CN116593414A - Method for rapidly detecting humic acid content based on TMB colorimetric method - Google Patents
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- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 title claims abstract description 132
- 239000004021 humic acid Substances 0.000 title claims abstract description 132
- 238000000034 method Methods 0.000 title claims abstract description 79
- 238000004737 colorimetric analysis Methods 0.000 title description 4
- 238000002835 absorbance Methods 0.000 claims abstract description 56
- 238000011161 development Methods 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
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- 229910021641 deionized water Inorganic materials 0.000 claims description 11
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- 239000011734 sodium Substances 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
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- 229960000583 acetic acid Drugs 0.000 claims description 4
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- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
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- 229950003937 tolonium Drugs 0.000 description 2
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000004566 building material Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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- 229910052760 oxygen Inorganic materials 0.000 description 1
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- 239000003208 petroleum Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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Abstract
The application discloses a method for rapidly detecting humic acid, which aims to provide a simple, sensitive and rapid detection method for quantifying HA by utilizing the oxidizing property of humic acid and TMB reaction color development principle, wherein the method is to detect standard humic acid solution with known concentration by using TMB method, measure absorbance value under 652nm wavelength under the condition of pH 4, establish a regression equation between the absorbance value and humic acid concentration, treat a sample to be detected, and detect absorbance value under 652nm wavelength by using TMB method after color development, and carry the absorbance value into the regression equation to calculate the concentration of humic acid in the sample to be detected; belongs to the technical field of detection.
Description
Technical Field
The application relates to a method for rapidly detecting humic acid content based on TMB colorimetric method, and belongs to the technical field of detection.
Background
Humic Acid (HA) is a main component in Humus (HS), is a macromolecular organic substance widely existing in nature, and is widely applied to various fields such as forests, pastures, agriculture, petroleum, chemical industry, building materials, medicine and health, environmental protection and the like. In addition, the presence of HA in wastewater also presents certain difficulties in wastewater treatment. There is a huge population growth trend and profound economic and social changes in the world, and wastewater treatment and purified water demand are increasing, but there are large deviations in supply and demand. Natural organic substances, which are one of the main pollutants present in wastewater, include humic acid, humus, etc., are widely present in various water sources. Organic matters such as humic acid with high concentration can cause harm to aquatic organisms, and if the human body ingests the organic matters indirectly or directly through a food chain, huge health problems can be caused to the human body.
Because of the complex definition and structure of HA, no absolute and accurate analysis method is available at present to verify the content of HA. The existing various analytical instruments and methods can carry out quantitative analysis on HA, including electrochemical methods, chromatographic methods, oxygen consumption measurement, flow injection chemiluminescence, ultraviolet and fluorescence methods, which can help to determine the humic acid content in a water sample, but the methods not only need specific instruments, but also have complicated operation process and low convenience and applicability. The ultraviolet method is used for detecting HA, the improved Lowry method is used as a standard, the detection range of the improved Lowry method is wide, the detection result is accurate and reliable, the reproducibility is good, the detection effect of other methods is often evaluated by being used as a standard method for detecting HA in water, and the HA quantization and the Toluidine Blue (TB) combination method are also carried out by using an ultraviolet absorption method. Therefore, there is a need to develop a new, simple, rapid, and sensitive quantitative analysis method for HA.
TMB is by far the most commonly used chromogenic substrate whose chromogenic reaction is based on H 2 O 2 Under the condition of being used as a catalyst, the oxidizing substance catalyzes and oxidizes TMB, and the reaction generates light blue to blue oxidation state TMB and has an ultraviolet visible light absorption peak at a specific wavelength (652 nm). Humic acid is a polyelectrolyte containing oxygen-containing functional groups such as hydroxyl, carboxyl, phenolic and carbonyl groups, and can be used as an oxidizing substance in the catalyst H 2 O 2 The chromogenic TMB is reduced under the action, and HA is rapidly and rapidly quantified by measuring the absorbance value of the TMB at a specific wavelength (652 nm).
Disclosure of Invention
Aiming at the defects of complex process, long time consumption, limited application range and the like of the existing HA detection method, the application aims to design a simple, sensitive and rapid detection method for quantifying HA by utilizing the oxidizing property of humic acid and the TMB reaction color development principle.
In order to achieve the above purpose, the present application provides the following technical solutions:
a method for quickly detecting humic acid comprises the steps of detecting standard humic acid solution with known concentration by using a TMB method, measuring absorbance value at 652nm wavelength under the condition of pH 4, establishing a regression equation between the absorbance value and the humic acid concentration, processing a sample to be detected, measuring the absorbance value at 652nm wavelength after developing by using the TMB method, and taking the absorbance value into the regression equation to calculate the humic acid concentration in the sample to be detected.
Further, the method for rapidly detecting humic acid specifically comprises the following steps:
(one) establishing a regression equation
1) Preparing a humic acid standard sample with the concentration of 1 g/L;
2) Preparing humic acid solutions with different concentrations by using acetic acid-sodium acetate solution with pH=4 as a background solution;
3) Respectively taking 1mL of humic acid solution with different concentrations into test tubes, and respectively adding TMB solution and H into the test tubes 2 O 2 Reacting the solution for 17min to obtain a color developing solution, respectively placing 1mL of the color developing solution into a micro cuvette, placing into an ultraviolet-visible-near infrared spectrophotometer, and respectively measuring the absorbance value of humic acid solution with each concentration under 652nm wavelength by the ultraviolet-visible-near infrared spectrophotometer;
4) Linearly fitting the concentration of humic acid solution with each concentration under 652nm wavelength with the corresponding absorbance value, and establishing a regression equation;
(II) calculating the concentration of humic acid in the sample to be measured
1) Filtering different water samples to prepare samples to be tested;
2) Taking 1mL of a sample to be tested, adding 500 mu L of an alkaline aqueous solution, mixing for 10min, vibrating, uniformly mixing to ensure that the sample is fully hydrolyzed, and regulating the pH to 4 by using a glacial acetic acid solution after the hydrolysis;
3) Taking 1mL of the solution treated in the step 2) into a 1.5mL test tube, and adding TMB solution and H into the tube respectively 2 O 2 Placing 1mL of color development liquid in a micro cuvette after 17min of reaction, wiping the two sides with clean paper towels, placing the cleaned paper towels in an ultraviolet-visible-near infrared spectrophotometer, and respectively measuring the absorbance value of humic acid solution of each sample under 652nm wavelength by the ultraviolet spectrophotometer;
4) Substituting the absorbance value measured in the step 3) into the regression equation in the step (one), and calculating to obtain the concentration of the humic acid in the sample to be measured.
Furthermore, in the above method for rapidly detecting humic acid, the regression equation is: y=0.01806x+0.08669 (R 2 ＝0.99312)。
Furthermore, in the method for rapidly detecting humic acid, the concentration of the humic acid solution is as follows: 0.1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55mg/L.
Further, in the method for rapidly detecting humic acid, the concentration of the TMB solution is 15mM.
Furthermore, the method for rapidly detecting humic acid comprises the step of H 2 O 2 The solution was 30% H 2 O 2 。
Furthermore, in the method for rapidly detecting humic acid, the filtering is that the filter paper with the aperture of 15mm is adopted for coarse filtering, and the filtered sample is further filtered through a 0.22um filter membrane to prepare the sample to be detected.
Furthermore, in the method for rapidly detecting humic acid, the method for preparing the alkaline aqueous solution comprises the following steps: firstly weighing 0.4g of NaOH, weighing 100mL of deionized water in a 150mL beaker by using a cylinder, adding the weighed NaOH to prepare 0.1M NaOH, and then adding 2g of Na 2 CO 3 (or 5.39g Na 2 CO 3 ·10H 2 And O), stirring and mixing uniformly by using a glass rod to obtain the required alkaline aqueous solution.
Furthermore, in the method for rapidly detecting humic acid, the configuration method of the humic acid standard sample comprises the following steps: weighing 0.8g of NaOH, dissolving in 100mL of deionized water, stirring uniformly by using a glass rod, dissolving 0.1g of humic acid in the solution, placing on an ultrasonic instrument, carrying out ultrasonic treatment for 15min, finally fixing the volume in a 100mL volumetric flask to prepare a 1g/L humic acid standard sample, and placing in a refrigerator at 4 ℃ for use.
Furthermore, in the method for rapidly detecting humic acid, the preparation method of the 15mM TMB solution comprises the following steps: 84.124mg of 3,3', 5' -tetramethylbenzidine is weighed, dissolved in a certain amount of absolute ethyl alcohol, stirred uniformly in a 100mL beaker by using a glass rod, placed on an ultrasonic instrument, subjected to ultrasonic treatment for 15min, and TMB solution is fixed in 50mL by using a rubber head dropper to prepare 15mM TMB color development liquid, and stored in a refrigerator at 4 ℃.
One of the above technical solutions of the present application has at least one of the following advantages or beneficial effects:
(1) The detection method provided by the application has higher sensitivity and good linearity, and compared with other ultraviolet absorption methods, the detection method has wider detection range, the effective detection range can reach 50mg/L, the detection limit is 0.98mg/L, and the standard curve linearity is good (R 2 ＞0.99)。
(2) Compared with a standard method (improved Lowry method), the detection method provided by the application has the advantages that the TMB method is simple, and special materials and instruments are not required to be used for detection. The HA concentration measured by these two methods is almost the same, and this work provides a new and effective method for HA quantification in terms of sensitivity and detection range.
(3) The detection method provided by the application can remove the interference of protein and polysaccharide substances on the color development result by using an alkaline solution hydrolysis method and film-passing pretreatment, and the interference of ions on HA measurement is negligible.
Drawings
FIG. 1 is a graph showing the influence of TMB colorimetric reaction condition optimization on a color development result, wherein (a) pH is shown in an illustration, color of a color development solution at a corresponding pH, (b) TMB concentration and (c) reaction time;
FIG. 2 is a graph of a TMB colorimetric stability assay in which (a) BSA, (b) SA, (c) BSA interference maximum allowable concentration assay, and (d) SA interference maximum allowable concentration assay are shown;
FIG. 3 is a graph showing the results of removing protein interference in the present application, wherein (a) the results of removing protein by an alkaline solution hydrolysis method and (b) the effects of a mixture of SA, BSA and HA on the color development results after the alkaline solution hydrolysis method is performed;
FIG. 4 is a graph of the results of the detection model establishment in the present application, wherein (a) ultraviolet spectra at 500-800nm after development of different HA concentrations, (b) linear regression equation of absorbance values and HA concentrations (N=3), (c) corresponding colors of HA color development solutions with different concentrations are photographed under a stable light source;
fig. 5 shows two standard curves (n=3) obtained in the modified Lowry method of the present application, wherein (a) standard curve 1 and (b) standard curve 2.
Detailed Description
The present application will be described in more detail with reference to examples, but the scope of the present application is not limited to the examples.
Example 1
The method for rapidly detecting the content of humic acid based on TMB colorimetric method provided by the embodiment specifically comprises the following steps:
(1) Establishing regression equations
The first step: weighing 0.8g of NaOH, dissolving in 100mL of deionized water, uniformly stirring by using a glass rod, dissolving 0.1g of Humic Acid (HA) in the solution, uniformly stirring by using the glass rod, placing on an ultrasonic instrument, performing ultrasonic treatment for 15min, finally fixing the volume in a 100mL volumetric flask to prepare a 1g/L humic acid standard sample, and placing in a refrigerator at 4 ℃ for use;
and a second step of: the method comprises the steps of preparing a certain amount of humic acid solution with known component concentration by taking acetic acid-sodium acetate solution with pH=4 as a background solution, wherein the humic acid solution concentrations are respectively as follows: 0.1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50mg/L;
and a third step of: separately taking 1mL of humic acid solution with different concentrations into 1.5mL small tubes, and respectively adding 100uL of 15mM TMB solution and 10uL of 30% H into the small tubes 2 O 2 Reaction of17min, 1mL of the color development liquid is respectively placed in a micro cuvette, and the two sides are wiped by a clean tissue and then placed in an ultraviolet-visible-near infrared spectrophotometer.
Fourth step: measuring the absorbance value of each concentration humic acid solution under 652nm wavelength by an ultraviolet spectrophotometer, wherein three groups of parallel samples are arranged in each measurement, and the absorbance value of each concentration humic acid solution under 652nm wavelength is obtained by respectively taking an average value;
fifth step: performing linear fitting on each concentration of humic acid solution under 652nm wavelength and a corresponding absorbance value, and establishing a regression equation;
(2) Calculating the concentration of humic acid in the sample to be measured
The first step: coarse filtering (filter paper aperture is 15 mm) is carried out on different water samples, and the filtered samples pass through a 0.22um filter membrane to prepare samples to be detected;
and a second step of: taking 1mL of a sample to be tested, adding 500 mu L of alkaline aqueous solution, mixing for 10min, vibrating, uniformly mixing to ensure that the sample is fully hydrolyzed, and regulating the pH to 4 by using glacial acetic acid solution after the hydrolysis.
And a third step of: taking 1mL of the solution treated in the second step into a small 1.5mL tube, and adding 100uL of 15mM TMB solution and 10uL of 30% H into the tube 2 O 2 And (3) reacting for 17min, respectively taking 1mL of the developing solution, placing the developing solution in a micro cuvette, wiping the two sides with a clean tissue, placing the micro cuvette into an ultraviolet-visible-near infrared spectrophotometer, ensuring that the absorbance value of the sample to be detected in the step (1) is within the range of the regression equation in the step (1), and if the absorbance value is not within the range of the regression equation in the step (1), diluting the solution, and measuring until the absorbance value is within the range of the regression equation in the step (1).
Fourth step: measuring the absorbance value of the humic acid solution of each sample at 652nm wavelength by an ultraviolet spectrophotometer; three groups of parallel samples are arranged in each measurement, and average values are respectively taken, so that absorbance values of samples to be measured under 652nm wavelength are obtained;
fifth step: substituting the measured absorbance value into the regression equation under the corresponding wavelength obtained in the step (1), and calculating to obtain the concentration of the humic acid in the sample to be detected.
1. In order to better understand the technical scheme provided by the application, the following provides a technical scheme research method provided by the application:
1. study on pH:
the pH values of the second step background solution of step (1) in example 1 were replaced with ph=2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, and the other steps and parameters were identical to those of example 1.
As a result, referring to FIG. 1 (a), the color development results of TMB are significantly affected by different pH conditions, referring to FIG. 1 (a). At reaction background ph=2.0, the TMB color developing solution turns yellow from colorless; at ph=3.0 and 4.0, the TMB color developing solution changes from colorless to blue, and at ph=4.0, the blue color is more obvious and easy to observe; at ph=5.0, 6.0, 7.0 and 8.0, the TMB color developing solution does not change color, but is colorless. The color of the reaction is therefore most pronounced at ph=4.0, which is more convenient to observe, with the wavelength corresponding to the maximum absorbance at 652 nm. The relative activity of the different pH was measured at a wavelength of 652nm, and the relative activity of the reaction to give a blue chromogenic product was gradually increased during the pH change from 2.0 to 4.0, reaching a maximum at ph=4.0. After the pH is 4.0, the relative activity suddenly drops, and after the pH reaches 6.0, the relative activity tends to be gentle and stable at about 0.1.
2. Study on development time
The third reaction time of step (1) in example 1 was replaced with 0 to 30min, and the other steps and parameters were identical to those of example 1.
As a result, referring to FIG. 1 (b), humic acid at pH=4.0, 10mg/L, 15mM TMB, 10uL of 30% H 2 O 2 The absorbance value and the change trend of TMB color developing liquid at 652nm within 0-30 min. The change of the absorbance value starts to rise from 0min, the rising amplitude is larger in the first 10min, the rising amplitude is increased from 0.3 to about 0.9, the speed is then increased slowly, and the absorbance value reaches the maximum in about 17 min. Therefore, the color development was optimal at a reaction time of 17 min.
3. Investigation of TMB concentration
The third-step TMB solution concentration of step (1) in example 1 was replaced with 1, 5, 10, 15, 20, 25mM, and the other steps and parameters were the same as in example 1.
The results are shown in FIG. 1 (c), humic acid at pH=4.0, 10mg/L, 10uL 30% H 2 O 2 Absorbance values and trends at 652nm for 1, 5, 10, 15, 20, 25mM TMB in a chromogenic background with a reaction time of 17 min. The absorbance value gradually increased during the increase of the TMB concentration from 1mM to 15mM, and reached a maximum value at a TMB concentration of 15mM, and began to decrease after 15mM. In the overall trend, the absorbance value of the reaction system was maximized when the TMB concentration was 15mM under the background condition. Therefore, the present study developed optimally at a TMB concentration of 15mM.
2. In order to prove the reliability of the technical scheme provided by the application, the experimental investigation of the stability of the TMB method of the technical scheme provided by the application is given below
1. Since proteins and polysaccharides are two widely occurring organic substances among dissolved organic substances (DOM), HA detection was explored, and BSA and SA were selected as representative proteins and polysaccharides, respectively, during the course of the study. 2 mg, 4mg, 6 mg, 8mg, 10mg, 12 mg, 14 mg, 16 mg, 20mg and 50mg of bovine serum albumin are respectively weighed, 100mL of deionized water is measured in a beaker by using a measuring cylinder, and a glass rod is uniformly stirred and fully dissolved. 20, 40, 60, 80, 100, 120, 140, 160, 200, 500mg/L BSA solutions were prepared, respectively. 2 mg, 4mg, 6 mg, 8mg, 10mg, 12 mg, 14 mg, 16 mg, 20mg and 50mg of sodium alginate are respectively weighed, 100mL of deionized water is weighed into a beaker by using a cylinder, and a glass rod is uniformly stirred and fully dissolved. 20, 40, 60, 80, 100, 120, 140, 160, 200, 500mg/L SA solutions were prepared, respectively.
The second step of step (1) in example 1 was changed to: the prepared 20, 40, 60, 80, 100, 120, 140, 160, 200, 500mg/L SA solution and 500. Mu.L of the 20, 40, 60, 80, 100, 120, 140, 160, 200, 500mg/L LBSA solution are respectively taken into small tubes with the concentration of 1.5mL, 500. Mu.L of pre-diluted 20mg/L humic acid is respectively added, the mixture and shaking are carried out, the mixture is placed on a vortex mixer, the mixture is rotated and shaken for at least more than 3 seconds, and the SA concentration of 10, 20, 30, 40, 50, 60, 70, 80, 100, 250mg/L and the BSA concentration of 10, 20, 30, 40, 50, 60, 70, 80, 100, 250mg/L humic acid solution are obtained, and other steps and parameters are consistent with those of example 1.
The results are shown in FIGS. 2 (a) and (b), where the absorbance of the 10mg/L HA solution was not significantly reduced after SA addition, but the absorbance of the 10mg/L HA solution was significantly reduced after BSA addition.
In order to integrally evaluate the interference of polysaccharide and protein on a detection system, an additional polysaccharide interference experiment taking 20mg/L as a background is carried out, because 20mg/L is approximately near the upper detection limit, the HA concentration in a water body generally fluctuates up and down at 20mg/L, and the concentration investigation detection model HAs great application significance and theoretical basis for polysaccharide selectivity. As shown in FIG. 2 (c), it was found that the absorbance value exhibited almost no difference from the standard case when the SA concentration was 60mg/L, indicating that the influence of the presence thereof could be disregarded when the SA concentration in water was 60mg/L or less. As shown in FIG. 2 (d), as the BSA concentration was decreased, the absorbance value was gradually increased, and when the BSA concentration was 1.25mg/L, the absorbance value was substantially equal to that of the standard HA solution under the same measurement conditions, i.e., even when the BSA concentration was 1.25mg/L or less, the interference was negligible with the measurement result of TMB color former. The DOM concentration in natural water is generally 0-10 mg/L, and the DOM concentration in wastewater is generally greater than this range. In order to ensure high accuracy and sensitivity of detection results, the method needs to consider the influence of protein substances in the application of the method in an actual water sample, and the influence is eliminated by an effective method.
3. The second step of step (1) in example 1 was changed to: and respectively taking 500 mu L of the prepared 20mg/L BSA solution into a small pipe with the volume of 1.5mL, respectively adding 500 mu L of pre-diluted 20mg/L humic acid, mixing, vibrating, placing on a vortex mixer, rotating and shaking for at least more than 3s, and obtaining the 10mg/L humic acid solution with the BSA concentration of 10 mg/L. Weighing 0.4g of NaOH, weighing 100mL of deionized water in a 150mL beaker by using a cylinder, adding the weighed NaOH to prepare 0.1M NaOH, and then adding 2g of Na 2 CO 3 (or 5).39g Na 2 CO 3 ·10H 2 And O), stirring and mixing uniformly by using a glass rod to obtain the alkaline water reagent. Adding 500 mu L of alkaline aqueous solution into 10mg/L humic acid solution containing 10mg/L BSA prepared above, mixing for 10min, shaking, and mixing uniformly to make hydrolysis complete. After hydrolysis, the pH was adjusted to about 4 with glacial acetic acid solution. Other steps and parameters were consistent with example 1.
As shown in FIG. 3 (a), the protein interference is removed by hydrolysis with an alkaline aqueous solution, and the absorbance value of the TMB chromogenic product after the hydrolysis operation is substantially similar to that of 10mg/L HA, so that the alkaline solution hydrolysis method can remove the interference of the protein on the TMB chromogenic liquid under most conditions, and the detection result is substantially similar to that under the interference-free background, and the method can be used for removing the protein interference. As shown in FIG. 3 (b), after BSA was removed by the alkaline solution hydrolysis method, their effect of the mixture was negligible. Thus, the stability of the detection method can be further verified.
4. Selective exploration
Considering various interference ions existing in water, simulating the ion environment in the wastewater, respectively measuring the absorbance value of each ion, and examining the detection result of the method under the most complex environment. 265.08mg NaHCO are weighed respectively 3 、37.36mg KOH、132.34mg KOH、129.6mg MgCl 2 、146.9mg Ca(OH) 2 Measuring 32.3uL of HF and 1.79uL of H 3 PO 4 、275.95uL HCl、58uL H 2 SO 4 、6.54uL HNO 3 Dissolving or mixing in 10mL deionized water, stirring with glass rod, transferring to 10mL small tube, and shaking on vortex mixer for more than 3 s. Respectively obtain Mg-containing 2+ 、Na + 、Ca 2+ 、K + 、HCO 3 - 、Cl - 、F - 、NO 3 - 、SO 4 2- 、PO 4 3- An interfering solution of ions. The second step of step (1) in example 1 was changed to: adding 10uL of the prepared solution containing different ions into 1.5mL small tubes, adding 990 uL of pre-diluted 20mg/L humic acid, mixing, vibrating, and placing into a vortex mixerRotating and shaking for more than 3s to obtain humic acid solution containing different interfering ions of 20 mg/L. Other steps and parameters were consistent with example 1.
As shown in Table 1, SO was studied 4 2- 、NO 3 - 、Cl - 、PO 4 3- 、HCO 3 - 、F - 、Mg 2+ 、K + 、Na + 、Ca 2+ The measurement of 20mg/L HA was interfered with by the matrix ions. SO (SO) 4 2- 、NO 3 - 、Cl - 、PO 4 3- 、HCO 3 - The plasma anions at the wastewater reference concentration have little interference with the HA assay using the method proposed in this work. Mg of 2+ 、K + 、Na + The interference of equimetallic cations under the interference of 10mg/L is negligible, especially Na + The ion, when the concentration is 38.050mg/L, has less than 5% interference (interference is 1.60%). Thus, the ion interference to HA measurements is negligible.
TABLE 1 Effect of interfering substances on 20mg/L HA assay
3. Establishment of detection model and feasibility verification experiment
A calibration model between absorbance values and HA concentration was established for determining the concentration of HA in unknown samples according to the experimental method of example 1. HA with different concentration (0-50 mg/L) is subjected to 3 times of parallel detection by adopting a TMB method. As shown in FIG. 4 (a), as the concentration of HA increases, the absorbance of the color-developing solution increases at 652 nm. As shown in FIG. 4 (c), the higher the HA concentration, the greater the color change of the sample, and the darker the blue color of the color-developing solution. As shown in figure 4 (b), a wider detection range is realized in the HA range of 0-50 mg/L, the HA concentration and the absorbance value have good linear relation, and a curve R is fitted 2 = 0.99312, the slope of the curve is 0.01806, and the lowest detection limit is 0.98mg/L by the detection limit calculation formula. When the HA concentration of the sample is in a linear range of 0-50 mg/L, the sample is detected to be a junctionThe method HAs high accuracy, and the sample can obtain the absorbance value at 652nm wavelength by the method and quickly calculate the concentration of HA in the sample by combining the regression equation after fitting. These results indicate that the method has potential for practical use.
This example is a feasibility verification with the modified Lowry method by using different concentrations of standard humic acid known.
The improved Lowry method is as follows:
adding 100mg/L humic acid 0, 0.2, 0.4, 0.6, 0.8 and 1mL to 10mL centrifuge tubes respectively, adding deionized water to 1mL, adding 4mL of reagent C respectively, mixing, placing on a vortex mixer, vibrating for 20s, standing for 10min, adding 0.5mL of Fu Lin Fen reagent, rapidly mixing, placing on the vortex mixer, vibrating for 20s, standing for 10min, measuring absorbance values of each sample at 750nm, and obtaining a standard curve 1. The correlation coefficient of the regression equation of the obtained standard curve 1 should be greater than 0.99.
Water was sampled in 1mL to 10mL centrifuge tubes. Adding 4mL of reagent C, mixing, placing on a vortex mixer, vibrating for 20s, standing for 10min, adding 0.5mL of Fu Lin Fen reagent, rapidly mixing, placing on the vortex mixer, vibrating for 20s, standing for 10min, and measuring the absorbance value of the sample at 750 nm. The experiment is repeated three times to obtain an average value, the absorbance value obtained by the measured water sample is substituted into a regression equation of the standard curve 1, and referring to fig. 5 (a), the concentration A total is calculated.
Adding 100mg/L humic acid 0, 0.2, 0.4, 0.6, 0.8 and 1mL to 10mL centrifuge tubes respectively, adding deionized water to 1mL, adding 4mL of reagent A respectively, mixing, placing on a vortex mixer, vibrating for 20s, standing for 10min, adding 0.5mL of Fu Lin Fen reagent, rapidly mixing, placing on the vortex mixer, vibrating for 20s, standing for 10min, and measuring absorbance at 735nm to obtain a standard curve. In the regression equation of the standard curve 2, referring to fig. 5 (b), the correlation coefficient should be greater than 0.99.
Water was sampled in 1mL to 10mL centrifuge tubes. Adding 4mL of reagent A, mixing, placing on a vortex mixer, vibrating for 20s, standing for 10min, adding 0.5mL of Fu Lin Fen reagent, rapidly mixing, placing on the vortex mixer, vibrating for 20s, standing for 10min, and measuring the absorbance value of the sample at 735 nm. The experiment is repeated for three times to obtain an average value, the absorbance value obtained by the measured water sample is substituted into a regression equation of the standard curve 2, and the concentration Abind is obtained through calculation.
The test for stability compared to the modified Lowry method was performed by measuring different known concentrations of standard humic acid according to the experimental method of example 1. As shown in fig. 5 (a) and (b), according to two standard curves (standard curve 1 and standard curve 2) obtained by measuring the modified Lowry method, the absorbance values (a blind and a total) of the samples under the corresponding standard curves are measured simultaneously, and finally, the HA concentration of each sample is calculated by using the formula C (HA) = (a blind-0.2A total)/0.8. The HA concentration measured by both methods is at substantially the same level as shown in table 2. The proposed method is indicated to be suitable for quantification of HA in natural water samples.
TABLE 2 comparison of TMB color development and Lowry results
The preferred embodiments of the present application have been described in detail above, but the present application is not limited thereto. Within the scope of the technical idea of the application, a number of simple variants of the technical solution of the application are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the application, all falling within the scope of protection of the application.
Claims (10)
1. A method for rapidly detecting humic acid is characterized in that a TMB method is used for detecting standard humic acid solution with known concentration, the absorbance value at 652nm wavelength is measured under the condition that the pH value is 4, a regression equation between the absorbance value and the humic acid concentration is established, a sample to be detected is processed, the absorbance value at 652nm wavelength is measured after color development by the TMB method, and the absorbance value is brought into the regression equation, so that the concentration of humic acid in the sample to be detected is calculated.
2. The method for rapidly detecting humic acid according to claim 1, wherein the method comprises the following steps:
(one) establishing a regression equation
1) Preparing a humic acid standard sample with the concentration of 1 g/L;
2) Preparing humic acid solutions with different concentrations by using acetic acid-sodium acetate solution with pH=4 as a background solution;
3) Respectively taking 1mL of humic acid solution with different concentrations into test tubes, and respectively adding TMB solution and H into the test tubes 2 O 2 Reacting the solution for 17min to obtain a color developing solution, respectively placing 1mL of the color developing solution into a micro cuvette, placing into an ultraviolet-visible-near infrared spectrophotometer, and respectively measuring the absorbance value of humic acid solution with each concentration under 652nm wavelength by the ultraviolet-visible-near infrared spectrophotometer;
4) Linearly fitting the concentration of humic acid solution with each concentration under 652nm wavelength with the corresponding absorbance value, and establishing a regression equation;
(II) calculating the concentration of humic acid in the sample to be measured
1) Filtering different water samples to prepare samples to be tested;
2) Taking 1mL of a sample to be tested, adding 500 mu L of an alkaline aqueous solution, mixing for 10min, vibrating, uniformly mixing to ensure that the sample is fully hydrolyzed, and regulating the pH to 4 by using a glacial acetic acid solution after the hydrolysis;
3) Taking 1mL of the solution treated in the step 2) into a 1.5mL test tube, and adding TMB solution and H into the tube respectively 2 O 2 Placing 1mL of color development liquid in a micro cuvette after 17min of reaction, wiping the two sides with clean paper towels, placing the cleaned paper towels in an ultraviolet-visible-near infrared spectrophotometer, and respectively measuring the absorbance value of humic acid solution of each sample under 652nm wavelength by the ultraviolet spectrophotometer;
4) Substituting the absorbance value measured in the step 3) into the regression equation in the step (one), and calculating to obtain the concentration of the humic acid in the sample to be measured.
3. The method for rapidly detecting humic acid according to claim 2 wherein the regression equation is: y=0.01806x+0.08669 (R 2 ＝0.99312)。
4. The method for rapidly detecting humic acid according to claim 2 wherein the concentration of the humic acid solution is: 0.1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55mg/L.
5. The method for rapidly detecting humic acid according to claim 2 wherein the concentration of TMB solution is 15mM.
6. The method for rapidly detecting humic acid according to claim 2 wherein H is 2 O 2 The solution was 30% H 2 O 2 A solution.
7. The method for rapidly detecting humic acid according to claim 2 wherein the filtration is carried out by coarse filtration with filter paper having a pore size of 15mm, and passing the filtered sample through a 0.22um filter membrane to obtain the sample to be detected.
8. The method for rapidly detecting humic acid according to claim 2 wherein the method for preparing the alkaline aqueous solution comprises the steps of: firstly weighing 0.4g of NaOH, weighing 100mL of deionized water in a 150mL beaker by using a cylinder, adding the weighed NaOH to prepare 0.1M NaOH, and then adding 2g of Na 2 CO 3 Or 5.39g Na 2 CO 3 ·10H 2 And (3) stirring and mixing uniformly by using a glass rod to obtain the required alkaline aqueous solution.
9. The method for rapidly detecting humic acid according to claim 2 wherein the method for preparing the standard sample of humic acid comprises the steps of: weighing 0.8g of NaOH, dissolving in 100mL of deionized water, stirring uniformly, dissolving 0.1g of humic acid in the solution, placing on an ultrasonic instrument, performing ultrasonic treatment for 15min, finally fixing the volume in a 100mL volumetric flask to prepare a 1g/L humic acid standard sample, and placing in a refrigerator at 4 ℃ for use.
10. The method for rapidly detecting humic acid according to claim 5 wherein the method for preparing the 15mM TMB solution comprises the following steps: 84.124mg of 3,3', 5' -tetramethylbenzidine is weighed, dissolved in a certain amount of absolute ethyl alcohol, stirred uniformly in a 100mL beaker by using a glass rod, placed on an ultrasonic instrument, subjected to ultrasonic treatment for 15min, and TMB solution is fixed in 50mL by using a rubber head dropper to prepare 15mM TMB color development liquid, and stored in a refrigerator at 4 ℃.
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