A non-colorimetric method for quantitative analysis of chitosan

Abstract

The present invention relates to the technical field of macromolecular detection, and in particular to a non-colorimetric spectrophotometric method for quantitatively analyzing chitosan. The present invention utilizes the fact that the absorbance value of a chitosan solution increases with the increase of the chitosan content within a certain concentration range at a wavelength of 190 to 195 nm, and presents a certain linear relationship, and uses this as the quantitative basis of chitosan to establish a non-colorimetric spectrophotometric method for analyzing chitosan. The method of the present invention does not require the addition of a color developer, reduces various interference factors in the color development link, has low requirements for instruments and equipment, is simple to operate, and ensures quantitative stability and reproducibility. In the absence of interference from other organic molecules or inorganic ligands, the method has excellent applicability for the quantitative analysis of chitosan in a controllable system (or pure system).

Description

Technical Field

[0001] The invention relates to the technical field of macromolecular detection, and in particular to a non-coloring spectrophotometric method for quantitatively analyzing chitosan. Background Art

[0002] Chitosan is a type of polyelectrolyte rich in amino and hydroxyl functional groups, with multifunctional natural activity. In the past two decades, it has attracted extensive attention from researchers in the fields of medicine, food, chemical industry, and environmental protection. Chitosan has many natural excellent properties, such as moisture absorption and breathability, biocompatibility, biodegradability, non-antigenicity, non-inflammatory, no harmful degradation products, adhesion, antibacterial properties, etc., and is therefore widely used in the textile industry, biomedicine, and daily environmental protection.

[0003] In the field of chitosan-solid interaction and chitosan modification, quantitative research on chitosan is necessary for in-depth exploration of the mechanism of related research, and its accurate quantitative method is worth exploring.

[0004] At present, the quantitative methods of chitosan mainly include colorimetric detection, size exclusion chromatography, high performance liquid chromatography, elemental analysis, ultraviolet spectroscopy, etc. (Table 1). Size exclusion chromatography, high performance liquid chromatography, elemental analysis, and ultraviolet spectroscopy have high requirements for instrument conditions or operating details. Relatively speaking, due to the high penetration rate of spectrophotometers, the ninhydrin colorimetric method is more commonly used. However, the ninhydrin colorimetric method includes the mixing and heating of ninhydrin for color development, and the color development results are easily affected by pH, temperature, and color development time, resulting in the condensation reaction of chitosan and ninhydrin, uneven heating, and photolysis over time. Based on this, it is very necessary to develop a chitosan non-color development quantitative method with strong applicability, simple operation, and high accuracy.

[0005] Table 1: Advantages and disadvantages of existing chitosan quantification methods

[0006]

[0007] Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a non-colorimetric method for quantitatively analyzing chitosan. The present invention utilizes that the absorbance value of chitosan solution increases with the increase of chitosan content within a certain concentration range at a wavelength of 190 to 195 nm, and presents a certain linear relationship. Based on this as the quantitative basis of chitosan, a non-colorimetric method for analyzing chitosan is established.

[0009] A non-colorimetric method for quantitatively analyzing chitosan comprises the following steps:

[0010] (1) Drawing a standard curve: accurately weigh a chitosan reference substance, soak and dissolve it with an aqueous solution of HClO4 having a concentration of 1M or more (preferably 1M, M is mol / L, the same below), then add ultrapure water to a concentration of HClO4 of 0.01M, shake well, and use it as a linear stock solution; dilute the linear stock solution with a 0.01M HClO4 aqueous solution to different chitosan concentrations (chitosan concentration is in the range of 0.1 to 50 mg / L) to use as a linear solution; at a wavelength of 190 to 195 nm, use ultrapure water as a reference solution, detect the absorbance of each linear solution, and adjust the absorbance value measured for the reference solution to 0; the absorbance of the solution containing chitosan is recorded as A, and a standard curve is drawn with the chitosan concentration as the horizontal coordinate and A as the vertical coordinate.

[0011] (2) Sample detection: Accurately weigh the sample, soak and dissolve it with an aqueous solution of HClO4 with a concentration of 1M or more (preferably 1M), then add ultrapure water to a concentration of 0.01M HClO4, and shake well; at a wavelength of 190-195nm, use ultrapure water as a reference solution, adjust the absorbance value of the reference solution to 0, and detect the absorbance of the sample solution, recorded as A. If A exceeds the linear range, dilute it with a 0.01M HClO4 aqueous solution to obtain a sample solution of appropriate concentration;

[0012] Substitute A into the standard curve equation in step (1) to obtain the chitosan concentration, then multiply it by the dilution factor, and multiply the obtained concentration (mass volume concentration) by its volume to obtain the mass of chitosan; then calculate the chitosan content in the test sample based on the mass of chitosan / the sample weight of the test sample.

[0013] The chitosan contained in the chitosan reference substance and the test substance is low molecular weight chitosan, the viscosity thereof is 20-300 cP, and the degree of deacetylation thereof is >75%;

[0014] Alternatively, the chitosan contained in the chitosan reference substance and the test substance is a medium molecular weight chitosan, the viscosity of which is 200-800 cP, and the degree of deacetylation thereof is greater than 75%;

[0015] Alternatively, the chitosan contained in the chitosan reference substance and the test substance is high molecular weight chitosan, the viscosity of which is >400 cP, and the degree of deacetylation of which is >75%;

[0016] In the method of the present invention, no other organic substances or inorganic ligands are present in the test sample.

[0017] Preferably, the sample is an adsorption system of an inorganic mineral adsorption material-chitosan solution, wherein the inorganic mineral adsorption material includes at least one of montmorillonite, kaolinite, illite, magnetite, etc.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Compared with the existing technology, this method does not need to add color developer, reduces various interference factors in the color development process, has low requirements for instruments and equipment, is simple to operate, and ensures quantitative stability and reproducibility. In the absence of interference from other organic molecules or inorganic ligands, it has excellent applicability for quantitative analysis of chitosan in a controllable system (or pure system). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the scanning spectrum of low molecular weight chitosan of different concentrations in the range of 190-300nm;

[0021] Figure 2 The scanning spectrum of different concentrations of medium molecular weight chitosan in the range of 190-300nm;

[0022] Figure 3 It is the scanning spectrum of high molecular weight chitosan with different concentrations in the range of 190-300nm;

[0023] Figure 4 is the linear relationship diagram of low molecular weight chitosan;

[0024] Figure 5 is the linear relationship diagram of medium molecular weight chitosan;

[0025] Figure 6 This is the linear relationship diagram of high molecular weight chitosan. DETAILED DESCRIPTION

[0026] The applicant will further describe the present invention in conjunction with the embodiments and drawings of the specification below, but the protection scope of the present invention is not limited to these embodiments. Those skilled in the art should understand that equivalent replacements or corresponding improvements made to the technical features of the present invention still fall within the protection scope of the present invention.

[0027] The experimental methods used in the following examples are all conventional methods unless otherwise specified; the water used is ultrapure water (resistivity>18.25 MΩ·cm); the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0028] In the following examples, low molecular weight (20-300 cP, deacetylation degree>75%), medium molecular weight (200-800 cP, deacetylation degree>75%), and high molecular weight (>400 cP, deacetylation degree>75%) chitosans were used as test objects. The low, medium, and high molecular weight chitosans were purchased from Sigma-Aldrich, and the product codes were 102428982, 102466447, and 102473231, respectively, and the production standards were all: CAS No.9012-76-4.

[0029] The equipment used in the following examples is a double-beam spectrophotometer (Beijing Puxi TU-1901, wavelength range 190-700nm).

[0030] Example 1: Analysis method establishment

[0031] 1) Wavelength selection

[0032] Saturated hydrocarbon derivatives containing heteroatoms such as O, N, S and halogen will produce light absorption at 150-250nm, which provides a basic basis for the non-colorimetric quantification of chitosan. First, use ultrapure water to scan the short-wave band of the machine to ensure that there is no obvious light absorption in the short-wave of ultrapure water. Using the ultrapure water spectrum as the baseline, scan the low, medium and high molecular weight chitosan solutions of different concentrations in the range of 190-300nm. Figure 1-3 .

[0033] Preparation process of chitosan solution: soak and dissolve 0.1g chitosan with 10mL 1M HClO4 aqueous solution, then add ultrapure water to the concentration of HClO4 to 0.01M, shake well, at this time the chitosan concentration is 100mg / L; then dilute the above chitosan solution to different concentrations with 0.01M HClO4 aqueous solution (low, medium and high molecular weight chitosan are prepared in the same way).

[0034] Depend on Figure 1-3 It can be seen that 190 nm is the absorption peak in the range, but considering that 190 nm is the critical detection wavelength of the instrument, the instrument may be unstable at this wavelength, so the present invention selects 195 nm as the wavelength for quantitative detection.

[0035] 2) Solvent selection (investigation of the influence of anions)

[0036] Under electromagnetic radiation, some inorganic substances may produce light absorption due to charge transfer transitions and coordination field transitions. Before realizing the short-wave colorless quantification of chitosan, it is necessary to clarify the effects of various ligands and other anion and cation coexistence conditions on the accurate quantification of chitosan.

[0037] Considering the range of chitosan absorbance, we believe that an absorbance error of 0.01 can be deducted by calibration, while an absorbance of 0.1 will affect the quantification of chitosan.

[0038] Aqueous solutions of sulfuric acid, nitric acid, hydrochloric acid, perchloric acid and acetic acid of different concentrations were prepared, ultrapure water was used as the reference solution, and the absorbance of each solution was measured at a wavelength of 195 nm to investigate the effect of the generation of acid radical ions (inorganic ligands). The results are shown in Tables 2 and 3 below:

[0039] Table 2: Concentration range of anion absorbance greater than 0.01

[0040]

[0041]

[0042] Table 3: Concentration range of anion absorbance greater than 0.1

[0043]

[0044] As shown in Tables 2 and 3, the light absorption coefficient of perchlorate is the smallest at a wavelength of 195 nm, while chitosan is insoluble in water, alkali and general organic solvents, but can be dissolved in dilute acid. In summary, the present invention selects 0.01 mol / L perchloric acid as the solvent.

[0045] 3) Investigation of the influence of cations

[0046] Aqueous solutions of KClO4, NaClO4, Ca(ClO4)2, and Mg(ClO4)2 of different concentrations were prepared, and ultrapure water was used as a reference solution. The absorbance of each solution was measured at a wavelength of 195 nm. The results are shown in Table 4 below:

[0047] Table 4: Concentration range of cation absorbance greater than 0.01 (minus 0.01M perchlorate absorbance)

[0048]

[0049] Conclusion: Under the condition of 195nm wavelength, common K, Na, Ca, Mg (perchlorate) cations have almost no interference to the method of the present invention.

[0050] 4) Investigation of the influence of pH

[0051] After testing, when the concentration of hydroxide ions is 0.00001mol / L (0.00001mol / L NaOH solution), it produces an absorbance of about 0.008 at 195nm; and the higher the concentration of hydroxide ions, the higher the absorbance (the reason is that hydroxide is a strong ligand, and under electromagnetic radiation, charge transfer transition occurs, resulting in light absorption). The absorbance of 0.01mol / L NaOH exceeds the detection limit of this method. In an acidic or neutral environment, there is almost no light absorption caused by hydrogen ions. Chitosan itself is protonated and soluble only below pH 6.3, so the influence of hydroxide can be ignored.

[0052] Example 2: Validation of analytical method

[0053] 1) Linearity test

[0054] Linear stock solution: Accurately weigh 0.1 g of chitosan of different molecular weights, add 10 mL of 1 M HClO4 aqueous solution to soak and dissolve, then add ultrapure water to 1 L, shake well, and use this as the linear stock solution.

[0055] Linear solution: Take an appropriate amount of linear stock solution and dilute it with 0.01M HClO4 aqueous solution to 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 mg / L respectively as linear solutions.

[0056] Ultrapure water was used as the reference solution, and its absorbance was adjusted to 0. The absorbance of each linear solution was detected at a wavelength of 195 nm and recorded as A. A standard curve was drawn with concentration as the horizontal axis and A as the vertical axis. Figure 4 , 5 , 6, the results are shown in Table 5 below (corresponding to Figure 4 )、6(corresponding Figure 5 )、7(corresponding to Figure 6 ):

[0057] Table 5: Linearity test results of low molecular weight chitosan

[0058]

[0059] Table 6: Linearity test results of medium molecular weight chitosan

[0060]

[0061]

[0062] Table 7: Linearity test results of high molecular weight chitosan

[0063]

[0064] 2) Repeatability test

[0065] Repeatability test solution: Prepare 10 mg / L low, medium and high molecular weight chitosan respectively (prepare the solution according to the linear test item of this embodiment). Take ultrapure water as the reference solution, adjust its absorbance value to 0, and measure the absorbance A of the above concentration samples at a wavelength of 195 nm. Substitute the absorbance A into the corresponding standard curve equation in 1) linear test to obtain the measured concentration. Recovery rate = (measured concentration * 100 / prepared concentration)%.

[0066] Table 8: Repeatability test results of chitosan (%)

[0067]

[0068] Conclusion: After repeating 5 times, the recovery rate was between 103.5% and 108.5%, and the RSD of the recovery rate was between 0.55 and 3.09. This method has good repeatability.

Claims

1. A non-colorimetric method for quantitative analysis of chitosan, characterized in that: The following steps are involved: (1) Drawing a standard curve: accurately weigh a chitosan reference substance, soak and dissolve it with an aqueous solution of HClO4 with a concentration of more than 1M, then add ultrapure water to a concentration of 0.01M HClO4, shake well, and use it as a linear stock solution; dilute the linear stock solution with a 0.01M HClO4 aqueous solution to different chitosan concentrations to use as linear solutions, wherein the concentration of chitosan is in the range of 0.1 to 50 mg / L; use ultrapure water as a reference solution, adjust the absorbance value measured by the reference solution to 0, and detect the absorbance of each linear solution at a wavelength of 190 to 195nm, which is recorded as A; use the concentration as the horizontal axis and A as the vertical axis to draw a standard curve; (2) Sample testing: Accurately weigh the sample to be tested, soak and dissolve it in a 1M or higher HClO4 aqueous solution, then add ultrapure water to a 0.01M HClO4 concentration, and shake well; Using ultrapure water as a reference solution, adjusting the absorbance value measured by the reference solution to 0, detecting the absorbance of the test solution at a wavelength of 190 to 195 nm, recorded as A; substituting A into the standard curve equation in step (1) to obtain the chitosan concentration, and then calculating the chitosan content in the test sample; The chitosan contained in the chitosan reference substance and the test substance is low molecular weight chitosan, the viscosity thereof is 20-300 cP, and the degree of deacetylation thereof is >75%; Alternatively, the chitosan contained in the chitosan reference substance and the test substance is a medium molecular weight chitosan, the viscosity of which is 200-800 cP, and the degree of deacetylation is greater than 75%; Alternatively, the chitosan contained in the chitosan reference substance and the test substance is high molecular weight chitosan, the viscosity of which is >400 cP, and the degree of deacetylation of which is >75%; The test sample is an adsorption system of an inorganic mineral adsorption material-chitosan solution, and the inorganic mineral adsorption material is one or more of montmorillonite, kaolinite, illite, and magnetite.

2. The non-colorimetric spectrophotometry method according to claim 1, characterized in that: The detection wavelength is 195 nm.

3. The non-colorimetric spectrophotometry method according to claim 1, characterized in that: The concentration of HClO4 used to dissolve the chitosan control and test samples was 1M.

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