A non-colorimetric spectrophotometric method for quantitative analysis of cysteine
By using colorless spectrophotometry and utilizing the linear relationship of absorbance of cysteine at a specific wavelength, the problems of complex operation and low accuracy in the existing technology are solved, and a simple and stable quantitative analysis of cysteine is realized.
Patent Information
- Application Number
- CN202310784138.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-28
- Publication Date
- 2025-01-14
- Estimated Expiration
- 2043-06-28
AI Technical Summary
Existing methods for quantifying cysteine rely on colorimetric reactions, which are easily affected by pH, temperature, and time. They are complex to operate and have low accuracy. There is a lack of quantitative analysis methods that are widely applicable, simple to operate, and accurate.
A colorless spectrophotometric method was adopted, which utilizes the linear relationship between the absorbance and concentration of cysteine at wavelengths of 190–195 nm. Quantitative analysis of cysteine was achieved by plotting a standard curve and detecting the sample, thus avoiding interference factors in the color development process.
It simplifies operation, reduces instrument and equipment requirements, minimizes interference factors, and ensures the stability and reproducibility of quantification. It is suitable for the accurate quantification of cysteine in systems without other organic substances or inorganic ligands.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of small molecule detection, and in particular to a non-colorimetric spectrophotometric method for quantitatively analyzing cysteine. Background Art
[0002] Cysteine is a physiologically active amino acid with an amino group and a thiol group. Among proteinogenic amino acids, cysteine may be the only amino acid that contains a thiol group on the side chain and has a cellular detoxification binding site. Cysteine can be used to synthesize MoS2 / graphene composites with excellent electrochemical properties, electrochemical sensors for heavy metal ions, and the removal of divalent heavy metal ions in non-woven geotextiles. In recent years, it has attracted extensive attention from researchers in the fields of medicine, food, chemical industry, and environmental protection.
[0003] In the field of the interaction between cysteine and the solid phase and the complex of cysteine, the quantitative study of cysteine is very necessary for in-depth exploration of the mechanism of related research, and its accurate quantitative method is worth exploring.
[0004] The current quantitative methods for cysteine are mainly based on the determination of its amino concentration, which are divided into colorimetric detection, 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 a mixed heating color development step, and the color development result is easily affected by pH, temperature, and color development time, resulting in the condensation reaction of cysteine and ninhydrin, uneven heating, and photolysis over time. Based on this, it is very necessary to develop a non-color development quantitative method for cysteine that is highly applicable, simple to operate, and highly accurate.
[0005] Table 1: Advantages and disadvantages of existing amino group quantitative methods
[0006] Summary of the invention
[0007] In order to solve the above technical problems, the present invention provides a non-colorimetric method for quantitatively analyzing cysteine. The present invention utilizes that the absorbance value of a cysteine solution at a wavelength of 190 to 195 nm increases with the increase of the cysteine content within a certain concentration range, and presents a certain linear relationship. Based on this as the quantitative basis of cysteine, a non-colorimetric method for analyzing cysteine is established.
[0008] A non-colorimetric method for quantitatively analyzing cysteine comprises the following steps:
[0009] (1) Drawing a standard curve: Accurately weigh different masses of cysteine reference substances, dissolve them in ultrapure water and dilute them to different concentrations (in the range of 0.1 to 50 mg / L) as linear solutions; use ultrapure water as the reference solution, and detect the absorbance A of each linear solution at a wavelength of 190 to 195 nm; draw a standard curve with concentration as the horizontal axis and A as the vertical axis.
[0010] (2) Sample detection: Accurately weigh a certain amount of the test sample, dissolve it in ultrapure water and make up to volume to prepare the test sample solution; use ultrapure water as the reference solution, and detect the absorbance A of the test sample solution at a wavelength of 190 to 195 nm; substitute the standard curve equation in step (1) to obtain the concentration of cysteine in mg / L, and calculate the content of cysteine in the test sample according to the formula: content (%) = concentration of cysteine / concentration of test sample solution*100%.
[0011] No other organic substances or inorganic ligands are present in the test sample of the method of the present invention.
[0012] Preferably, the sample is an adsorption system of an inorganic mineral adsorption material-cysteine solution, wherein the inorganic mineral adsorption material includes at least one of montmorillonite, kaolinite, illite, magnetite, etc.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] 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 the quantitative analysis of cysteine in a controllable system (or pure system). BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1a , Figure 1b The scanning spectrum of cysteine aqueous solution with different concentrations in the range of 190-500nm, where Figure 1b This is an enlarged view of the scanning spectrum at 190-250 nm;
[0016] Figure 2 is the linear relationship diagram of cysteine. DETAILED DESCRIPTION
[0017] 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.
[0018] The experimental methods used in the following examples are all conventional methods unless otherwise specified; the water used is ultrapure water (resistivity>18.25MΩ); the materials, reagents, etc. used are all commercially available unless otherwise specified.
[0019] The equipment used in the following examples is a double-beam spectrophotometer (Beijing Puxi TU-1901, wavelength range 190-700nm), and the L-cysteine used is from Sinopharm Group, Shanghai Trial BR, product number 62007234, batch number 20201215.
[0020] Example 1: Analysis method establishment
[0021] 1) Wavelength selection
[0022] Unsaturated functional groups of organic matter will produce characteristic absorption bands in the wavelength range of 190nm-1000nm, such as C=C, C=O, N=N, etc., which are called chromophores. Some groups containing n electrons, such as -OH, -NH2, etc., do not have color-producing functions themselves (cannot absorb light > 190nm), but when they are connected to chromophores, n-π conjugation will occur, enhancing the color-producing ability of the chromophore (the absorption wavelength moves to the long-wave direction, and the absorption intensity increases). This provides a basic basis for the non-colorimetric quantification of cysteine.
[0023] First, use ultrapure water to scan the spectrum to test the stability of 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 spectrum of cysteine aqueous solutions of different concentrations in the range of 190-500nm. Figure 1a .
[0024] Preparation process of cysteine aqueous solution: weigh 0.1g of cysteine stored in the dark, dissolve it in 100mL ultrapure water to obtain 1000mg / L cysteine, and then dilute it with ultrapure water to obtain the required concentration.
[0025] Depend on Figure 1b It can be seen that the absorption peaks of cysteine at different concentrations are between 190-195 nm, and 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.
[0026] 2) Investigation of the influence of anions
[0027] 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 cysteine, it is necessary to clarify the effects of various ligands and other anion and cation coexistence conditions on the accurate quantification of cysteine.
[0028] Considering the range of cysteine absorbance, we believe that an absorbance error of 0.01 can be deducted by calibration, while an absorbance error of 0.1 will affect the quantification of cysteine.
[0029] 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. The results are shown in Tables 2 and 3 below:
[0030] Table 2: Concentration range of anion absorbance greater than 0.01
[0031]
[0032] Table 3: Concentration range of anion absorbance greater than 0.1
[0033]
[0034] In summary, common anions (ligands) in this method, such as sulfate, acetate, nitrate, chloride and perchlorate, can produce absorbances greater than 0.01 at concentrations of about 10, 2, 10, 10, and 100 μmol / L, respectively. This indicates that anionic ligands (except perchlorate) interfere with the quantification of cysteine at 195 nm. When using this assay method, high concentrations of common coordination ions such as sulfate, nitrate, chloride, and acetate should be avoided in the test solution.
[0035] 3) Investigation of the influence of cations
[0036] 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:
[0037] Table 4: Concentration range of cation absorbance greater than 0.01 (minus 0.01M perchlorate absorbance)
[0038]
[0039] 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.
[0040] 4) Investigation of the influence of pH
[0041] 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. The aqueous solution of cysteine is acidic, and the influence of hydroxide can be ignored.
[0042] Example 2: Validation of analytical method
[0043] 1) Linearity test
[0044] Linear stock solution: Accurately weigh 0.1 g of cysteine, dissolve it in ultrapure water and dilute it to 1 L, shake well, and use it as the linear stock solution.
[0045] Linear solution: Take an appropriate amount of linear stock solution and dilute it with ultrapure water to 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 mg / L respectively as linear solutions.
[0046] Using ultrapure water as the reference solution, the absorbance A of each linear solution was detected at a wavelength of 195 nm. A standard curve was drawn with concentration as the horizontal axis and A as the vertical axis. Figure 2 The results are shown in Table 5 below:
[0047] Table 5: Linearity test results for cysteine
[0048]
[0049]
[0050] 2) Repeatability test
[0051] Repeatability test solution: Prepare 10 mg / L cysteine. Use ultrapure water as the reference solution and measure the absorbance A of the sample with the above concentration at a wavelength of 195 nm. Substitute A into the standard curve equation in the linear test in 1) to obtain the measured concentration. Recovery rate = (measured concentration * 100 / prepared concentration)%.
[0052] Table 6: Cysteine repeatability test results (%)
[0053]
[0054] Conclusion: After repeated 5 times, the recovery rate was between 104.2% and 107.9%, and the RSD of the recovery rate was 1.27%. The repeatability of this method was good.
Claims
1. A non-colorimetric method for quantitative analysis of cysteine, characterized in that: The following steps are involved: (1) Drawing a standard curve: Accurately weigh different masses of cysteine reference substances, dissolve them in ultrapure water and dilute them to different concentrations as linear solutions with a concentration range of 0.1 to 50 mg / L; use ultrapure water as the reference solution and detect the absorbance A of each linear solution at a wavelength of 195 nm; draw a standard curve with concentration as the horizontal axis and A as the vertical axis; (2) Sample detection: Accurately weigh a certain amount of the test sample, dissolve it in ultrapure water and make up to volume to prepare the test sample solution; use ultrapure water as the reference solution and detect the absorbance A of the test sample solution at a wavelength of 195 nm; substitute the standard curve equation in step (1) to obtain the concentration of cysteine in mg / L, and calculate the content of cysteine in the test sample according to the formula: content = concentration of cysteine / concentration of test sample solution*100%; The test sample is an adsorption system of an inorganic mineral adsorption material-cysteine solution, and the inorganic mineral adsorption material is one or more of montmorillonite, kaolinite, illite, and magnetite.