A method for rapid determination of humic acid content based on TMB colorimetric method

Abstract

This invention discloses a rapid detection method for humic acid, aiming to provide a simple, sensitive, and rapid detection method for quantifying HA by utilizing the oxidizing properties of humic acid combined with the TMB reaction colorimetric principle. The method involves using the TMB method to detect a standard humic acid solution of known concentration. Under pH 4 conditions, the absorbance value at a wavelength of 652 nm is measured, and a regression equation is established between the absorbance value and the humic acid concentration. After processing the sample to be tested, the absorbance value at a wavelength of 652 nm is measured using the TMB method, and the result is substituted into the regression equation to calculate the concentration of humic acid in the sample. This method belongs to the field of detection technology.

Description

Technical Field

[0001] This invention relates to methods for detecting humic acid content, specifically to a method for rapid detection of humic acid content based on the TMB colorimetric method, belonging to the field of detection technology. Background Technology

[0002] Humic acid (HA) is a major component of humic substances (HS). Humic acid is a large-molecule organic substance widely found in nature and widely used in forestry, animal husbandry, agriculture, petroleum, chemical industry, building materials, medicine, and environmental protection. However, the presence of HA in wastewater also poses challenges to wastewater treatment. The world is currently experiencing a massive population growth trend and profound economic and social changes, leading to a surge in demand for wastewater treatment and purified water, but a significant imbalance exists between supply and demand. Natural organic matter, including humic acid and humic substances, is one of the main pollutants in wastewater and is widely present in various water sources. High concentrations of humic acid and other organic matter can harm aquatic organisms, and if ingested by humans through the food chain or indirectly or directly, it can cause significant health problems.

[0003] Due to the complexity of humic acid's definition and structure, there is currently no absolutely precise analytical method to determine its content. Various analytical instruments and methods exist for quantifying humic acid, including electrochemical methods, chromatography, oxygen consumption measurement, flow injection chemiluminescence, ultraviolet (UV) and fluorescence methods. These methods can help determine the content of humic acid in water samples, but they require specific instruments, are cumbersome to operate, and lack versatility. Among UV methods for HA detection, the modified Lowry method is the standard. The modified Lowry method has a wide detection range, accurate and reliable results, and good reproducibility, and is often used as the standard method for HA detection in water to evaluate the detection effectiveness of other methods. Another UV absorption method for HA quantification is the toluidine blue (TB) binding method. This method is rapid and simple, and proteins do not interfere with it, but the presence of ions can interfere with the method, requiring the addition of masking agents to eliminate interference, and its detection range is relatively small. Therefore, it is necessary to develop a new, simple, rapid, and sensitive quantitative analysis method for HA.

[0004] TMB is the most commonly used chromogenic substrate to date. Its colorimetric reaction is based on the catalytic oxidation of TMB by an oxidizing agent under the condition of H2O2 as a catalyst, producing a pale blue to blue oxidized TMB with an absorption peak in the ultraviolet-visible light at a specific wavelength (652nm). Humic acid is a high molecular weight electrolyte containing oxygen-containing functional groups such as hydroxyl, carboxyl, phenolic, and carbonyl groups. It can be used as an oxidizing agent to reduce and colorimetric TMB under the action of the catalyst H2O2. By measuring the absorbance value of TMB at a specific wavelength (652nm), HA can be quickly and easily quantified. Summary of the Invention

[0005] To address the shortcomings of existing HA detection methods, such as complex processes, long processing times, and limited application scope, the purpose of this application is to design a simple, sensitive, and rapid detection method for quantifying HA by utilizing the oxidizing properties of humic acid combined with the TMB reaction colorimetric principle.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A rapid method for detecting humic acid is proposed. This method utilizes the TMB method to detect standard humic acid solutions of known concentrations. Under pH 4 conditions, the absorbance value at a wavelength of 652 nm is measured, and a regression equation is established between the absorbance value and the humic acid concentration. After processing the sample to be tested, the absorbance value at a wavelength of 652 nm is measured using the TMB method and then substituted into the regression equation to calculate the concentration of humic acid in the sample.

[0008] Furthermore, the above-mentioned rapid detection method for humic acid specifically includes the following steps:

[0009] (I) Establishing the regression equation

[0010] 1) Prepare a 1 g / L humic acid standard sample;

[0011] 2) Using an acetic acid-sodium acetate solution with pH=4 as the background solution, humic acid solutions of different concentrations were prepared.

[0012] 3) Take 1 mL of humic acid solution of different concentrations into test tubes, and add TMB solution and H2O2 solution to the test tubes respectively. React for 17 min to obtain colorimetric solution. Take 1 mL of colorimetric solution and place it in a micro-volume cuvette. Place it in a UV-Vis-NIR spectrophotometer and measure the absorbance value of each concentration of humic acid solution at a wavelength of 652 nm using the UV-Vis-NIR spectrophotometer.

[0013] 4) Linearly fit the concentrations of humic acid solutions at various concentrations at a wavelength of 652 nm with the corresponding absorbance values ​​to establish a regression equation;

[0014] (ii) Calculate the concentration of humic acid in the sample to be tested.

[0015] 1) Different water samples were filtered to prepare the test samples;

[0016] 2) Take 1 mL of the sample to be tested, add 500 μL of alkaline aqueous solution, mix for 10 min, shake and mix well to ensure complete hydrolysis, and adjust the pH to 4 with glacial acetic acid solution after hydrolysis.

[0017] 3) Take 1 mL of the solution prepared in step 2) into a 1.5 mL test tube, add TMB solution and H2O2 to the tube respectively, react for 17 min, take 1 mL of the colorimetric solution and place it in a micro-volume cuvette, wipe both sides with a clean paper towel and place it in a UV-Vis-NIR spectrophotometer, and measure the absorbance value of the humic acid solution of each sample at a wavelength of 652 nm using a UV spectrophotometer.

[0018] 4) Substitute the absorbance value obtained in step 3) into the regression equation in step (I) above to calculate the concentration of humic acid in the sample to be tested.

[0019] Furthermore, in the aforementioned rapid detection method for humic acid, the regression equation is: Y = 0.01806X + 0.08669(R0). 2 =0.99312).

[0020] Furthermore, in the above-mentioned rapid detection method for humic acid, the concentration of the humic acid solution is: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 mg / L.

[0021] Furthermore, in the above-described rapid detection method for humic acid, the TMB solution concentration is 15 mM.

[0022] Furthermore, in the above-mentioned rapid detection method for humic acid, the H2O2 solution is 30% H2O2.

[0023] Furthermore, in the aforementioned rapid detection method for humic acid, the filtration process involves first using filter paper with a pore size of 15 mm for coarse filtration, and then passing the filtered sample through a 0.22 μm filter membrane to prepare the sample to be tested.

[0024] Furthermore, in the above-mentioned rapid detection method for humic acid, the alkaline aqueous solution is prepared as follows: First, weigh 0.4g of NaOH, measure 100mL of deionized water into a 150mL beaker using a graduated cylinder, add the weighed NaOH to obtain 0.1M NaOH, then add 2g of Na2CO3 (or 5.39g of Na2CO3·10H2O), stir and mix evenly with a glass rod to obtain the desired alkaline aqueous solution.

[0025] Furthermore, in the above-mentioned rapid detection method for humic acid, the preparation method of the humic acid standard sample is as follows: Weigh 0.8g of NaOH and dissolve it in 100mL of deionized water, stir evenly with a glass rod, then dissolve 0.1g of humic acid in the above solution, place it on an ultrasonic instrument, sonicate for 15min, and finally dilute to 100mL in a volumetric flask to obtain a 1g / L humic acid standard sample, and store it in a refrigerator at 4℃ for use.

[0026] Furthermore, in the above-mentioned rapid detection method for humic acid, the preparation method of the 15mM TMB solution is as follows: Weigh 84.124mg of 3,3′,5,5′-tetramethylbenzidine, dissolve it in a certain amount of anhydrous ethanol, stir it evenly with a glass rod in a 100mL beaker, place it on an ultrasonic instrument, sonicate for 15min, and use a dropper to make up the TMB solution to 50mL to obtain the 15mM TMB colorimetric solution, and store it in a refrigerator at 4℃.

[0027] One of the above-described technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0028] (1) The detection method provided by this invention has high sensitivity and good linearity. Compared with other ultraviolet absorption methods, it has a wider detection range, with an effective detection range of up to 50 mg / L and a detection limit of 0.98 mg / L. Within the detection range, the standard curve exhibits good linearity (R0). 2 >0.99).

[0029] (2) Compared with the standard method (modified Lowry method), the TMB method provided by this invention is simpler and does not require special materials and instruments. The HA concentrations measured by these two methods are almost identical. In terms of sensitivity and detection range, this work provides a new and effective method for HA quantification.

[0030] (3) The detection method provided by the present invention utilizes alkaline solution hydrolysis and membrane pretreatment to remove the interference of proteins and polysaccharides on the colorimetric results, and the interference of ions on HA measurement can be ignored. Attached Figure Description

[0031] Figure 1 This is a graph showing the effect of optimized reaction conditions of the TMB colorimetric method on the color development results in this invention, where (a) pH, the inset shows the color of the colorimetric solution at the corresponding pH, (b) TMB concentration, and (c) reaction time.

[0032] Figure 2 These are experimental figures investigating the stability of the TMB colorimetric method in this invention, where (a) BSA, (b) SA, (c) investigation of the maximum allowable concentration of BSA interference, and (d) investigation of the maximum allowable concentration of SA interference.

[0033] Figure 3 These are the results of removing protein interference in this invention, including (a) the results of removing protein using alkaline solution hydrolysis, and (b) the effect of the mixture of SA, BSA and HA on the color development results after alkaline solution hydrolysis.

[0034] Figure 4The results of establishing the detection model in this invention are shown in the figure, where (a) the ultraviolet spectrum at 500-800nm ​​after color development with different HA concentrations, (b) the linear regression equation of absorbance value and HA concentration (N=3), and (c) the corresponding colors of HA color development solution with different concentrations under a stable light source.

[0035] Figure 5 These are two standard curves (N=3) obtained from the improved Lowry method in this invention, where (a) is standard curve 1 and (b) is standard curve 2. Detailed Implementation

[0036] The present invention will be described in more detail below with reference to the embodiments, but the scope of protection of the present invention is not limited to the scope shown in the embodiments.

[0037] Example 1

[0038] This embodiment provides a method for rapid detection of humic acid content based on the TMB colorimetric method, which specifically includes the following steps:

[0039] (1) Establishing the regression equation

[0040] Step 1: Weigh 0.8g of NaOH and dissolve it in 100mL of deionized water. Stir well with a glass rod. Then dissolve 0.1g of humic acid (HA) in the above solution and stir well with a glass rod. Place the solution on an ultrasonic instrument and sonicate for 15min. Finally, dilute to 100mL in a volumetric flask to obtain a 1g / L humic acid standard sample. Store the sample in a refrigerator at 4℃ for use.

[0041] Step 2: Using an acetic acid-sodium acetate solution with pH=4 as the background solution, prepare a certain amount of humic acid solution with known concentrations of each component. The concentrations of the humic acid solution are: 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / L.

[0042] Step 3: Take 1 mL of humic acid solution of different concentrations into 1.5 mL tubes, add 100 μL of 15 mM TMB solution and 10 μL of 30% H2O2 to the tubes respectively, react for 17 min, take 1 mL of the colorimetric solution and place it in a micro-volume cuvette, wipe both sides with a clean paper towel and then place it in a UV-Vis-NIR spectrophotometer.

[0043] Step 4: Measure the absorbance of each concentration of humic acid solution at a wavelength of 652 nm using a UV spectrophotometer. Three parallel samples are set up for each measurement, and the average value is taken to obtain the absorbance value of each concentration of humic acid solution at a wavelength of 652 nm.

[0044] Step 5: Linearly fit the concentrations of the humic acid solution at a wavelength of 652 nm with the corresponding absorbance values ​​to establish a regression equation;

[0045] (2) Calculate the concentration of humic acid in the sample to be tested.

[0046] Step 1: Perform coarse filtration on different water samples (filter paper pore size 15mm), and then pass the filtered samples through a 0.22um filter membrane to prepare the test samples;

[0047] Step 2: Take 1 mL of the sample to be tested, add 500 μL of alkaline aqueous solution, mix for 10 min, shake and mix well to ensure complete hydrolysis, and then adjust the pH to 4 with glacial acetic acid solution.

[0048] Step 3: Take 1 mL of the solution prepared in step 2 into a 1.5 mL tube, add 100 μL of 15 mM TMB solution and 10 μL of 30% H2O2 to the tube respectively, react for 17 min, take 1 mL of the colorimetric solution and place it in a micro-volume cuvette, wipe both sides with a clean paper towel and then place it in a UV-Vis-NIR spectrophotometer to ensure that the absorbance value of the sample to be tested is within the range of the regression equation in step (1) above. If it is not, dilute the solution and measure it until the absorbance obtained is within the range of the regression equation in step (1).

[0049] Step 4: Measure the absorbance of the humic acid solution of each sample at a wavelength of 652 nm using a UV spectrophotometer. Each measurement is performed in triplicate, and the average value of each sample is taken to obtain the absorbance value of the sample at a wavelength of 652 nm.

[0050] Step 5: Substitute the measured absorbance value into the regression equation for the corresponding wavelength obtained in step (1) above to calculate the concentration of humic acid in the sample to be tested.

[0051] I. To better understand the technical solution provided in this application, the research method for the technical solution provided in this application is given below:

[0052] 1. Research on pH value:

[0053] Replace the pH value of the background solution in step (1) of Example 1 with pH = 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, and the other steps and parameters are the same as in Example 1.

[0054] The results are attached. Figure 1 (a) Different pH conditions have a significant impact on the TMB colorimetric results. See Appendix. Figure 1(a) At a background pH of 2.0, the TMB chromogenic solution changed from colorless to yellow; at pH 3.0 and 4.0, the TMB chromogenic solution changed from colorless to blue, with the blue color being more obvious and easier to observe at pH 4.0; at pH 5.0, 6.0, 7.0, and 8.0, the TMB chromogenic solution remained colorless. Therefore, the color of the reaction was most obvious and easier to observe at pH 4.0, with the maximum absorbance corresponding to a wavelength of 652 nm. The relative activity at different pH values ​​at 652 nm was measured. As the pH changed from 2.0 to 4.0, the relative activity of the blue chromogenic product gradually increased, reaching its maximum at pH 4.0. After pH 4.0, the relative activity dropped sharply, and after pH 6.0, the relative activity leveled off and stabilized at around 0.1.

[0055] 2. Research on color development time

[0056] Replace the reaction time in step (1) of Example 1 with 0-30 min, and keep the other steps and parameters the same as in Example 1.

[0057] The results are attached. Figure 1 (b) The absorbance values ​​and trends of the TMB colorimetric solution at 652 nm within 0–30 min under a colorimetric background of pH 4.0, 10 mg / L humic acid, 15 mM TMB, and 10 μL 30% H2O2. The absorbance value started to increase from 0 min, with a significant increase in the first 10 min, jumping from approximately 0.3 to 0.9. The rate of increase then slowed, reaching its maximum at approximately 17 min. Therefore, the optimal colorimetric development time is 17 min.

[0058] 3. Research on TMB concentration

[0059] Replace the TMB solution concentration in the third step of step (1) in Example 1 with 1, 5, 10, 15, 20, 25 mM, and keep the other steps and parameters the same as in Example 1.

[0060] The results are attached. Figure 1(c) The absorbance values ​​and trends of 1, 5, 10, 15, 20, and 25 mM TMB at 652 nm under a color development background of pH 4.0, 10 mg / L humic acid, 10 μL 30% H₂O₂, and a reaction time of 17 min. The absorbance values ​​gradually increased as the TMB concentration increased from 1 mM to 15 mM, reaching a maximum at 15 mM. After 15 mM, the absorbance values ​​began to decrease. Overall, the absorbance of the reaction system reached its maximum at a TMB concentration of 15 mM under the background conditions. Therefore, this study found the optimal color development result to be a TMB concentration of 15 mM.

[0061] II. To demonstrate the reliability of the technical solution provided in this application, the following is an experimental study on the stability of the TMB method of the technical solution provided in this application.

[0062] 1. Since proteins and polysaccharides are two widely present organic compounds in dissolved organic matter (DOM), the detection of HA was explored. Therefore, BSA and SA were selected as representative proteins and polysaccharides, respectively, in this study. Bovine serum albumin (BSA) solutions of 2, 4, 6, 8, 10, 12, 14, 16, 20, and 50 mg were weighed out, and 100 mL of deionized water was added to each beaker using a graduated cylinder. The solutions were stirred thoroughly with a glass rod to obtain concentrations of 20, 40, 60, 80, 100, 120, 140, 160, 200, and 500 mg / L. Sodium alginate (SA) solutions of 2, 4, 6, 8, 10, 12, 14, 16, 20, and 50 mg were weighed out, and 100 mL of deionized water was added to each beaker using a graduated cylinder. The solutions were stirred thoroughly with a glass rod to obtain concentrations of 20, 40, 60, 80, 10, 12, 14, 16, 20, and 50 mg were weighed out, and 100 mL of deionized water was added to each beaker using a graduated cylinder. The solutions were stirred thoroughly with a glass rod to obtain concentrations of 20, 40, 60, 8 ...0, 10, 12, 14, 16, 20, and 50 mg were weighed out, and 100 mL of deionized water was added to each beaker. SA solutions of 20, 40, 60, 80, 100, 120, 140, 160, 200, and 500 mg / L were prepared.

[0063] The second step of step (1) in Example 1 is modified as follows: Take the prepared concentrations of 20, 40, 60, 80, 100, 120, 140, 160, 200, and 500 mg / L respectively. Add 500 μL of SA solution and 20, 40, 60, 80, 100, 120, 140, 160, 200, and 500 mg / L BSA solution to 1.5 mL tubes. Then add 500 μL of pre-diluted 20 mg / L humic acid to each tube. Mix and shake, place on a vortex mixer, and rotate and shake for at least 3 seconds to obtain 10 mg / L humic acid solutions containing SA concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 100, and 250 mg / L and BSA concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 100, and 250 mg / L. Other steps and parameters are the same as in Example 1.

[0064] The results are attached. Figure 2 In (a) and (b), the absorbance of the 10 mg / L HA solution did not decrease significantly after the addition of SA, but the absorbance of the 10 mg / L HA solution decreased significantly after the addition of BSA.

[0065] To comprehensively evaluate the interference of polysaccharides and proteins on the detection system, an additional polysaccharide interference experiment was conducted with a background concentration of 20 mg / L. Since 20 mg / L is roughly near the upper limit of detection, and the concentration of HA in water generally fluctuates around 20 mg / L, examining the selectivity of the detection model for polysaccharides at this concentration has significant practical value and theoretical basis. (Appendix) Figure 2 As shown in (c), further reducing the SA concentration revealed that when the SA concentration was 60 mg / L, its absorbance value was almost identical to that under standard conditions. This indicates that when the SA concentration in water is below 60 mg / L, the effect of its presence can be disregarded when using this method. (Appendix) Figure 2 As shown in (d), the absorbance value gradually increases as the BSA concentration decreases. When the BSA concentration is 1.25 mg / L, its absorbance value is basically equal to that of the standard HA solution under the same measurement conditions. This means that even when the BSA concentration is below 1.25 mg / L, its interference with the determination of TMB chromogenic substances is negligible. The concentration of DOM in natural water is generally 0–10 mg / L, while the concentration of DOM in wastewater is generally greater than this range. To ensure high accuracy and sensitivity of the detection results, the application of this method in actual water samples needs to consider the influence of proteinaceous substances and use effective methods to eliminate their influence.

[0066] 3. To remove protein interference by hydrolysis with alkaline aqueous solution, the second step of step (1) in Example 1 is modified as follows: Take 500 μL of the prepared 20 mg / L BSA solution into 1.5 mL tubes, and then add 500 μL of pre-diluted 20 mg / L humic acid to each tube. Mix and shake, place on a vortex mixer, and rotate and shake for at least 3 seconds to obtain a 10 mg / L humic acid solution containing 10 mg / L BSA. Weigh 0.4 g of NaOH, measure 100 mL of deionized water into a 150 mL beaker using a graduated cylinder, add the weighed NaOH to obtain 0.1 M NaOH, and then add 2 g of Na2CO3 (or 5.39 g of Na2CO3·10H2O). Stir and mix evenly with a glass rod to obtain an alkaline aqueous reagent. Add 500 μL of alkaline aqueous solution to the prepared 10 mg / L humic acid solution containing 10 mg / L BSA and mix for 10 min. Shake and mix thoroughly to ensure complete hydrolysis. After hydrolysis, adjust the pH to approximately 4 with glacial acetic acid solution. Other steps and parameters are the same as in Example 1.

[0067] The results are attached. Figure 3As shown in (a), protein interference was removed by hydrolysis with alkaline aqueous solution. After hydrolysis, the absorbance value of the TMB chromogenic product was basically similar to that of 10 mg / L HA. Therefore, alkaline hydrolysis can remove protein interference to the TMB chromogenic solution in most cases, and the detection results are basically similar to those under no interference background. This method can be used to remove protein interference. (Appendix) Figure 3 As shown in (b), after removing BSA using the alkaline solution hydrolysis method, the effect of the mixture is negligible. This further verifies the stability of the detection method.

[0068] 4. Selective Inquiry

[0069] Considering the various interfering ions present in water, the ionic environment in wastewater was simulated, and the absorbance values ​​under the presence of each ion were measured. The detection results of this method were investigated under the most complex environment. 265.08 mg NaHCO3, 37.36 mg KOH, 132.34 mg KOH, 129.6 mg MgCl2, and 146.9 mg Ca(OH)2 were weighed out, and 32.3 μL HF, 1.79 μL H3PO4, 275.95 μL HCl, 58 μL H2SO4, and 6.54 μL HNO3 were dissolved or mixed in 10 mL of deionized water. The solutions were stirred thoroughly with a glass rod, transferred to 10 mL tubes, and vortexed for at least 3 seconds. The solutions containing Mg were then obtained. 2+ Na + Ca 2+ K + HCO3 - Cl - F - NO3 - SO4 2- PO4 3- Interference solution of ions. The second step of step (1) in Example 1 is modified as follows: Take 10 μL of the prepared solution containing different ions into a 1.5 mL tube, and then add 990 μL of pre-diluted 20 mg / L humic acid to each tube. Mix and shake, place on a vortex mixer, and rotate and shake for more than 3 seconds to obtain a humic acid solution containing different interfering ions at 20 mg / L. Other steps and parameters are the same as in Example 1.

[0070] As shown in Appendix Table 1, SO4 was studied. 2- NO3 - Cl - PO4 3- HCO3 - F - Mg 2+ K + Na + Ca2+ Interfering matrix ions were used in the determination of 20 mg / L HA. SO4 2- NO3 - Cl - PO4 3- HCO3 - The method proposed in this work showed almost no interference with HA determination when anions were present at the reference concentration in wastewater. Mg 2+ K + Na + The interference from metal cations at a concentration of 10 mg / L is negligible and can be ignored, especially for Na. + The interference from ions at a concentration of 38.050 mg / L is less than 5% (interference rate is 1.60%). Therefore, the interference of ions on HA measurement is negligible.

[0071] Table 1. Effects of interfering substances on the determination of 20 mg / L HA

[0072]

[0073] III. Establishment and Feasibility Verification Experiment of the Detection Model

[0074] Following the experimental method in Example 1, a calibration model was established between absorbance values ​​and HA concentrations for determining the concentration of HA in unknown samples. The TMB method was used to perform three parallel measurements of HA at different concentrations (0–50 mg / L). (See attached image.) Figure 4 As shown in (a), the absorbance of the chromogenic solution at 652 nm increases with increasing HA concentration. (See attached image.) Figure 4 As shown in (c), the higher the HA concentration, the greater the color change, and the deeper the blue color of the developing solution becomes. (See attached image.) Figure 4 As shown in (b), a wide detection range was achieved within the range of 0–50 mg / L HA, and a good linear relationship was observed between HA concentration and absorbance value. The fitted curve R 2 =0.99312, with a curve slope of 0.01806. Using the detection limit calculation formula, the lowest detection limit is found to be 0.98 mg / L. When the sample HA concentration is within the linear range of 0–50 mg / L, the detection results are highly accurate. The absorbance value at 652 nm wavelength can be obtained using this method, and the HA concentration in the sample can be quickly calculated by combining it with the fitted regression equation. These results indicate that this method has the potential for practical application.

[0075] This embodiment demonstrates the feasibility of the improved Lowry method by using known standard humic acids of different concentrations.

[0076] The improved Lowry method is as follows:

[0077] Take 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of 100 mg / L humic acid into 10 mL centrifuge tubes, respectively, and add deionized water to a final volume of 1 mL. Add 4 mL of reagent C to each tube and mix. Vortex for 20 s, let stand for 10 min, then add 0.5 mL of Folin-Ciocalteu reagent, mix quickly, vortex for 20 s, let stand for 10 min, and measure the absorbance at 750 nm to obtain standard curve 1. The correlation coefficient of the regression equation for standard curve 1 should be greater than 0.99.

[0078] Transfer 1 mL of water sample to a 10 mL centrifuge tube. Add 4 mL of reagent C, mix, and vortex for 20 s. Let stand for 10 min, then add 0.5 mL of Folin-Ciocalteu reagent, mix quickly, vortex for 20 s, and let stand for 10 min. Measure the absorbance of the sample at 750 nm. Repeat the experiment three times and obtain the average value. Substitute the measured absorbance values ​​of the water sample into the regression equation of standard curve 1, as shown in the reference. Figure 5 (a) The concentration A total is calculated.

[0079] Take 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of 100 mg / L humic acid into 10 mL centrifuge tubes, respectively, and add deionized water to a final volume of 1 mL. Add 4 mL of reagent A to each tube and mix. Vortex for 20 s, let stand for 10 min, then add 0.5 mL of Folin-Ciocalteu reagent, mix quickly, vortex for 20 s, let stand for 10 min, and measure the absorbance at 735 nm to obtain the standard curve. Refer to the regression equation for the obtained standard curve 2. Figure 5 (b) The correlation coefficient should be greater than 0.99.

[0080] Take 1 mL of water sample and transfer it to a 10 mL centrifuge tube. Add 4 mL of reagent A and mix. Vortex for 20 s, let stand for 10 min, then add 0.5 mL of Folin-Ciocalteu reagent, mix quickly, vortex for 20 s, let stand for 10 min, and then measure the absorbance of the sample at 735 nm. Repeat the experiment three times and obtain the average value. Substitute the measured absorbance values ​​of the water sample into the regression equation of standard curve 2 to calculate the concentration Ablind.

[0081] Following the experimental method of Example 1, stability tests were conducted on standard humic acids of different known concentrations, compared to the modified Lowry method. (Appendix) Figure 5As shown in (a) and (b), two standard curves (standard curve 1 and standard curve 2) were obtained using the modified Lowry method. Simultaneously, the absorbance values ​​(Ablind and A total) of the samples under the corresponding standard curves were measured. Finally, the HA concentration of each sample was calculated using the formula C(HA) = (A blind - 0.2A total) / 0.8. Table 2 shows that the HA concentrations measured by the two methods are basically at the same level. This indicates that the proposed method is suitable for the quantification of HA in natural water samples.

[0082] Table 2 Comparison of results between TMB colorimetric method and Lowry method

[0083]

[0084] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for rapid detection of humic acid, characterized by, The method specifically comprises the following steps: (I) establishing a regression equation 1) configure 1 g / L of humic acid standard sample; 2) use acetic acid-sodium acetate solution with pH=4 as a background solution to prepare humic acid solutions with different concentrations; 3) take 1 mL of humic acid solution with different concentrations in a test tube, respectively, and add TMB solution and H2O2 solution in the test tube, react for 17 min to obtain a color developing solution, take 1 mL of the color developing solution into a microcolorimetric cell, and place it into an ultraviolet-visible-near infrared spectrophotometer to measure the absorbance value of each concentration of humic acid solution at 652 nm wavelength by the ultraviolet-visible-near infrared spectrophotometer; 4) linearly fit the concentration of each concentration of humic acid solution at 652 nm wavelength with the corresponding absorbance value to establish a regression equation; (II) calculating the concentration of humic acid in the sample to be measured 1) filter different water samples to prepare samples to be measured; 2) take 1 mL of the sample to be measured, add 500 μL of alkaline aqueous solution, mix for 10 min, shake and mix well to fully hydrolyze, and then adjust the pH to 4 with glacial acetic acid solution after hydrolysis; 3) take 1 mL of the solution treated in step 2) into a 1.5 mL test tube, add TMB solution and H2O2 in the tube, react for 17 min, take 1 mL of the color developing solution into a microcolorimetric cell, wipe both sides with a clean paper towel, and then place it into an ultraviolet-visible-near infrared spectrophotometer to measure the absorbance value of each sample of humic acid solution at 652 nm wavelength by the ultraviolet spectrophotometer; 4) put the absorbance value measured in step 3) into the regression equation in step (I) above to calculate the concentration of humic acid in the sample to be measured.

2. The method for rapid detection of humic acid according to claim 1, characterized in that, The regression equation is: Y=0.01806X+0.08669 (R 2 =0.99312).

3. The method for rapid detection of humic acid according to claim 2, characterized in that, The concentration of the humic acid solution is 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 mg / L.

4. The method for rapid detection of humic acid according to claim 1, characterized in that, The concentration of the TMB solution is 15 mM.

5. The method for rapid detection of humic acid according to claim 1, characterized in that, The H2O2 solution is a 30% H2O2 solution.

6. The method for rapid detection of humic acid according to claim 1, characterized in that, The filtration is first coarse filtration with filter paper with a pore size of 15 mm, and then the filtered sample is filtered through a 0.22 um filter membrane to prepare the sample to be measured.

7. The method for rapid detection of humic acid according to claim 1, characterized in that, The preparation method of the alkaline aqueous solution is as follows: first, weigh 0.4 g of NaOH, measure 100 mL of deionized water in a 150 mL beaker with a graduated cylinder, add the weighed NaOH, prepare 0.1 M NaOH, then add 2 g of Na2CO3 or 5.39 g of Na2CO3·10H2O, and mix well with a glass rod to obtain the required alkaline aqueous solution.

8. The method for rapid detection of humic acid according to claim 1, characterized in that, The preparation method of the humic acid standard sample is as follows: weigh 0.8 g of NaOH and dissolve it in 100 mL of deionized water, stir well, then dissolve 0.1 g of humic acid in the above solution, place it in an ultrasonic instrument, ultrasonic for 15 min, and finally dilute to 100 mL in a volumetric flask to prepare 1 g / L of humic acid standard sample, which is placed in a 4℃ refrigerator for use.

9. The method for rapid detection of humic acid according to claim 4, wherein the 15 mM TMB solution is prepared by weighing 84.124 mg of 3,3',5,5'-tetramethylbenzidine, dissolving it in a certain amount of anhydrous ethanol, stirring it uniformly in a 100 mL beaker with a glass rod, placing it in an ultrasonic instrument, ultrasonicating it for 15 min, using a rubber bulb dropper to make the TMB solution up to 50 mL, preparing 15 mM TMB chromogenic solution, and storing it in a refrigerator at 4°C.

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