods, such as a spectroscopic method, an electrochemical method, a titration method, a chromatography method and the like.
The Resonance Rayleigh Scattering (RRS) method is that when Rayleigh Scattering is located at or close to its molecular absorption band, electrons absorb electromagnetic wave frequency the same as Scattering frequency, electrons strongly absorb energy of light due to Resonance and generate re-Scattering, and this absorption-re-Scattering process is called Resonance Rayleigh Scattering, and its Scattering intensity is increased by several orders of magnitude compared with simple Rayleigh Scattering, so that Rayleigh law is not followed any more. The resonance rayleigh scattering method is a molecular spectroscopic analysis method which has been developed in recent years and has high sensitivity and is easy to operate. At present, the resonance Rayleigh scattering method is widely applied to the researches in the aspects of biomacromolecule analysis, inorganic matter and medicine analysis, trace inorganic ion determination, nano ion characterization and the like. The method is applied to the analysis of saccharides, a novel resonance Rayleigh scattering method for measuring the chitosan is established, and the appropriate reaction conditions and the influence factors of the reaction are researched.
Brilliant blue is an anionic dye, under the weak acid condition, the Brilliant blue with negative charges and chitosan with positive charges are subjected to ion association reaction, the ion association complex of the Brilliant blue generates two new characteristic peaks at 345nm and 445nm, and the scattering intensity and the chitosan content are in a linear relation within a certain concentration range. According to the characteristics, a resonance Rayleigh scattering method with strong anti-interference capability is established, and the method is suitable for measuring and quantitatively analyzing the chitosan content in the health care products.
Disclosure of Invention
In order to overcome the technical defects that the content of chitosan in a finished product is difficult to quantify and the operation technology is complicated in the prior art, the invention provides a method for measuring the content of chitosan by using a dual-wavelength resonance Rayleigh scattering method. Under the selected conditions, the chitosan concentration is in the range of 0.005-1.8 mug/mL and has good linear relation with the resonance Rayleigh scattering intensity, and accordingly a dual-wavelength resonance Rayleigh scattering method for measuring the chitosan content is established. The method is influenced by the molecular weight and the deacetylation degree of the chitosan, and has the advantages of cheap reagent, high sensitivity, good reproducibility, strong anti-interference capability, simple and convenient operation and the like.
A method for measuring the content of chitosan by a dual-wavelength resonance Rayleigh scattering method comprises the following steps:
1) drawing a standard curve of delta I and chitosan concentration with different molecular weights
Adding 1.0mL of chitosan standard solution with a certain concentration gradient and 1.0mL of buffer solution into a 10mL colorimetric tube, wherein the concentration of 1.0X 10 is 1.0mL-3Fixing volume of mol/L brilliant blue solution to scale with triple distilled water, shaking thoroughly, placing at room temperature for 10min, adding 1ml mixed solution into quartz cuvette, placing on F-2500 type fluorescence spectrophotometer, and measuring with lambdaex＝λemSynchronous scanning is carried out, and resonance scattering intensity values I of systems at the maximum resonance Rayleigh scattering wavelength lambda of 345nm of each detection system containing chitosan are respectively recorded1Wherein the resonance scattering intensity value I of the reagent blank system at lambda-345 nm01And calculating Δ I1＝I1-I01(ii) a Simultaneously recording the resonance Rayleigh scattering intensity I of each detection system containing chitosan at the position of the secondary large resonance Rayleigh scattering wavelength lambda of 445nm2And the resonant Rayleigh scattering intensity I of the blank reagent02Calculating the difference Delta I of the resonant Rayleigh scattering intensity2＝I2-I02Wherein Δ I ═ Δ I1+ΔI2. Establishing a standard curve C1 of the delta I and the concentration of the low molecular chitosan; replacing a low-molecular-weight chitosan solution with a high-molecular-weight chitosan solution, and establishing a standard curve C2 of delta I and the concentration of 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 I and the concentration of high-molecular-weight chitosan;
under the optimal experimental conditions, the measurement results of the system on chitosan with different molecular weights are 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.
2) Preparation of 10. mu.g/mL sample working solution: and (3) removing the capsule shell of the chitosan capsule, weighing 0.04g of chitosan capsule into a 100mL volumetric flask, dissolving the chitosan 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 the delta I 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, determining the molecular weight of the chitosan in the sample according to a regression equation of the delta I of the sample and the standard curve of the sample working solution, and determining the used regression equation according to the molecular weight of the corresponding chitosan;
4) determination of chitosan content in the sample: sampling 1mL of working solution, determining according to the detection method in the step 1), and determining the difference delta I of the resonant Rayleigh scattering intensity at the wavelength of 345nm and 445nm1And Δ I2And calculating Δ I ═ Δ I1+ΔI2Substituting the delta I into a linear regression equation to obtain the content of the chitosan in the sample, and simultaneously performing a standard addition recovery test.
The method for measuring the chitosan content by the double-wavelength resonance Rayleigh scattering method comprises the following steps of (1) determining the chitosan content by using the buffer solution, wherein the buffer solution is one of a B-R buffer solution, a HAc-NaAc buffer solution, a glycine-hydrochloric acid buffer solution and a citric acid-disodium hydrogen phosphate buffer solution; the applicant finds out through experiments that the acidity of the buffer solution has a remarkable influence on the reaction system, and the scattering value is large at the pH value of 3.0; and the system has the highest sensitivity and the largest scattering value in citric acid-sodium citrate buffer solution, so the optimal buffer solution is selected to be citric acid-disodium hydrogen phosphate buffer solution with the pH value of 3.0.
According to the method for measuring the chitosan content by the dual-wavelength resonance Rayleigh scattering method, in the step 1, the adding sequence of the reagent is firstly adding the citric acid-disodium hydrogen phosphate buffer solution, secondly adding the brilliant blue solution, thirdly adding the sodium dodecyl sulfate anionic surfactant solution and finally adding the chitosan solution.
The method for measuring the chitosan content by the double-wavelength resonance Rayleigh scattering method comprises the step 1) of measuring the concentration of the chitosan in a range of 0.005-1.8 mu g/mL.
Sample pretreatment: and (3) removing the capsule shell of the chitosan capsule, weighing 0.04g of chitosan capsule into a 100mL volumetric flask, dissolving the chitosan 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.
Sampling 1mL of working solution, determining according to the detection method in the step 1), and determining the difference delta I of the resonant Rayleigh scattering intensity at the wavelength of 345nm and 445nm1And Δ I2And calculating Δ I ═ Δ I1+ΔI2Substituting the delta I into a linear regression equation to obtain the content of the chitosan in the sample, and simultaneously performing a standard addition recovery test.
Test example:
1. resonance Rayleigh scattering map of brilliant blue-chitosan system
After the solution was prepared according to the experimental method, the resonance rayleigh scattering spectrum was scanned simultaneously on a fluorescence spectrophotometer model F-2500 (see fig. 1). Fig. 1 shows the resonance rayleigh scattering spectra of blank tubes of brilliant blue solution-buffer solution and different amounts of chitosan and brilliant blue associate, and the results show that: the resonance Rayleigh scattering signals of the brilliant blue solution and the buffer solution under the measurement condition are weaker (curve 0 mL); when chitosan is added into a bright blue-buffer solution system (under an acidic condition), a new resonance Rayleigh scattering spectrum with two obvious scattering peaks (345nm and 445nm) is generated, and the resonance Rayleigh scattering intensity of the spectrum is enhanced along with the increase of the concentration of the chitosan and is in a linear relation within a certain mass concentration range; two peaks are on the spectrum, each peak is increased linearly, but the two wavelengths are adopted to enable R2And better, the experimental results are both dual-wavelength determination.
2. Influence of different buffer solutions and pH of the buffer solution on system resonance Rayleigh scattering intensity
The Δ I of the system was first measured over the pH range 2-6, and as can be seen from FIG. 2, the Δ I exhibited a rising-to-falling trend with a peak between 2.5 and 3.5, thus establishing a pH of 3.0.
The influence of the B-R buffer solution, the HAc-NaAc buffer solution, the glycine-hydrochloric acid buffer solution and the citric acid-disodium hydrogen phosphate buffer solution on a brilliant blue-chitosan system is respectively tested. The results are shown in FIG. 3, and show that the strength is higher in the citric acid-disodium hydrogen phosphate buffer solution, so the citric acid-disodium hydrogen phosphate buffer solution with pH of 3.0 is selected to control the acidity of the system, and the optimal dosage is 1.0 mL.
3. Influence of brilliant blue concentration and addition amount thereof on system resonance Rayleigh scattering intensity
The influence of the brilliant blue concentration and the addition amount thereof on the system is examined, and the brilliant blue concentration is set to be 2 multiplied by 10-4mol/mL; the addition amount (mL) was: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, the results are shown in FIG. 4, showing that Δ I is increasing, and therefore, it is considered that the brilliant blue concentration is increased to 8X 10-4mol/mL, the addition amount (mL) is: 0.5, 1, 1.5, 2, 2.5, and 3, and the results showed that Δ I had a peak at 1.5mL and that the brilliant blue concentration was 1X 10-3mol/L, 1mL 1.0X 10 selected because the system Δ I reached the maximum at 1mL-3And (3) a bright blue solution in mol/L to obtain higher sensitivity.
Effect of buffer solution on brilliant blue-chitosan system. The result shows that the strength is higher in the citric acid-disodium hydrogen phosphate buffer solution, so the citric acid-disodium hydrogen phosphate buffer solution with the pH value of 3.0 is selected to control the acidity of the system, and the optimal dosage is 1.0 mL.
4. Effect of reagent addition sequence on System resonance Rayleigh Scattering Strength
The influence of 3 different reagent adding sequences on the reaction system is tested, the test result is shown in figure 5, the optimal sequence is chitosan-citric acid-disodium hydrogen phosphate buffer solution-brilliant blue solution, but due to the poor linearity, the adding sequence of the citric acid-disodium hydrogen phosphate buffer solution-brilliant blue solution-chitosan is not greatly different from the adding sequence delta I of the chitosan-citric acid-disodium hydrogen phosphate buffer solution-brilliant blue solution and is more optimal than the linearity, so the adding sequence is selected to be citric acid-disodium hydrogen phosphate buffer solution-brilliant blue solution-chitosan.
5. Influence of reaction temperature on System resonant Rayleigh Scattering Strength
The influence of the system on the system is examined at different temperatures, the heated system has no color change in the experimental process, the resonance Rayleigh scattering intensity is as shown in figure 6, the delta I is the highest at 40 ℃, however, the linear relation of the standard curve is poor at 40 ℃, and the slope of the standard curve is not much different from that of the system at normal temperature, so the system can be determined at normal temperature.
6. Effect of stabilization time on System resonant Rayleigh Scattering Strength
Under the condition of room temperature, the stability of the system within 240min is discussed, the result is shown in fig. 7, the result shows that the delta I is relatively stable within 45min after the reaction of the brilliant blue solution and the chitosan, and the delta I shows a sharp decline trend after 45min begins. Therefore, the experiment should be completed within 45 min.
7. Influence of the Ionic Strength
Different NaCl solutions are prepared and added into the system, the ionic strength allowed by the system is observed, the result is shown in figure 8, and as can be seen from figure 8, when the ionic strength of the chitosan-brilliant blue system is less than 0.01mol/L, the system is relatively stable; when the concentration is more than 0.01mol/L, the effect is larger as the concentration is higher.
8. Anti-interference experiment
The interference of 20 substances on the method is considered, and when the concentration of the chitosan is 1 mu g/mL and the relative error is less than or equal to +/-5 percent, the allowable times of the coexisting substances are shown in Table 1.
TABLE 1 Effect of coexisting materials
Figure BDA0001535475930000051
Figure BDA0001535475930000061
As shown in Table 1, iron, copper, calcium and aluminum have great influence on reminding, and need to be masked by masking agents, 10 times of aluminum can be masked by 0.01mol/LEDTA masking agents, 10 times of calcium ions are masked by 1mL of 100 mu g/mL and 0.4% of tartaric acid, and errors generated in the determination process are reduced by adding the two masking agents before determining the sample.
9. Effect of different degrees of deacetylation on the System
Under the optimal experimental conditions, the difference of the measurement results of the system on chitosan with different deacetylation degrees is examined. Chitosan having a degree of deacetylation of 85% and 90% and a molecular weight of 60 was examined, respectively. The result is shown in fig. 9, and through statistical analysis, there is no significant difference in chitosan results with different deacetylation degrees, i.e. chitosan with different deacetylation degrees has no influence on the system.
Compared with the prior art, the invention has the following technical advantages:
1) good linearity and low detection limit: under the best experimental condition, determining the corresponding delta I of the chitosan with different concentrations according to the experimental method, drawing a standard curve, and the result shows that the chitosan has good linear relation with the delta I within the range of 0.005-1.8 mu g/mL L, the linear range is wide, and the detection limit is 5.48 multiplied by 10-2μg/mL。
2) The method for determining the content of the chitosan by the dual-wavelength resonance Rayleigh scattering method is established, the determination is influenced by the molecular weight of the chitosan, in the actual sample determination, a standard substance with similar molecular weight needs to be considered to be used as a quantitative standard, and the method has the advantages of low reagent price, high sensitivity, strong anti-interference capability, good reproducibility, simplicity and convenience in operation and the like.
Drawings
Figure 1 resonance rayleigh scattering spectrum of brilliant blue-chitosan system.
FIG. 2 is a graph showing the effect of pH of a buffer solution on the resonant Rayleigh scattering intensity of a system.
FIG. 3 is a graph showing the effect of different buffer solutions on the resonant Rayleigh scattering intensity of a system.
FIG. 4 is a graph showing the effect of the amount of brilliant blue added on the resonant Rayleigh scattering intensity of the system.
FIG. 5 is a graph showing the effect of the order of addition of reagents on the resonant Rayleigh scattering intensity of a system.
FIG. 6 is a graph showing the effect of reaction temperature on the system resonance Rayleigh scattering intensity.
FIG. 7 is a graph showing the effect of settling time on the resonant Rayleigh scattering intensity of a system.
FIG. 8 is a graph of the effect of ionic strength on system resonance Rayleigh scattering strength.
FIG. 9 is a graph showing the effect of chitosan deacetylation on the resonant Rayleigh scattering intensity of a system.
Detailed Description
The invention is further described below by means of specific examples, but said invention is not in any way restricted to the scope of the invention as claimed.
Examples
A method for measuring the content of chitosan by a dual-wavelength resonance Rayleigh scattering method comprises the following steps:
1) drawing a standard curve of delta I and chitosan concentration with different molecular weights
Adding 1.0mL of chitosan standard solution with a certain concentration gradient, 1.0mL of citric acid-disodium hydrogen phosphate buffer solution with pH of 3.0, and 1.0mL of 1.0 × 10-3Fixing volume of mol/L brilliant blue solution to scale with triple distilled water, shaking thoroughly, placing at room temperature for 10min, adding 1ml mixed solution into quartz cuvette, placing on F-2500 type fluorescence spectrophotometer, and measuring with lambdaex＝λemSynchronous scanning is carried out, and resonance scattering intensity values I of systems at the maximum resonance Rayleigh scattering wavelength lambda of 345nm of each detection system containing chitosan are respectively recorded1Wherein the resonance scattering intensity value I of the reagent blank system at lambda-345 nm01And calculating Δ I1＝I1-I01(ii) a Simultaneously recording the resonance Rayleigh scattering intensity I of each detection system containing chitosan at the position of the secondary large resonance Rayleigh scattering wavelength lambda of 445nm2And the resonant Rayleigh scattering intensity I of the blank reagent02Calculating the difference Delta I of the resonant Rayleigh scattering intensity2＝I2-I02Wherein Δ I ═ Δ I1+ΔI2. Establishing a standard curve C1 of the delta I and the concentration of the low molecular chitosan; replacing a low-molecular-weight chitosan solution with a high-molecular-weight chitosan solution, and establishing a standard curve C2 of delta I and the concentration of medium-molecular-weight chitosan; and replacing the low-molecular-weight chitosan solution with the high-molecular-weight chitosan solution to establish a standard curve C3 of the delta I and the concentration of the high-molecular-weight chitosan.
TABLE 2 results for chitosans of different molecular weights
Figure BDA0001535475930000071
2) Preparation of 10. mu.g/mL sample working solution: and (3) removing the capsule shell of the chitosan capsule, weighing 0.04g of chitosan capsule into a 100mL volumetric flask, dissolving the chitosan 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 the delta I 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, determining the molecular weight of the chitosan in the sample according to a regression equation of the delta I of the sample and the standard curve of the sample working solution, and determining the used regression equation according to the molecular weight of the corresponding 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 that delta I is 2014.20c +30.05, the correlation coefficient is 0.9958, the result is close to the chitosan standard curve with the medium molecular weight, and the standard curve with the medium molecular weight is used as a quantitative standard curve.
4) Determination of chitosan content in the sample: sampling 1mL of working solution, determining according to the detection method in the step 1), and determining the difference delta I of the resonant Rayleigh scattering intensity at the wavelength of 345nm and 445nm1And Δ I2And calculating Δ I ═ Δ I1+ΔI2Substituting the delta I into a linear regression equation to obtain the content of the chitosan in the sample, and simultaneously performing a standard addition recovery test.
Sampling 1mL of working solution, measuring according to an experimental method, and measuring the difference delta I of the resonant Rayleigh scattering intensity at the wavelength of 345nm and 445nm1And Δ I2And calculating Δ I ═ Δ I1+ΔI2Substituting the delta I 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 Haibao chitosan capsule is 251.4mg/g, and the RSD is 1.1%. The recovery rates are shown in Table 3.
TABLE 3 recovery of chitosan by addition of standard
Figure BDA0001535475930000081
In the experiment, 1.0mL of a 1.0 × 10 citric acid-disodium hydrogen phosphate buffer solution having a ph of 3.0 was sequentially added to a 10mL colorimetric cylinder, and the concentration was 1.0mL-3Adding 1.0mL of chitosan solution with concentration of 10 μ g/mL and mol/L brilliant blue solution into distilled water to constant volume, shaking thoroughly, standing at room temperature for 10min, adding 1mL of mixed solution into quartz cuvette, placing on F-2500 type fluorescence spectrophotometer at lambdaex＝λemSynchronous scanning is carried out, and the determination is completed within 45 min. Respectively recording the resonance scattering intensity value I of each detection system containing chitosan at the position of maximum resonance Rayleigh scattering wavelength lambda being 345nm1Wherein the resonance scattering intensity value I of the reagent blank system at lambda-345 nm01And calculating Δ I1＝I1-I01(ii) a Simultaneously recording the resonance Rayleigh scattering intensity I of each detection system containing chitosan at the position of the secondary large resonance Rayleigh scattering wavelength lambda of 445nm2And the resonant Rayleigh scattering intensity I of the blank reagent02Calculating the difference Delta I of the resonant Rayleigh scattering intensity2＝I2-I02Wherein Δ I ═ Δ I1+ΔI2. . The chitosan has good linear relation with delta I within the concentration range of 0.005-1.8 mu g/mL, the linear range is wide, and the detection limit is 5.48 multiplied by 10-2μ g/mL. The invention establishes a method for measuring the chitosan content by a dual-wavelength resonance Rayleigh scattering method, the measurement of the method is influenced by the molecular weight of chitosan, in the actual sample measurement, a standard substance with similar molecular weight is taken as a quantitative standard, and the method has the advantages of cheap reagent, high sensitivity, strong anti-interference capability, good reproducibility, simple and convenient operation and the like.
Linear range and detection limit
Under the best experimental condition, measuring corresponding delta I of chitosan with different concentrations according to an experimental method, drawing a working curve by using the delta I to the chitosan concentration, wherein the chitosan mass concentration is in a good linear relation with the delta I within the range of 0.005-1.8 mu g/mL, a linear regression equation is that y is 2014.20x +30.05, and a correlation coefficient R is2Is 0.9958. According to the experimental methodThe blank and the minimum tube were measured in parallel 13 times each, and the detection limit was 5.48 × 10, as determined from CL ═ 3s/k (s is the standard deviation, and k is the slope of the regression equation)-2μg/mL。

Claims (1)

1. A method for measuring the content of chitosan by a dual-wavelength resonance Rayleigh scattering method is characterized by comprising the following steps:
1) standard curves were drawn for Δ I and chitosan concentration at different molecular weights: adding 1.0mL of chitosan standard solution with a certain concentration gradient and 1.0mL of buffer solution into a 10mL colorimetric tube, wherein the concentration of 1.0X 10 is 1.0mL-3Fixing volume of mol/L brilliant blue solution to scale with triple distilled water, shaking thoroughly, placing at room temperature for 10min, adding 1ml mixed solution into quartz cuvette, placing on F-2500 type fluorescence spectrophotometer, and measuring with lambdaex＝λemSynchronous scanning is carried out, and resonance scattering intensity values I of systems at the maximum resonance Rayleigh scattering wavelength lambda of 345nm of each detection system containing chitosan are respectively recorded1Wherein the resonance scattering intensity value I of the reagent blank system at lambda-345 nm01And calculating Δ I1＝I1-I01(ii) a Simultaneously recording the resonance Rayleigh scattering intensity I of each detection system containing chitosan at the position of the secondary large resonance Rayleigh scattering wavelength lambda of 445nm2And the resonant Rayleigh scattering intensity I of the blank reagent02Calculating the difference Delta I of the resonant Rayleigh scattering intensity2＝I2-I02Wherein Δ I ═ Δ I1+ΔI2(ii) a Establishing a standard curve C1 of the delta I and the concentration of the low molecular chitosan; replacing a low-molecular-weight chitosan solution with a high-molecular-weight chitosan solution, and establishing a standard curve C2 of delta I and the concentration of 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 I and the concentration of high-molecular-weight chitosan;
2) preparation of 10. mu.g/mL sample working solution: removing the capsule shell of the chitosan capsule, weighing 0.04g of chitosan capsule into a 100mL volumetric flask, dissolving the chitosan capsule in 0.5mol/L glacial acetic acid, and fixing the volume to obtain a sample stock solution; filtering the stock solution by using absorbent cotton of a funnel, centrifuging the filtrate for 20min at 6000r/min by using a centrifugal machine, 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: drawing a standard curve of the delta I 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, determining the molecular weight of the chitosan in the sample according to a regression equation of the delta I of the sample and the standard curve of the sample working solution, and determining the used regression equation according to the molecular weight of the corresponding chitosan;
4) determination of chitosan content in the sample: sampling 1mL of working solution, determining according to the detection method in the step 1), and determining the difference delta I of the resonant Rayleigh scattering intensity at the wavelength of 345nm and 445nm1And Δ I2And calculating Δ I ═ Δ I1+ΔI2Substituting the delta I into a linear regression equation to obtain the content of the chitosan in the sample, and simultaneously performing a standard addition recovery test;
the buffer solution is citric acid-disodium hydrogen phosphate buffer solution; the pH value of the citric acid-sodium dihydrogen phosphate buffer solution is 3.0; the adding sequence of the reagents in the step 1 is that firstly, citric acid-disodium hydrogen phosphate buffer solution is added, secondly, brilliant blue solution is added, thirdly, lauryl sodium sulfate anionic surfactant solution is added, and finally, chitosan solution is added; the concentration range of chitosan in step 1) is 0.005-1.8. mu.g/mL.
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