Water sample pretreatment reagent for total nitrogen detection in water bodies, total nitrogen detection kit and detection method

Abstract

The present application discloses a water sample pretreatment reagent, a total nitrogen detection kit and a detection method for the detection of total nitrogen in water bodies. The water sample pretreatment reagent includes reagent A, reagent B and reagent C; reagent A is a nitrogen fixation alloy; reagent B is a urease extract; reagent C is a sodium potassium tartrate-sodium hydroxide solution; the preparation of the urease extract includes: mixing a stabilizer and glycerol, then adding soybean powder, stirring and extracting at room temperature, taking the supernatant and filtering, and taking the filtrate. The total nitrogen detection kit includes an ammonia nitrogen detection reagent and a pretreatment reagent. The detection method includes: taking a detection water sample, adding reagent A and mixing evenly, then adding reagent B and mixing evenly, carrying out an enzymatic hydrolysis reaction for 4 to 6 minutes, finally adding reagent C, mixing evenly and reacting to obtain a pretreatment solution, adding an ammonia nitrogen detection reagent to the pretreatment solution, and detecting after color development. The present application can measure the sum of urea nitrogen and inorganic total nitrogen in water bodies, and is particularly suitable for the determination of total nitrogen in swimming pool water and the determination of total nitrogen in relevant river channels of farmland where urea is applied.

Description

Technical Field

[0001] The present invention relates to the technical field of total nitrogen detection in water bodies, and particularly to a water sample pretreatment reagent, a total nitrogen detection kit and a detection method for detecting the total nitrogen in water bodies containing urea nitrogen and inorganic nitrogen. Background Art

[0002] The total nitrogen in water bodies refers to the total nitrogen content in nitrogen compounds such as organic nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, which is one of the internationally recognized important indicators for measuring the eutrophication degree of water bodies. The Ministry of Ecology and Environment of the country has listed total nitrogen as one of the main on-line determination factors in surface water and industrial and mining enterprise sewage emissions.

[0003] The methods for determining total nitrogen include ultraviolet spectrophotometry for determining the total nitrogen content, high-temperature oxidation-chemiluminescence method for determining the total nitrogen content, and other improved detection methods. For example, the Chinese invention patent document with the publication number CN 115508179 A discloses a method for detecting total nitrogen in water bodies. The organic nitrogen and inorganic nitrogen compounds in the water sample are digested with alkaline potassium persulfate under high-temperature and high-pressure conditions. In the digestion reaction, various nitrogen-containing compounds are finally converted into nitrates. After digestion, in the hydrochloric acid medium, the reducing agent rapidly reduces and decomposes the nitrates to generate gaseous NO, and NO reacts with O3 to generate excited NO 2* When the excited NO 2* transitions to the ground state, photons are emitted, and the intensity is proportional to the total nitrogen content in the sample. Therefore, the total nitrogen content in the sample can be determined by measuring the intensity of chemiluminescence.

[0004] The potassium persulfate used in the national standard method or the above patent often has a high nitrogen content, and the reagent may become invalid due to improper operation during the preparation process, failing to meet the standard requirements, resulting in it being difficult for laboratories to effectively control its blank background value. The ultraviolet spectrophotometry method of potassium persulfate in the national standard requires that the absorbance ratio (A275 / A220)*100% should be less than 20%, and the smaller the better. However, in actual operation, the above ratio often exceeds 20%, leading to measurement failure and it is difficult to analyze the reasons. Moreover, in the case of a more complex water environment, the accuracy is often greatly challenged. In addition, the potassium persulfate method for total nitrogen determination must have a high-temperature digestion step, and the safety of the experimental operation needs to be constantly monitored. Summary of the Invention

[0005] The present application provides a water sample pretreatment reagent, a total nitrogen detection kit and a detection method for detecting the total nitrogen in water bodies, which can measure the sum of urea nitrogen and total inorganic nitrogen in water bodies, and is particularly suitable for the determination of total nitrogen in swimming pool water and the total nitrogen determination in relevant river channels of farmland where urea is applied.

[0006] A water sample pretreatment reagent for total nitrogen detection in water bodies, comprising reagent A, reagent B, and reagent C; said reagent A is a nitrogen fixation alloy; said reagent B is a urease extract; said reagent C is a sodium potassium tartrate-sodium hydroxide solution, by weight percentage, sodium potassium tartrate is 1%-10%, sodium hydroxide concentration is 0.1%-10%, and the solvent is water;

[0007] The preparation of the urease extract includes:

[0008] Mix the stabilizer and glycerol evenly, then add soybean powder, stir at room temperature for 0.5 - 1.5 h, then let it stand at 0 - 5 °C for 20 - 25 h, take the supernatant and filter it, and the obtained filtrate is the urease extract;

[0009] The mass ratio of the stabilizer, glycerol, and soybean powder is 60 - 100:5 - 50:10;

[0010] The stabilizer, calculated by 250 mL, contains 1 - 10 g of anhydrous citric acid, 1 - 10 g of anhydrous disodium hydrogen phosphate, and 1 - 10 g of EDTA-2Na, the solvent is water, and the pH is 5 - 7.

[0011] In the pretreatment reagent of the present application, the nitrogen fixation alloy converts the inorganic nitrogen (such as nitrate nitrogen) in the water sample into ammonium nitrogen, the urease extract converts the urea nitrogen in the water sample into ammonium nitrogen, and after both the urea nitrogen and inorganic total nitrogen in the water sample are converted into ammonium nitrogen, a color reaction is carried out with the ammonia nitrogen detection reagent, and the sum of urea nitrogen and inorganic total nitrogen in the water body can be measured. In a quantitative water sample, an appropriate amount of nitrogen fixation alloy is beneficial to enhancing the urease activity in the urease extract, and the two cooperate with each other, and the urease extract of the present application can maintain its activity for at least 1 year at room temperature.

[0012] Optionally, in the preparation of the urease extract, stir at room temperature for 1 h and then let it stand at 0 - 5 °C for 24 h.

[0013] Optionally, in the preparation of the urease extract, the mass ratio of the stabilizer, glycerol, and soybean powder is 80 - 100:5 - 15:10.

[0014] Optionally, in the preparation of the urease extract, the stabilizer, calculated by 250 mL, contains 1 - 2 g of anhydrous citric acid, 3 - 6 g of anhydrous disodium hydrogen phosphate, and 5 - 10 g of EDTA-2Na, the solvent is water, and the pH is 5 - 7.

[0015] Optionally, in the sodium potassium tartrate-sodium hydroxide solution, by weight percentage, sodium potassium tartrate is 3%-5%, sodium hydroxide concentration is 0.1%-5%, and the solvent is water.

[0016] Most preferably,

[0017] The method for preparing the urease extract is as follows:

[0018] Stabilizer preparation: 1g anhydrous citric acid, 5g anhydrous disodium hydrogen phosphate, 7g EDTA-2Na, first dissolve in 200mL pure water, then adjust the pH to 5-7 with sodium hydroxide solution, and add pure water to make up to 250mL;

[0019] Preparation of soybean powder: grind soybeans with a traditional Chinese medicine grinder, and pass through a 100-mesh sieve for later use;

[0020] Preparation of urease extract: 90g stabilizer, add 10g glycerol and stir evenly, then add 10g soybean powder and stir for 1h to extract at room temperature, place in the refrigerator and let stand for 24h, take the supernatant and filter it with a 500-mesh sieve, and keep the filtrate for later use.

[0021] In the sodium potassium tartrate-sodium hydroxide solution, by weight percentage, the sodium potassium tartrate concentration is 5%, the sodium hydroxide concentration is 4%, and the solvent is water.

[0022] The present application also provides a water sample pretreatment method for detecting total nitrogen in water using the water sample pretreatment reagent, comprising:

[0023] Take the test water sample, add the reagent A and mix, then add the reagent B and mix, enzymatically react for 4 to 6 minutes, then add the reagent C, mix and react for 8 to 12 minutes to obtain the pretreatment solution.

[0024] Optionally, based on 1 mL of the test water sample, the added amounts of reagent A, reagent B and reagent C are 30-60 mg, 100-200 uL and 3-5 mL respectively.

[0025] Furthermore, based on 1 mL of the test water sample, the added amounts of the reagent A, reagent B and reagent C are 50 mg, 3 drops (about 150 uL) and 4 mL respectively.

[0026] In a quantitative water sample, in the presence of a urease stabilizer, an appropriate amount of nitrogen alloy has the effect of enhancing the urease activity in the urease extract and catalyzing the reaction; however, when the ratio of the nitrogen alloy and urease is not coordinated, the urea conversion rate may be insufficient or even worse, it may have an inhibitory effect. Therefore, the appropriate reagent addition ratio is also one of the keys to the pretreatment method of this application.

[0027] The present application also provides a detection kit for total nitrogen in water, characterized in that it comprises an ammonia nitrogen detection reagent and the water sample pretreatment reagent.

[0028] Optionally, the ammonia nitrogen detection reagent is an ammonia nitrogen detection reagent with a detection concentration range of 0-20 mg / L.

[0029] The ammonia nitrogen detection reagent itself can be a commercially available product. For example, optionally, the reagent in the ammonia nitrogen colorimetric tube / test kit with a range of 0-20 mg / L from Zhejiang Luheng Environmental Technology Co., Ltd. can be used. Or the ammonia nitrogen detection reagent disclosed in the patent with the publication number CN112858271 B can also be used.

[0030] This application also provides a method for detecting total nitrogen in water using the above-mentioned detection kit, including:

[0031] Take the water sample to be detected, add the reagent A and mix well, then add the reagent B and mix well, perform enzymatic hydrolysis reaction for 4-6 minutes, and then add the reagent C, mix well and react for 8-12 minutes to obtain a pretreated solution;

[0032] Filter the obtained pretreated solution and take the filtrate. After diluting in proportion, add the ammonia nitrogen detection reagent, let it stand for reaction for 4-6 minutes, and then detect the absorbance value at 620 nm;

[0033] Substitute the obtained absorbance value into the standard curve, and calculate the total nitrogen concentration in the water sample to be detected through calculation.

[0034] Optionally, when the water sample to be detected is 1 mL, the addition amounts of the reagent A, reagent B and reagent C are 30-60 mg, 100-200 μL and 3-5 mL respectively. Further, when the water sample to be detected is 1 mL, the addition amounts of the reagent A, reagent B and reagent C are 50 mg, 3 drops (about 150 μL) and 4 mL respectively.

[0035] The addition amount of the ammonia nitrogen detection reagent can be added according to the dosage when the ammonia nitrogen detection reagent is used to detect ammonia nitrogen, without special requirements.

[0036] Compared with the prior art, this application has at least one of the following beneficial effects:

[0037] (1) At present, the national standard method for determining total nitrogen is potassium persulfate oxidation-ultraviolet spectrophotometry, but the experimental process is relatively cumbersome, and the requirements for experimental conditions, reagents, utensils, etc. are also relatively strict. And when measuring actual water samples, especially when the ammonia nitrogen in the water sample accounts for a relatively high proportion of the total nitrogen, the measured total nitrogen value is often lower than the ammonia nitrogen, indicating that there are certain defects in this method. This method is based on the ammonia nitrogen detection reagent to directly convert both inorganic nitrogen and urea nitrogen into ammonia nitrogen for determination, which can avoid the above problems.

[0038] (2) This method can measure the sum of urea nitrogen and inorganic total nitrogen in water, and is especially suitable for the determination of total nitrogen in swimming pool water and the determination of total nitrogen in relevant river channels of farmland where urea is applied.

[0039] (3) The potassium persulfate oxidation method involves a high-temperature and high-pressure digestion process. This method avoids high-temperature and high-pressure operations, reduces experimental condition limitations, and can be used as a portable reagent, facilitating the direct determination of total nitrogen in the field.

[0040] (4) The test reagent has good stability, a long shelf life, and can be stored at room temperature. The urease extraction solution of this application can maintain its activity for at least 1 year at room temperature, while the urease purchased on the conventional market needs to be stored at -20°C and is difficult to circulate on the market. Description of the Drawings

[0041] Figure 1 It is the color development result diagram in Example 1;

[0042] Figure 2 It is the standard curve diagram in Example 1;

[0043] Figure 3 It is the color development result diagram in Example 5. Detailed Implementation Modes

[0044] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application in this specification are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0046] Example 1

[0047] Total nitrogen standard stock solution (100 mg / L): Weigh 0.7218 g of potassium nitrate (dried at 105 - 110°C for 2 h and cooled to room temperature in a desiccator), dissolve it in an appropriate amount of water, transfer it to a 1000 mL volumetric flask, and dilute it to the mark with water and mix well. Prepare it immediately before use. The preparation of the total nitrogen standard stock solution refers to the national standard: "Determination of Total Nitrogen in Water - Alkaline Potassium Persulfate Digestion - Ultraviolet Spectrophotometry" (HJ636 - 2012).

[0048] The total nitrogen standard solution (100 mg / L) is prepared into a standard gradient (calibration gradient) in the following manner: Among them, the standard gradient solution is prepared from high concentration to low concentration.

[0049] Table 1

[0050]

[0051] (2)Detection reagent

[0052] Reagent A: Nitrogen - fixing alloy;

[0053] Reagent B: Urease extract, the preparation method is as follows:

[0054] Preparation of stabilizer: 1g of anhydrous citric acid, 5g of anhydrous disodium hydrogen phosphate, 7g of EDTA - 2Na, first dissolve in 200mL of pure water, then adjust the pH to 5 - 7 with sodium hydroxide solution, and make up the volume to 250mL with pure water.

[0055] Preparation of soybean powder: After grinding soybeans into powder with a traditional Chinese medicine grinder, sieve through a 100 - mesh sieve for standby.

[0056] Preparation of urease extract: 90g of stabilizer, add 10g of glycerol and stir evenly, then add 10g of soybean powder and extract at room temperature for 1h with stirring. After standing in the refrigerator for 24h, take the supernatant and filter it through a 500 - mesh sieve, and keep the filtrate for use.

[0057] Reagent C: Sodium potassium tartrate - sodium hydroxide solution (mass percentage concentration), 5% sodium potassium tartrate, 4% sodium hydroxide concentration, and the solvent is water.

[0058] Ammonia nitrogen detection reagent: The detection range is 0 - 20mg / L, which can be obtained commercially. Here, take the ammonia nitrogen colorimetric tube / test package with a range of 0 - 20mg / L in the water quality rapid detection series provided by Zhejiang Luheng Environmental Technology Co., Ltd. as an example.

[0059] (3)Determination steps

[0060] Take 1 mL of total nitrogen standard solutions with different concentrations (0, 1mg / L, 5mg / L, 10mg / L, 25mg / L, 50mg / L, 75mg / L, 100mg / L), add 50mg of Reagent A, shake well, add 3 drops of Reagent B (using a conventional dropper in the laboratory, about 150 uL), shake well, enzymatically hydrolyze for 5min, add 4mL of Reagent C and shake well, react for 10min, filter with fast qualitative filter paper, take 1mL of the filtrate, add 4mL of pure water, shake well, add the reagent in the whole tube of ammonia nitrogen colorimetric tube, stand and react for 5min, and measure the absorbance value at 620nm with a multi - parameter water quality detector (multi - parameter water quality detector LH - T725, Zhejiang Luheng Environmental Technology Co., Ltd.).

[0061] Each concentration is tested in 6 parallel tests.

[0062] (4)Determination results

[0063] The results after the reagent in the ammonia nitrogen colorimetric tube shows color as Figure 1 shown.

[0064] The detection results of absorbance values are shown in Table 2.

[0065] Table 2

[0066]

[0067] Taking the absorbance value as the abscissa and the total nitrogen concentration (mg / L) as the ordinate, a standard curve is plotted as Figure 2 shown: y = 29.989x - 0.7496, R 2 = 0.9981, where x is the absorbance value and y is the total nitrogen concentration, with the unit of mg / L.

[0068] Example 2 Screening of the dosage of Reagent A

[0069] Using the detection reagent and detection method of Example 1, with the total nitrogen standard substance potassium nitrate as the benchmark, the conversion of the dosage of Reagent A to the nitrogen content in the urea solution was explored. Taking 90% - 110% as the qualified conversion rate, the results are shown in Table 3. From the results in Table 3, it can be seen that when the dosage of Reagent A is less than 30 mg, the conversion rates of urea nitrogen above 5 mg / L are all lower than 85.86%, and the lowest conversion rate is only 71.41%; when the dosage of Reagent A is higher than 60 mg, the conversion rates of urea nitrogen all exceed 110%. Therefore, 30 - 60 mg is appropriate.

[0070] Table 3

[0071]

[0072] Example 3 Screening of the dosage of Reagent B

[0073] Using the detection reagent and detection method of Example 1, with the total nitrogen standard substance potassium nitrate as the benchmark, the conversion of the dosage of Reagent B to the nitrogen content in the urea solution was explored. Taking 90% - 110% as the qualified conversion rate, the results are shown in Table 4. From the results in Table 4, it can be seen that when the dosage of Reagent B is 1 drop (about 50 μL), the conversion rates of urea nitrogen are all lower than 90.86%, and the lowest conversion rate is only 74.83%; when the dosage of Reagent B is 5 drops (about 250 μL), the conversion rates of 100 mg / L urea nitrogen all exceed 126%. Therefore, a dosage of 2 - 4 drops (about 100 - 200 μL) is appropriate, and the dropper used is a conventional dropper in the laboratory.

[0074] Table 4

[0075]

[0076] Example 4 Influence of the reagent addition sequence

[0077] Take 1 mL of urea working solutions with different concentrations (in terms of nitrogen, the total nitrogen concentration equivalents are 0, 1 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L respectively). Using the detection reagent of Example 1, compared with the determination steps of Example 1, the difference is that reagent C is added first, and then reagents A and B are added for enzymatic reaction. The results are as Figure 3 shown, and there is no color difference at each concentration gradient.

[0078] Spiked recovery of actual water samples in Example 5

[0079] Using the detection reagent and standard curve of Example 1, following the determination steps of Example 1, take actual water samples including purified water, tap water, river water and seawater for spiked recovery tests. The results are shown in Table 5, and the spiked recovery rates are between 91.89% - 108.70%.

[0080] Table 5

[0081]

[0082] Example 6 Comparison of anti-interference ability

[0083] Compared with 2 foreign total nitrogen detection reagents, according to the relevant instructions or test results, the anti-interference ability of relevant ions is equal to or better than that of total nitrogen reagent (1). For the anti-interference ability of relevant ions, except for copper ions and aluminum ions, the anti-interference ability of other ions is equal to or better than that of total nitrogen reagent (2). The results are shown in Table 6, and the blank parts in the following table are unknown items.

[0084] Table 6

[0085]

[0086] The above-described embodiments merely represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A method for detecting total nitrogen in water body, characterized in that, Comprising: Take the test water sample, add reagent A and mix well, then add reagent B and mix well, carry out enzymatic hydrolysis reaction for 4 - 6 min, then add reagent C, mix well and react for 8 - 12 min to obtain the pretreated solution; Calculated based on 1 mL of the test water sample, the addition amounts of the reagent A, reagent B and reagent C are 30 - 60 mg, 100 - 200 μL and 3 - 5 mL respectively; Filter the obtained pretreated solution, take the filtrate, dilute it in proportion and add ammonia nitrogen detection reagent, after standing and reacting for 4 - 6 min, detect the absorbance value at 620 nm; Substitute the obtained absorbance value into the standard curve, and calculate the total nitrogen concentration in the test water sample through calculation; The reagent A is a nitrogen - fixing alloy; The reagent B is a urease extraction solution; The reagent C is a sodium potassium tartrate - sodium hydroxide solution, by weight percentage, sodium potassium tartrate is 1% - 10%, sodium hydroxide is 0.1% - 10%, and the solvent is water; The preparation of the urease extraction solution includes: Mix the stabilizer and glycerol, then add soybean powder, stir at room temperature for 0.5 - 1.5 h and then stand at 0 - 5 °C for 20 - 25 h, take the supernatant and filter, and the obtained filtrate is the urease extraction solution; The mass ratio of the stabilizer, glycerol and soybean powder is 80 - 100:5 - 15:10; Based on 250 mL, the stabilizer contains 1 - 2 g of anhydrous citric acid, 3 - 6 g of anhydrous disodium hydrogen phosphate and 5 - 10 g of EDTA - 2Na, the solvent is water, and the pH is 5 - 7; The ammonia nitrogen detection reagent is an ammonia nitrogen detection reagent with an ammonia nitrogen detection concentration range of 0 - 20 mg / L.

2. The detection method according to claim 1, characterized in that, In the preparation of the urease extraction solution, stir at room temperature for 1 h and then stand at 0 - 5 °C for 24 h.

3. The detection method according to claim 1, wherein In the sodium potassium tartrate - sodium hydroxide solution, by weight percentage, sodium potassium tartrate is 3% - 5%, sodium hydroxide is 0.1% - 5%, and the solvent is water.

4. A detection kit for total nitrogen in water bodies, characterized in that, Including ammonia nitrogen detection reagent and water sample pretreatment reagent; The ammonia nitrogen detection reagent is an ammonia nitrogen detection reagent with an ammonia nitrogen detection concentration range of 0 - 20 mg / L; The water sample pretreatment reagent includes reagent A, reagent B and reagent C; The reagent A is a nitrogen - fixing alloy; The reagent B is a urease extraction solution; The reagent C is a sodium potassium tartrate - sodium hydroxide solution, by weight percentage, sodium potassium tartrate is 1% - 10%, sodium hydroxide is 0.1% - 10%, and the solvent is water; The preparation of the urease extraction solution includes: Mix the stabilizer and glycerol, then add soybean powder, stir at room temperature for 0.5 - 1.5 h and then stand at 0 - 5 °C for 20 - 25 h, take the supernatant and filter, and the obtained filtrate is the urease extraction solution; The mass ratio of the stabilizer, glycerol and soybean powder is 80 - 100:5 - 15:10; Based on 250 mL, the stabilizer contains 1 - 2 g of anhydrous citric acid, 3 - 6 g of anhydrous disodium hydrogen phosphate and 5 - 10 g of EDTA - 2Na, the solvent is water, and the pH is 5 - 7.

5. The detection kit according to claim 4, characterized in that, In the preparation of the urease extraction solution, stir at room temperature for 1 h and then stand at 0 - 5 °C for 24 h.

6. The detection kit according to claim 4, characterized in that, In the sodium potassium tartrate - sodium hydroxide solution, by weight percentage, sodium potassium tartrate is 3% - 5%, sodium hydroxide is 0.1% - 5%, and the solvent is water.

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