CN111426665A - A Fluorescence Analysis Method for Determining the Concentration of Total Nitrogen in Water - Google Patents

Abstract

The invention provides a fluorescence analysis method for measuring the concentration of total nitrogen in a water body, comprising the following steps: preparing a first blank solution, and measuring the first fluorescence intensity; preparing a standard working solution with the first blank solution and a nitrite standard solution, and Measure the second fluorescence intensity; draw a standard curve according to the first fluorescence intensity, the second fluorescence intensity and the corresponding nitrogen concentration; perform ammonia nitrogen oxidation treatment and nitrate reduction treatment on the sample to be tested to obtain the sample to be tested after reduction; prepare a second blank solution , and measure the third fluorescence intensity; add the second blank solution to the sample to be tested after reduction, and measure the fourth fluorescence intensity; calculate the total nitrogen concentration according to the fourth fluorescence intensity, the third fluorescence intensity and the standard curve. The fluorescence analysis method for measuring the total nitrogen concentration in water provided by the present invention measures the total nitrogen concentration in the sample to be tested by the fluorescence quenching degree of the fluorescent probe.

Description

A Fluorescence Analysis Method for Determining the Concentration of Total Nitrogen in Water

technical field

The invention relates to the technical field of chemical analysis and detection, in particular to a fluorescence analysis method for measuring the concentration of total nitrogen in a water body.

Background technique

Nitrogen is one of the main factors leading to eutrophication of water bodies, and it mainly exists in water bodies in the form of ammonia nitrogen and nitrate. Under aerobic conditions, ammonia nitrogen in water can be converted into nitrite under the action of nitrifying bacteria, and finally oxidized into nitrate. Nitrate is the highest stable valence state of nitrogen in water. Under the condition of insufficient oxygen, nitrate can be reduced to nitrite by denitrifying bacteria, and further reduced to ammonia nitrogen. Nitrogen-containing organic matter mainly derived from domestic sewage is decomposed into ammonia nitrogen under the action of microorganisms, and enters the cycle of nitrogen elements. Ammonia nitrogen has toxic and side effects on fish in water, which can lead to the death of aquatic organisms and the poisoning and death of water-drinking organisms. Nitrite can combine with protein in the human body to form a nitrosamine with strong carcinogenic effect. If you drink it for a long time, it will have extremely adverse effects on your health. Therefore, the determination of the total nitrogen concentration in the water body is helpful to evaluate the water pollution and "self-purification" status, and the total nitrogen concentration is an important indicator of water environment quality monitoring in my country.

At present, the total nitrogen concentration is mainly measured by high-temperature oxidation of alkaline potassium persulfate, and then by spectrophotometry such as ultraviolet, phenolic disulfonic acid or thymol; some methods also use hydrazine sulfate and cadmium columns to oxidize the nitric acid obtained by oxidation. After salt reduction, analysis was performed by spectrophotometry. In these determination methods, not only the oxidation process of high-temperature digestion is complicated, but also the reducing agents such as hydrazine sulfate and cadmium column used are carcinogenic and toxic, which is not conducive to the green development of pollutant detection methods.

Therefore, it is very necessary to establish a detection method for the determination of total nitrogen concentration in water that meets the needs of green development.

SUMMARY OF THE INVENTION

The problem solved by the present invention is that the current detection method for measuring the total nitrogen concentration in the water body does not meet the requirements of green development.

In order to solve the above problems, the present invention provides a fluorescence analysis method for measuring the concentration of total nitrogen in a water body, comprising the following steps:

S1: prepare a first blank solution with hydrochloric acid solution, potassium bromide solution and fluorescent probe solution, and measure the first fluorescence intensity F1 of the first blank solution at a specific excitation wavelength and a specific emission wavelength;

S2: Mix the first blank solution and a series of nitrite standard solutions with different concentrations to prepare a series of standard working solutions, and respectively measure a series of standard working solutions at the specific excitation wavelength and the specific emission wavelength The second fluorescence intensity F2;

S3: Draw a standard curve according to the first fluorescence intensity F1, the second fluorescence intensity F2 of a series of the standard working solutions, and a series of nitrogen concentrations in the nitrite standard solution;

S4: sequentially adding an oxidant and a reducing agent to the sample to be tested, and subjecting the sample to be tested to ammonia nitrogen oxidation treatment and nitrate reduction treatment to obtain a reduced sample to be tested;

S5: Add the oxidizing agent and the reducing agent to the first blank solution to prepare a second blank solution, and measure the third blank solution of the second blank solution at the specific excitation wavelength and the specific emission wavelength Fluorescence intensity F3;

S6: adding the second blank solution to the sample to be tested after reduction to obtain a pretreated sample, and measuring the fourth fluorescence intensity F4 of the pretreated sample at the specific excitation wavelength and the specific emission wavelength ;

S7: Calculate the total nitrogen concentration in the sample to be tested according to the fourth fluorescence intensity F4, the third fluorescence intensity F3 and the standard curve.

Optionally, the fluorescent probe solution includes an amino acid solution.

Optionally, the specific excitation wavelength is 278 nm; the specific emission wavelength is 465 nm.

Optionally, preparing a series of standard working solutions in step S2 includes:

S21: prepare a series of standard solutions of nitrite with different concentrations;

S22: After adding the first blank solution to a series of different concentrations of the nitrite standard solution, the volume is fixed, mixed and allowed to stand for 10 minutes to obtain a series of the standard working solutions.

Optionally, step S3 includes:

S31: Calculate the difference between the first fluorescence intensity F1 and a series of the second fluorescence intensity F2, respectively, to obtain a series of first fluorescence intensity difference ΔF1;

S32: Draw the standard curve with the concentration of nitrogen in the series of the nitrite standard solutions as the abscissa and the series of the first fluorescence intensity difference ΔF1 as the ordinate.

Optionally, the standard curve is a straight line.

Optionally, step S4 includes:

S41: adding potassium bromate solution and sodium hydroxide solution to the sample to be tested, and performing an oxidation reaction at room temperature to obtain the sample to be tested after oxidation;

S42: Add sodium borohydride solution to the sample to be tested after oxidation, shake well and perform a reduction reaction at room temperature to obtain the sample to be tested after reduction.

Optionally, the time of the oxidation reaction in step S41 is 30min.

Optionally, the time range of the restoration in step S42 is 5 min to 10 min.

Optionally, step S7 includes:

S71: Calculate the difference between the third fluorescence intensity F3 and the fourth fluorescence intensity F4 to obtain the second fluorescence intensity difference ΔF2;

S72: Find the total nitrogen concentration in the sample to be tested from the standard curve according to the second fluorescence intensity difference ΔF2.

Compared with the prior art, the fluorescence analysis method for measuring the concentration of total nitrogen in water provided by the present invention has the following advantages:

The fluorescence analysis method for measuring the concentration of total nitrogen in water provided by the present invention, after the sample to be tested is subjected to ammonia nitrogen oxidation treatment, nitrate reduction treatment and diazotization reaction with a fluorescent probe, the fluorescence quenching degree of the fluorescent probe is used to determine the fluorescence quenching degree. The total nitrogen concentration in the sample to be tested has a wide linear range, high sensitivity, few types of reagents, weak toxicity, small dosage, simple experimental operation, and meets the green development requirements of the detection method.

Description of drawings

Fig. 1 is a graph showing the change of fluorescence spectrum of amino J acid in the determination of total nitrogen concentration according to the present invention.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

In order to solve the problem that the current detection method for measuring the total nitrogen concentration in the water body does not meet the needs of green development, the present invention provides a fluorescence analysis method for measuring the total nitrogen concentration in the water body, comprising the following steps:

S1: prepare a first blank solution with hydrochloric acid solution, potassium bromide solution and fluorescent probe solution, and measure the first fluorescence intensity F1 of the first blank solution at a specific excitation wavelength and a specific emission wavelength;

S2: Mix the first blank solution and a series of standard solutions of nitrite with different concentrations to prepare a series of standard working solutions, and respectively measure the second fluorescence intensity F2 of a series of standard working solutions at specific excitation wavelength and specific emission wavelength ;

S3: draw a standard curve according to the first fluorescence intensity F1, the second fluorescence intensity F2 of a series of standard working solutions, and the concentration of nitrogen in a series of nitrite standard solutions;

S4: sequentially adding an oxidizing agent and a reducing agent to the sample to be tested, and subjecting the sample to be tested to ammonia nitrogen oxidation treatment and nitrate reduction treatment to obtain a reduced sample to be tested;

S5: adding the oxidizing agent and reducing agent in step S4 to the above-mentioned first blank solution to prepare a second blank solution, and measuring the third fluorescence intensity F3 of the second blank solution at a specific excitation wavelength and a specific emission wavelength;

S6: adding a second blank solution to the sample to be tested after reduction to obtain a pretreated sample, and measuring the fourth fluorescence intensity F4 of the pretreated sample at a specific excitation wavelength and a specific emission wavelength;

S7: Calculate the total nitrogen concentration in the sample to be tested according to the fourth fluorescence intensity F4, the third fluorescence intensity F3 and the standard curve.

The fluorescent probe solution can undergo a diazo reaction with nitrite. After the diazo reaction, the fluorescence of the fluorescent probe is quenched, and the fluorescence intensity decreases; this application calculates the degree of decrease in the fluorescence intensity of the fluorescent probe solution after the diazo reaction. Total nitrogen concentration in the water body.

In order to facilitate the carrying out of the diazo reaction, the application adds hydrochloric acid solution and potassium bromide solution to the reaction system; wherein the function of the hydrochloric acid solution is to adjust the pH value of the reaction system to acidity, so as to facilitate the carrying out of the diazo reaction; this application Preferably, the concentration of hydrochloric acid added in step S1 is 2.0 mol/L, and the amount of hydrochloric acid solution added ranges from 1.6 mL to 1.8 mL; the function of adding potassium bromide solution is to promote the rapid diazo reaction at room temperature, thereby simplifying the experimental operation. Shorten the measurement time; it is preferred in the present application that the concentration of the potassium bromide solution in step S1 is 2.5 mol/L, and the addition amount of the potassium bromide solution is 1.7 mL.

Specifically, in order to facilitate the determination of the total nitrogen concentration in the water body, the present invention prepares a first blank solution and a plurality of nitrite standard solutions with different nitrite concentrations, and further uses the first blank solution and the nitrite standard solution. The solution prepares a series of standard working solutions, and then respectively measures the fluorescence intensity of the first blank solution and multiple standard working solutions; the preferred nitrite in this application is sodium nitrite; wherein, there is no nitrite in the first blank solution , therefore, no diazo reaction occurred in the first blank solution; and since a series of standard working solutions were obtained by mixing the first blank solution with a series of nitrite standard solutions with different concentrations, the first blank solution was mixed with nitrous acid During the mixing process of the salt solution, the fluorescent probe in the first blank solution undergoes a diazotization reaction with nitrite, resulting in fluorescence quenching of the fluorescent probe; therefore, a series of standard working solutions are composed of the first blank solution. The fluorescent probe is obtained after reacting with a series of nitrite solutions with different concentrations, and the fluorescent probe in the standard working solution has different degrees of fluorescence quenching; in different nitrite standard solutions, the concentration of nitrite is different, The amount of fluorescent probe participating in the diazo reaction is different, and the degree of fluorescence quenching of the fluorescent probe is also different. Specifically, the higher the concentration of nitrite, the more the amount of fluorescent probe participating in the reaction. The greater the degree of fluorescence quenching, the smaller the value of the measured second fluorescence intensity F2 in the standard working solution.

Since the concentration of nitrogen in each nitrite standard solution is known, according to the first fluorescence intensity F1 of the first blank solution, the second fluorescence intensity F2 of each standard working solution and the nitrogen concentration in each nitrite standard solution, namely The nitrogen concentration in the nitrite participating in the diazo reaction is performed by curve fitting and a standard curve is drawn; the standard curve can reflect the difference between the nitrogen concentration corresponding to the nitrite ion in the nitrite standard solution and the fluorescence intensity of the standard working solution. relation.

Since the obtained standard curve reflects the relationship between the nitrogen concentration corresponding to nitrite in the nitrite standard solution and the fluorescence intensity of the standard working solution, and the nitrogen element mainly exists in the water body in the form of ammonia nitrogen and nitrate, it is necessary to pass the The curve obtains the total nitrogen concentration in the sample to be tested. Before testing the sample to be tested, the sample to be tested, that is, the water body to be tested, is subjected to ammonia nitrogen oxidation treatment, and the ammonia nitrogen and most of the organic nitrogen in the sample to be tested are oxidized to nitrous acid. salt to obtain the sample to be tested after oxidation; then the sample to be tested after oxidation is subjected to nitrate reduction treatment to reduce the nitrate in the sample to be tested after oxidation to nitrite, and at the same time, it can also reduce the residual oxidant introduced in the ammonia nitrogen oxidation treatment Remove to obtain the sample to be tested after reduction; after ammonia nitrogen oxidation treatment and nitrate reduction treatment, all nitrogen elements in the sample to be tested are converted into nitrite, that is, all nitrogen elements in the sample to be tested after reduction are nitrite. The nitrogen concentration corresponding to the nitrite in the sample to be tested after the reduction is measured, that is, the total nitrogen concentration in the sample to be tested.

In order to measure the nitrite concentration in the sample to be tested after reduction, the oxidant required for ammonia nitrogen oxidation treatment and the required oxidant for nitrate reduction treatment were added to the first blank solution prepared with hydrochloric acid solution, potassium bromide solution and fluorescent probe solution. The reducing agent of , obtains the second blank solution; because the second blank solution does not contain nitrite, the diazotization reaction does not occur in the second blank solution; measure the second blank solution at the specific excitation wavelength and the specific emission wavelength. The third fluorescence intensity F3.

The second blank solution is prepared by adding an oxidant and a reducing agent to the first blank solution, in order to eliminate the measurement error caused by excessive reducing agent and other substances in the sample to be tested after reduction, and improve the accuracy of the detection results .

A second blank solution is added to the sample to be tested after reduction, the nitrite in the sample to be tested after reduction undergoes a diazotization reaction with the fluorescent probe solution, and the fluorescence of the fluorescent probe is quenched to obtain a pretreated sample; The fourth fluorescence intensity F4 of the processed sample at a specific excitation wavelength and a specific emission wavelength; since the standard curve is obtained according to the first fluorescence intensity F1, the second fluorescence intensity F2 and the concentration of nitrogen in the nitrite standard solution, the standard curve It reflects the relationship between the concentration of nitrogen in the nitrite standard solution and the fluorescence intensity of the standard working solution. Combining the measured third fluorescence intensity F3, fourth fluorescence intensity F4 and the standard curve, it is possible to obtain the participation in the diazo reaction. The nitrogen concentration of nitrite, that is, the nitrogen concentration in the nitrite in the sample to be tested after reduction is obtained, and the nitrogen concentration of the nitrite is the total nitrogen concentration in the sample to be tested.

The fluorescence analysis method for measuring the concentration of total nitrogen in water provided by the present invention, after the sample to be tested is subjected to ammonia nitrogen oxidation treatment, nitrate reduction treatment and diazotization reaction with a fluorescent probe, the fluorescence quenching degree of the fluorescent probe is used to determine the fluorescence quenching degree. The total nitrogen concentration in the sample to be tested has few types of reagents, weak toxicity, small dosage, simple experimental operation, and meets the green development requirements of the detection method.

In the present application, the total nitrogen concentration in the water body is determined by the fluorescence analysis method, the detection process is green, the equipment used is moderately expensive, the instrument is easy to operate, and the measurement process is highly sensitive, and can be widely used in the fields of food, environment and biology.

In the present application, the fluorescent probe solution preferably includes an amino acid solution, that is, a 6-amino-1,3-naphthalenedisulfonic acid solution.

The amino acid molecule contains 2 sulfonic acid groups, which not only has good water solubility, but also has little harm to the environment. It is a fluorescent probe with excellent performance; using amino J acid as a fluorescent probe, the fluorescent signal is strong and stable, with a wide detection linear range, low detection limit and high sensitivity , the advantage of high accuracy.

The specific excitation wavelength and specific emission wavelength in the present invention refer to the optimal excitation wavelength and optimal emission wavelength of the fluorescent probe. Specifically, in this application, the specific excitation wavelength is preferably 278 nm and the specific emission wavelength is 465 nm.

Referring to Figure 1, 1 is the excitation spectrum of amino J acid in the blank solution, 1' is the emission spectrum of amino J acid in the blank solution; 2 is the concentration of amino J acid under the action of 0.096mg/L nitrogen. Excitation spectrum, 2' is the emission spectrum of amino J acid under the action of nitrogen concentration of 0.096 mg/L.

The preparation of a series of standard working solutions in step S2 of this application includes:

S21: prepare a series of standard solutions of nitrite with different concentrations;

S22: After adding the first blank solution to a series of standard solutions of nitrite with different concentrations, the volume is fixed, mixed and allowed to stand for 10 minutes to obtain a series of standard working solutions.

After mixing the nitrite standard solution and the first blank solution, the fluorescent probe and the nitrite undergo diazotization reaction to obtain a series of standard working solutions.

In the process of the diazotization reaction in the present application, the reaction conditions are mild, and the reaction can be performed at room temperature; and the operation is simple, just mixing and standing; the reaction is rapid, and the reaction is complete after standing for 10 minutes.

Step S3 of this application includes:

S31: Calculate the difference between the first fluorescence intensity F1 and a series of second fluorescence intensities F2, respectively, to obtain a series of first fluorescence intensity difference ΔF1;

S32: Draw a standard curve with the concentration of nitrogen in a series of nitrite standard solutions as the abscissa and a series of first fluorescence intensity differences ΔF1 as the ordinate.

Specifically, the standard curve in this application is a straight line; the first fluorescence intensity difference ΔF1 has a linear relationship with the nitrogen concentration in the range of 0.0008 mg/L to 0.176 mg/L, that is, the total nitrogen in the water body is determined by the method provided in this application. The concentration fluorescence analysis method can measure the total nitrogen concentration in the water body in the range of 0.0008mg/L～0.176mg/L; further, the correlation coefficient R ² of the standard curve obtained in this application is 0.9972; this application can also Further, a standard curve equation is obtained according to the standard curve, and the standard curve equation is a binary linear equation with the difference between the nitrogen concentration of nitrite and the fluorescence intensity as a variable, and then the total nitrogen concentration in the water body is calculated according to the obtained standard curve equation. calculate.

Step S4 in this application specifically includes:

S41: adding potassium bromate solution and sodium hydroxide solution to the sample to be tested, and performing oxidation reaction at room temperature to obtain the sample to be tested after oxidation;

S42 : adding sodium borohydride solution to the sample to be tested after oxidation, shaking well and performing a reduction reaction at room temperature to obtain the sample to be tested after reduction.

Wherein the concentration of potassium bromate solution in step S41 is 0.15mmol/L, and the range of addition is 1.8mL-2.0mL; the concentration of sodium hydroxide solution is 0.05mol/L, and the range of addition is 0.3mL-0.6mL.

The potassium bromate solution added in this application is used as an oxidant to oxidize ammonia nitrogen and organic nitrogen in the sample to be tested to nitrite; wherein, in the alkaline solution, the electrode reaction of bromate is:

BrO ₃ ^- +3H ₂ O+6e=Br ^- +6OH ^- , φ ^θ =0.61V;

In an acidic solution, the electrode reaction of bromate is:

BrO ₃ ^- +6H ⁺ +6e=Br ^- +3H ₂ O, φ ^θ =1.423V;

That is, the acidity of the solution is enhanced, and the oxidative property of the bromate is significantly enhanced; therefore, the application uses potassium bromate as the oxidant, which can not only directly prepare the potassium bromate standard solution, but also easily control its oxidative properties; The ammonia nitrogen and organic nitrogen in the sample are oxidized to nitrite, and the oxidation reaction is carried out in a certain concentration of sodium hydroxide solution.

Wherein, the addition amount of the oxidant potassium bromate is suitable to be sufficient to oxidize all the ammonia nitrogen whose concentration range is in the linear range in the sample to be tested to nitrite.

In the prior art, the oxidation process of ammonia nitrogen concentration is measured, usually using hypobromite oxidation method and hypochlorite oxidation method; To generate elemental bromine, and then to generate hypobromite under strong alkali to be used as an oxidant, not only is the process cumbersome, but also requires a large amount of strong alkali solution. The hypochlorite oxidation method often uses sodium hypochlorite as the oxidant, but the solution stability is poor, and its effective chlorine content needs to be calibrated with sodium thiosulfate solution, and the process is complicated.

In the ammonia nitrogen oxidation treatment process of the present application, the alkalinity of the solution is adjusted by adding sodium hydroxide solution to ensure that the oxidation strength of potassium bromate meets the needs of oxidizing ammonia nitrogen to nitrite, and no toxic by-products are generated during the reaction process, which is in line with green development. need.

Wherein, the oxidation reaction conditions in step S41 are mild, and can take place at room temperature; the reaction is rapid, and the oxidation reaction time is 30 minutes.

The application uses potassium bromate as the oxidant, which has stable properties and can directly prepare standard solutions without calibration; the oxidative strength of potassium bromate can be adjusted by changing the pH of the solution to meet the needs of oxidizing all ammonia nitrogen to nitrite nitrogen.

In step S42, the concentration of the sodium borohydride solution is 0.1 mol/L, and the addition amount ranges from 2.5 mL to 2.8 mL; sodium borohydride is used as a reducing agent to reduce nitrate in the sample to be tested after oxidation to nitrite, At the same time, potassium bromate, the oxidant remaining after the oxidation reaction in step S41, can also be removed.

The addition amount of the reducing agent sodium borohydride solution is enough to remove all the remaining potassium bromate in the oxidation process, so as to eliminate the influence of the residual potassium bromate oxidizing the amino acid to reduce its fluorescence intensity and improve the accuracy of the measurement results; The nitrates in the range are all converted into nitrites, and the residual sodium borohydride in the solution will not have any effect on the fluorescence intensity of amino J acid.

The 0.1 mol/L sodium borohydride solution used in the above experiments was added to 1 mL of 10 mol/L sodium hydroxide solution for preservation.

The reduction reaction is carried out at room temperature, the operation is convenient, the reaction is rapid, and the time range of shaking and standing is 5 min to 10 min to complete the reaction.

In this application, sodium borohydride plays a dual role, which can not only reduce nitrate to nitrite, but also eliminate excess oxidant in the oxidation process. At the same time, the residual sodium borohydride in the solution will not produce the fluorescent signal of amino acid. Any influence; the use of sodium borohydride avoids secondary pollution during the measurement.

Step S7 of this application includes:

S71: Calculate the difference between the third fluorescence intensity F3 and the fourth fluorescence intensity F4 to obtain the second fluorescence intensity difference ΔF2;

S72: According to the second fluorescence intensity difference ΔF2, the total nitrogen concentration in the sample to be tested is obtained from the obtained standard curve.

In this step, the second fluorescence intensity difference ΔF2 can also be substituted into the standard curve equation obtained according to the standard curve, that is, the binary linear equation, and the total nitrogen concentration in the sample to be tested can be obtained by solving the standard curve equation.

The fluorescence analysis method for measuring the concentration of total nitrogen in water provided by the present application has the advantages of wide linear range, high sensitivity, and simple and quick detection process.

For ease of understanding, the present application describes the fluorescence analysis method of total nitrogen concentration in water by way of specific examples.

Example 1

This embodiment provides a fluorescence analysis method for measuring the concentration of total nitrogen in a water body, and the method includes the following steps:

1. Preparation of ammonia nitrogen, nitrite and nitrate standard solutions.

Accurately weigh 38.22 mg of analytically pure ammonium chloride, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a 100.0 mg (NH ₄ ⁺ -N)/L ammonia nitrogen standard solution, that is, ammonia nitrogen standard solution. The concentration of 100.0mg/L.

Accurately weigh 49.29 mg of analytically pure sodium nitrite, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a 100.0 mg (NO ₂ ^- -N)/L nitrite stock solution, that is, the sodium nitrite stock solution. The concentration of nitrogen in the nitrate stock solution was 100.0 mg/L. Dilute the stock solution step by step to prepare standard nitrite solutions of different concentrations, namely, prepare standard solutions of nitrite with different nitrogen concentrations.

Accurately weigh 60.72 mg of analytically pure sodium nitrate, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a nitrate standard solution of 100.0 mg (NO ₃ ^- -N)/L, that is, the nitrate standard solution. The concentration of nitrogen in the solution was 100.0 mg/L.

2. Drawing of the standard curve.

A series of 25mL volumetric flasks were added with different concentrations of nitrite standard solutions, and 1.6mL of 2.0mol/L hydrochloric acid solution and 1.7mL of 2.5mol/L hydrochloric acid solution were successively added to the different concentrations of nitrite standard solutions. Potassium bromide solution and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L, and set the volume to the mark to prepare a series of standard working solutions; mix well and let stand to make nitrite and amino acid recombine at room temperature Nitrogen reaction, after 10 min, the second fluorescence intensity F2 of the standard working solution was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm.

Take another 25mL volumetric flask without adding the nitrite standard solution, repeat the above steps to obtain the first blank solution; measure the first fluorescence intensity F1 of the first blank solution at the excitation wavelength of 278nm and the emission wavelength of 465nm.

Calculate the difference between the first fluorescence intensity F1 and a series of second fluorescence intensities F2 respectively, and obtain a series of first fluorescence intensity differences ΔF1; take the nitrogen concentration in a series of nitrite standard solutions as the abscissa, and take a series of first fluorescence intensity differences as the abscissa. A fluorescence intensity difference ΔF1 is the ordinate, and a standard curve is drawn.

3. Preparation and determination of low-concentration mixed simulated water samples.

Take a certain amount of ammonia nitrogen standard solution and nitrate standard solution and dilute with tap water to prepare a mixed simulated water sample containing 0.20 mg/L ammonia nitrogen and 0.10 mg/L nitrate nitrogen. Take a certain amount of mixed simulated water sample, add 1.8 mL potassium bromate solution with a concentration of 0.15 mmol/L and 0.3 mL sodium hydroxide solution with a concentration of 0.05 mol/L in turn, shake it and let it stand for oxidation at room temperature for 30 min, to obtain an oxidized sample to be tested.

Add 2.5 mL of sodium borohydride solution with a concentration of 0.1 mol/L to the sample to be tested after oxidation, shake well and let stand for reduction for 10 min, eliminate excess potassium bromate and reduce the nitrate in the water sample to obtain the sample to be tested after reduction.

After reduction, add 1.6 mL of hydrochloric acid solution with a concentration of 2.0 mol/L, 1.7 mL of potassium bromide solution with a concentration of 2.5 mol/L, and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L, and determine After 10 minutes, the fourth fluorescence of the pretreated sample was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm. Intensity F4.

Without adding the sample to be tested, repeat the above steps, namely 1.8mL potassium bromate solution with a concentration of 0.15mmol/L, 0.3mL sodium hydroxide solution with a concentration of 0.05mol/L, and 2.5mL sodium borohydride solution with a concentration of 0.1mol/L , 1.6mL of hydrochloric acid solution with concentration of 2.0mol/L, 1.7mL of potassium bromide solution with concentration of 2.5mol/L and 5.0mL of amino acid solution with concentration of 10.0mg/L were mixed to obtain the second blank solution; The third fluorescence intensity F3 of the second blank solution was measured at a wavelength of 278 nm and an emission wavelength of 465 nm.

Calculate the difference between the third fluorescence intensity F3 and the fourth fluorescence intensity F4 to obtain the second fluorescence intensity difference ΔF2; substitute the second fluorescence intensity difference ΔF2 into the standard curve to calculate the total nitrogen concentration.

Using the above method to measure 11 times in parallel, the average value of the total nitrogen concentration of the measured water sample is 0.302 mg/L, and the relative standard deviation is 1.74%, which proves that the method for measuring the total nitrogen concentration provided by the present invention has high accuracy and reproducibility. good sex.

Embodiment 2

1. Preparation of ammonia nitrogen, nitrite and nitrate standard solutions.

Accurately weigh 38.22 mg of analytically pure ammonium chloride, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a 100.0 mg (NH ₄ ⁺ -N)/L ammonia nitrogen standard solution, that is, ammonia nitrogen standard solution. The concentration of nitrogen was 100.0 mg/L.

Accurately weigh 49.29 mg of analytically pure sodium nitrite, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a 100.0 mg (NO ₂ ^- -N)/L nitrite stock solution, namely nitrous acid The concentration of nitrogen in the salt stock solution was 100.0 mg/L. Dilute the stock solution step by step to prepare standard nitrite solutions of different concentrations, namely, prepare standard solutions of nitrite with different nitrogen concentrations.

Accurately weigh 60.72 mg of analytically pure sodium nitrate, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a nitrate standard solution of 100.0 mg (NO ₃ ^- -N)/L, that is, nitrate standard solution The nitrogen concentration was 100.0 mg/L.

2. Drawing of the standard curve.

A series of 25mL volumetric flasks were added with different concentrations of nitrite standard solutions, and 1.8mL of 2.0mol/L hydrochloric acid solution and 1.7mL of 2.5mol/L hydrochloric acid solution were added to the different concentrations of nitrite standard solutions. Potassium bromide solution and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L, and set the volume to the mark to prepare a series of standard working solutions; mix well and let stand to make nitrite and amino acid recombine at room temperature Nitrogen reaction, after 10 min, the second fluorescence intensity F2 of the standard working solution was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm.

Take another 25mL volumetric flask without adding sodium nitrite standard solution, repeat the above steps to obtain the first blank solution; measure the first fluorescence intensity F1 of the first blank solution at the excitation wavelength of 278nm and the emission wavelength of 465nm.

Calculate the difference between the first fluorescence intensity F1 and a series of second fluorescence intensities F2 respectively, and obtain a series of first fluorescence intensity differences ΔF1; take the concentration of nitrogen in a series of nitrite standard solutions as the abscissa, and take a series of first fluorescence intensity differences as the abscissa. A fluorescence intensity difference ΔF1 is the ordinate, and a standard curve is drawn.

3. Preparation and determination of high-concentration mixed simulated water samples.

Take a certain amount of ammonia nitrogen standard solution and nitrate standard solution and dilute with tap water to prepare a mixed simulated water sample containing 1.20 mg/L ammonia nitrogen and 1.40 mg/L nitrate nitrogen. Take a certain amount of mixed simulated water sample solution, add 2.0 mL of potassium bromate solution with a concentration of 0.15 mmol/L and 0.6 mL of sodium hydroxide solution with a concentration of 0.05 mol/L in turn, shake and let stand for oxidation at room temperature for 30 min to obtain oxidation sample to be tested.

Add 2.8 mL of sodium borohydride solution with a concentration of 0.1 mol/L to the sample to be tested after oxidation, shake well and let stand for reduction for 10 min, eliminate excess potassium bromate and reduce nitrate in the water sample to obtain the sample to be tested after reduction.

After the reduction, 1.8 mL of hydrochloric acid solution with a concentration of 2.0 mol/L, 1.7 mL of potassium bromide solution with a concentration of 2.5 mol/L and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L were added to the sample. After 10 minutes, the fourth fluorescence of the pretreated sample was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm. Intensity F4.

Without adding the sample to be tested, repeat the above steps, namely 2.0mL potassium bromate solution with a concentration of 0.15mmol/L, 0.6mL sodium hydroxide solution with a concentration of 0.05mol/L, and 2.8mL sodium borohydride solution with a concentration of 0.1mol/L. , 1.8mL of hydrochloric acid solution with a concentration of 2.0mol/L, 1.7mL of potassium bromide solution with a concentration of 2.5mol/L and 5.0mL of amino acid solution with a concentration of 10.0mg/L were mixed to obtain a second blank solution; The third fluorescence intensity F3 of the second blank solution was measured at a wavelength of 278 nm and an emission wavelength of 465 nm.

Calculate the difference between the third fluorescence intensity F3 and the fourth fluorescence intensity F4 to obtain the second fluorescence intensity difference ΔF2; substitute the second fluorescence intensity difference ΔF2 into the standard curve to calculate the total nitrogen concentration.

Using the above method to measure 11 times in parallel, the average value of the total nitrogen concentration of the measured water sample is 2.586 mg/L, and the relative standard deviation is 0.86%, which proves that the measuring method of the total nitrogen concentration provided by the present invention has high accuracy and reproducibility. good sex.

Embodiment 3

1. Preparation of nitrite standard solution.

Accurately weigh 49.29 mg of analytically pure sodium nitrite, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a 100.0 mg (NO ₂ ^- -N)/L nitrite stock solution, namely nitrous acid The concentration of nitrogen in the salt stock solution was 100.0 mg/L. Dilute the stock solution step by step to prepare standard nitrite solutions of different concentrations, namely, prepare standard solutions of nitrite with different nitrogen concentrations.

2. Drawing of standard working curve.

A series of 25mL volumetric flasks were added with different concentrations of nitrite standard solutions, and 1.7mL of 2.0mol/L hydrochloric acid solution and 1.7mL of 2.5mol/L hydrochloric acid solution were added to the different concentrations of nitrite standard solutions. Potassium bromide solution and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L, and set the volume to the mark to prepare a series of standard working solutions; mix well and let stand to make nitrite and amino acid recombine at room temperature Nitrogen reaction, after 10 min, the second fluorescence intensity F2 of the standard working solution was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm.

Take another 25mL volumetric flask without adding sodium nitrite standard solution, repeat the above steps to obtain the first blank solution; measure the first fluorescence intensity F1 of the first blank solution at the excitation wavelength of 278nm and the emission wavelength of 465nm.

Calculate the difference between the first fluorescence intensity F1 and a series of second fluorescence intensities F2 respectively, and obtain a series of first fluorescence intensity differences ΔF1; take the concentration of nitrogen in a series of nitrite standard solutions as the abscissa, and take a series of first fluorescence intensity differences as the abscissa. A fluorescence intensity difference ΔF1 is the ordinate, and a standard curve is drawn.

3. Treatment and determination of surface water samples.

After the water collector was washed three times with surface water, 200 mL of fresh surface water was collected, allowed to stand, and the insolubles were removed by filtration to obtain a fresh surface water sample solution.

Take a certain amount of fresh surface water sample solution, add 1.9 mL of potassium bromate solution with a concentration of 0.15 mmol/L and 0.5 mL of sodium hydroxide solution with a concentration of 0.05 mol/L in turn, shake and let stand for oxidation at room temperature for 30 minutes to obtain oxidation sample to be tested.

Add 2.7 mL of sodium borohydride solution with a concentration of 0.1 mol/L to the sample to be tested after oxidation, shake well and let stand for reduction for 10 min, eliminate excess potassium bromate and reduce possible nitrate in the sample to nitrite to obtain reduction sample to be tested.

After the reduction, 1.7 mL of hydrochloric acid solution with a concentration of 2.0 mol/L, 1.7 mL of potassium bromide solution with a concentration of 2.5 mol/L and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L were added to the sample. After 10 minutes, the fourth fluorescence of the pretreated sample was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm. Intensity F4.

Do not add the sample to be tested, repeat the above steps, namely 1.9 mL of potassium bromate solution with a concentration of 0.15 mmol/L, 0.5 mL of sodium hydroxide solution with a concentration of 0.05 mol/L, and 2.7 mL of sodium borohydride solution with a concentration of 0.1 mol/L. , 1.7mL of hydrochloric acid solution with concentration of 2.0mol/L, 1.7mL of potassium bromide solution with concentration of 2.5mol/L and 5.0mL of amino acid solution with concentration of 10.0mg/L were mixed to obtain the second blank solution; The third fluorescence intensity F3 of the second blank solution was measured at a wavelength of 278 nm and an emission wavelength of 465 nm.

Calculate the difference between the third fluorescence intensity F3 and the fourth fluorescence intensity F4 to obtain the second fluorescence intensity difference ΔF2; substitute the second fluorescence intensity difference ΔF2 into the standard curve to calculate the total nitrogen concentration.

Five parallel determinations were carried out by the standard addition method, and the recovery rate of total nitrogen was between 93.5% and 103.1%, which met the requirements. The relative standard deviation is between 0.44% and 0.95%, and the reproducibility is good. It is proved that the measuring method of the total nitrogen concentration provided by the present invention has high measurement result accuracy.

Embodiment 4

1. Preparation of nitrite standard solution.

Accurately weigh 49.29 mg of analytically pure sodium nitrite, add an appropriate amount of secondary distilled water to dissolve, and put it in a 100.0 mL brown volumetric flask to prepare a 100.0 mg (NO ₂ ^- -N)/L nitrite stock solution, namely nitrous acid The concentration of nitrogen in the salt stock solution was 100.0 mg/L. Dilute the stock solution step by step to prepare standard nitrite solutions of different concentrations, namely, prepare standard solutions of nitrite with different nitrogen concentrations.

2. Drawing of standard working curve.

A series of 25mL volumetric flasks were added with different concentrations of nitrite standard solutions, and 1.7mL of 2.0mol/L hydrochloric acid solution and 1.7mL of 2.5mol/L hydrochloric acid solution were added to the different concentrations of nitrite standard solutions. Potassium bromide solution and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L, and set the volume to the mark to prepare a series of standard working solutions; mix well and let stand to make nitrite and amino acid recombine at room temperature Nitrogen reaction, after 10 min, the second fluorescence intensity F2 of the standard working solution was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm.

Take another 25mL volumetric flask without adding sodium nitrite standard solution, repeat the above steps to obtain the first blank solution; measure the first fluorescence intensity F1 of the first blank solution at the excitation wavelength of 278nm and the emission wavelength of 465nm.

Calculate the difference between the first fluorescence intensity F1 and a series of second fluorescence intensities F2 respectively, and obtain a series of first fluorescence intensity differences ΔF1; take the concentration of nitrogen in a series of nitrite standard solutions as the abscissa, and take a series of first fluorescence intensity differences as the abscissa. A fluorescence intensity difference ΔF1 is the ordinate, and a standard curve is drawn.

3. Collection and determination of rainwater samples.

Collect the rainfall of a certain day in a clean and dry container, take 200 mL of the collected rainwater, let it stand, and remove the insoluble matter by filtration to obtain a fresh rainwater sample solution.

Take a certain amount of fresh rainwater sample solution, add 1.9 mL of potassium bromate solution with a concentration of 0.15 mmol/L and 0.4 mL of sodium hydroxide solution with a concentration of 0.05 mol/L in turn, shake it and let it stand for oxidation at room temperature for 30 min, to obtain an oxidized sample to be tested.

After oxidation, add 2.6 mL of sodium borohydride solution with a concentration of 0.1 mol/L to the sample to be tested, shake well and let stand for reduction for 10 min to eliminate excess potassium bromate and reduce possible nitrate in the sample to nitrite to obtain reduction. sample to be tested.

After the reduction, 1.7 mL of hydrochloric acid solution with a concentration of 2.0 mol/L, 1.7 mL of potassium bromide solution with a concentration of 2.5 mol/L and 5.0 mL of amino acid solution with a concentration of 10.0 mg/L were added to the sample. After 10 minutes, the fourth fluorescence of the pretreated sample was measured at the excitation wavelength of 278 nm and the emission wavelength of 465 nm. Intensity F4.

Do not add the sample to be tested, repeat the above steps, namely 1.9 mL of potassium bromate solution with a concentration of 0.15 mmol/L, 0.4 mL of sodium hydroxide solution with a concentration of 0.05 mol/L, and 2.6 mL of sodium borohydride solution with a concentration of 0.1 mol/L , 1.7mL of hydrochloric acid solution with a concentration of 2.0mol/L, 1.7mL of potassium bromide solution with a concentration of 2.5mol/L and 5.0mL of amino acid solution with a concentration of 10.0mg/L were mixed to obtain a second blank solution; The third fluorescence intensity F3 of the second blank solution was measured at a wavelength of 278 nm and an emission wavelength of 465 nm.

Calculate the difference between the third fluorescence intensity F3 and the fourth fluorescence intensity F4 to obtain the second fluorescence intensity difference ΔF2; substitute the second fluorescence intensity difference ΔF2 into the standard curve to calculate the total nitrogen concentration.

The standard addition method was used for five parallel determinations, and the recovery rate of total nitrogen was between 97.5% and 100.5%, which met the requirements. The relative standard deviation is between 0.17% and 1.36%, and the reproducibility is good. It is proved that the measuring method of the total nitrogen concentration provided by the present invention has high measurement result accuracy.

Although the present disclosure is disclosed above, the scope of protection of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims (10)

1. a fluorescence analysis method of measuring total nitrogen concentration in water body, is characterized in that, comprises the steps: S1: prepare a first blank solution with hydrochloric acid solution, potassium bromide solution and fluorescent probe solution, and measure the first fluorescence intensity F1 of the first blank solution at a specific excitation wavelength and a specific emission wavelength; S2: Mix the first blank solution and a series of nitrite standard solutions with different concentrations to prepare a series of standard working solutions, and respectively measure a series of standard working solutions at the specific excitation wavelength and the specific emission wavelength The second fluorescence intensity F2; S3: Draw a standard curve according to the first fluorescence intensity F1, the second fluorescence intensity F2 of a series of the standard working solutions, and a series of nitrogen concentrations in the nitrite standard solution; S4: sequentially adding an oxidant and a reducing agent to the sample to be tested, and subjecting the sample to be tested to ammonia nitrogen oxidation treatment and nitrate reduction treatment to obtain a reduced sample to be tested; S5: Add the oxidizing agent and the reducing agent to the first blank solution to prepare a second blank solution, and measure the third blank solution of the second blank solution at the specific excitation wavelength and the specific emission wavelength Fluorescence intensity F3; S6: adding the second blank solution to the sample to be tested after reduction to obtain a pretreated sample, and measuring the fourth fluorescence intensity F4 of the pretreated sample at the specific excitation wavelength and the specific emission wavelength ; S7: Calculate the total nitrogen concentration in the sample to be tested according to the fourth fluorescence intensity F4, the third fluorescence intensity F3 and the standard curve. 2 . The fluorescence analysis method for measuring the concentration of total nitrogen in water according to claim 1 , wherein the fluorescent probe solution comprises an amino acid solution. 3 . 3 . The fluorescence analysis method for measuring the concentration of total nitrogen in water according to claim 2 , wherein the specific excitation wavelength is 278 nm; the specific emission wavelength is 465 nm. 4 . 4. the fluorescence analysis method of measuring total nitrogen concentration in water body as claimed in claim 1 is characterized in that, in step S2, preparing a series of standard working solutions comprises: S21: prepare a series of standard solutions of nitrite with different concentrations; S22: After adding the first blank solution to a series of different concentrations of the nitrite standard solution, the volume is fixed, mixed and allowed to stand for 10 minutes to obtain a series of the standard working solutions. 5. The fluorescence analysis method for measuring total nitrogen concentration in water body as claimed in claim 1, wherein step S3 comprises: S31: Calculate the difference between the first fluorescence intensity F1 and a series of the second fluorescence intensity F2, respectively, to obtain a series of first fluorescence intensity difference ΔF1; S32: Draw the standard curve with the concentration of nitrogen in the series of the nitrite standard solutions as the abscissa and the series of the first fluorescence intensity difference ΔF1 as the ordinate. 6 . The fluorescence analysis method for measuring the concentration of total nitrogen in a water body according to claim 5 , wherein the standard curve is a straight line. 7 . 7. The fluorescence analysis method for measuring total nitrogen concentration in water body as claimed in claim 1, wherein step S4 comprises: S41: adding potassium bromate solution and sodium hydroxide solution to the sample to be tested, and performing an oxidation reaction at room temperature to obtain the sample to be tested after oxidation; S42: Add sodium borohydride solution to the sample to be tested after oxidation, shake well, and perform a reduction reaction at room temperature to obtain the sample to be tested after reduction. 8 . The fluorescence analysis method for measuring the concentration of total nitrogen in a water body according to claim 7 , wherein the oxidation reaction time in step S41 is 30 min. 9 . 9 . The fluorescence analysis method for measuring the concentration of total nitrogen in a water body according to claim 7 , wherein the reduction time range in step S42 is 5 min to 10 min. 10 . 10. The fluorescence analysis method for measuring the concentration of total nitrogen in a water body according to any one of claims 1 to 9, wherein step S7 comprises: S71: Calculate the difference between the third fluorescence intensity F3 and the fourth fluorescence intensity F4 to obtain the second fluorescence intensity difference ΔF2; S72: Find the total nitrogen concentration in the sample to be tested from the standard curve according to the second fluorescence intensity difference ΔF2.

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