CN114354594A - A method for the determination of ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample - Google Patents

Abstract

The invention provides a method for measuring ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample, belonging to the technical field of nitrogen content measurement. When the ammonium nitrogen is measured, an oxidant (sodium hypochlorite and sodium chloroisocyanurate) and a color developing agent (salicylic acid) system are adopted, the ammonium nitrogen is oxidized into monochloramine under the action of the oxidant, the formed monochloramine and the color developing agent form indophenol, and the measurement is carried out by measuring the ultraviolet absorbance at 660 nm; when the nitric nitrogen, the nitrous nitrogen and the total dissolved nitrogen are measured, a new first Griess reagent (N- (1-naphthyl) ethylenediamine dihydrochloride) and a new second Griess reagent system (sulfanilamide and vanadium chloride) are adopted for measurement, the detection limit is low, and the sensitivity is high. The color reaction speed is stable and uniform, and the color reaction can be performed on a 96-well plate.

Description

Determination method of ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample

This application is a division of the application date of August 23, 2019, the application number of 201910782806.3, and the title of the invention "Determination of Ammonium Nitrogen, Nitrate Nitrogen, Nitrite Nitrogen and Total Dissolved Nitrogen in a Sample". Application.

technical field

The invention relates to the technical field of nitrogen content detection, in particular to a method for determining ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample.

Background technique

The methods for determining nitrogen content in soil in the prior art mainly rely on common UV-Vis spectrophotometers, which can only measure a limited number of samples at a time. Moreover, limited by the speed and uniformity of the reaction during the assay, it cannot be achieved on a 96-well plate (microplate reader). In addition, simply relying on ordinary UV-Vis spectrophotometer for measurement, the detection limit and accuracy need to be improved.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a method for measuring ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample. The 96-well plate (microplate reader) was used to determine various forms of nitrogen in the sample, and the method had better detection limit and accuracy, and realized the high-throughput determination of nitrogen.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a method for determining ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample, comprising the following steps:

The sample to be tested is prepared into a sample solution to be tested, the sample solution to be tested is mixed with an oxidant and a chromogenic agent, and the color is developed, the absorbance at 660 nm is measured, and the absorbance of the sample solution to be tested at 660 nm obtained according to the measurement and the predetermined ammonium nitrogen concentration-absorbance standard curve to obtain the ammonium nitrogen content in the sample to be tested;

Described oxidizing agent is the aqueous solution of sodium hypochlorite and chloroacetonitrile sodium urate, and described color developing agent is the sodium hydroxide solution of salicylic acid;

The mass ratio of sodium hypochlorite to sodium chloroisocyanurate in the oxidant is 6.5-7.2:0.025-0.035;

Mix the sample solution to be tested with the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, according to the measured absorbance at 540 nm of the sample solution to be tested and the predetermined nitrite nitrogen concentration -Absorbance standard curve to obtain the nitrite nitrogen content in the sample to be tested;

Mix the sample solution to be tested with nitrate reductase, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain the absorbance at 540 nm of the sample solution to be tested according to the measurement and the predetermined value. The nitrate nitrogen concentration-absorbance standard curve was obtained to obtain the apparent nitrate nitrogen content of the sample;

The first Griess reagent is an aqueous solution of N-(1-naphthyl)ethylenediamine dihydrochloride, and the second Griess reagent is an aqueous hydrochloric acid solution of sulfonamide and vanadium chloride;

The mass ratio of sulfonamide and vanadium chloride in the second Griess reagent is 4.5-5.5:0.10-0.20;

The solute of the nitrogen nitrate standard solution is potassium nitrate;

Mix the sample solution to be tested with persulfate oxidant and water to obtain a digestion solution; mix the digestion solution, nitrate reductase, the first Griess reagent and the second Griess reagent, develop color, and measure the absorbance at 540 nm , according to the measured absorbance of the sample solution to be tested at 540 nm and the predetermined total dissolved nitrogen concentration-absorbance standard curve, obtain the total dissolved nitrogen content in the sample to be tested;

The persulfate oxidant is a mixture of potassium persulfate, potassium permanganate, sodium hydroxide and boric acid;

The solute of the total dissolved nitrogen standard solution is glycine;

The difference between the apparent nitrate nitrogen content of the sample and the nitrite nitrogen content in the sample to be tested is the nitrate nitrogen content in the sample to be tested;

Establishment of standard curve: the standard curve includes ammonium nitrogen concentration-absorbance standard curve, nitrate nitrogen concentration-absorbance standard curve, nitrite nitrogen concentration-absorbance standard curve and total dissolved nitrogen concentration-absorbance standard curve;

The establishment of the ammonium nitrogen concentration-absorbance standard curve includes the following steps:

Prepare an ammonium nitrogen standard solution, mix the ammonium nitrogen standard solution, an oxidant and a color developer, develop a color, measure the absorbance at 660 nm, and obtain an ammonium nitrogen concentration-absorbance standard curve;

The solute of the ammonium nitrogen standard solution is ammonium chloride;

The establishment of the nitrate nitrogen concentration-absorbance standard curve includes the following steps:

Prepare a nitrate nitrogen standard solution, mix the nitrate nitrogen standard solution, nitrate reductase, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain a nitrate nitrogen concentration-absorbance standard curve ;

The establishment of the nitrite nitrogen concentration-absorbance standard curve includes the following steps:

Prepare a nitrite nitrogen standard solution, mix the nitrite nitrogen standard solution, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain a nitrite nitrogen concentration-absorbance standard curve; The solute of the nitrite nitrogen standard solution is sodium nitrite;

The establishment of the total dissolved nitrogen concentration-absorbance standard curve includes the following steps:

Prepare a total dissolved nitrogen standard solution, mix the total dissolved nitrogen standard solution, persulfate oxidant and water to obtain a digestion solution; combine the digestion solution, nitrate reductase, the first Griess reagent and the second Griess reagent Mix and develop color, measure the absorbance at 540nm, and obtain the standard curve of total dissolved nitrogen concentration-absorbance.

Preferably, the mass ratio of the glycine to the persulfate oxidant is 0.02-1.00:2500-3500.

Preferably, the mass ratio of potassium persulfate, potassium permanganate, sodium hydroxide and boric acid in the persulfate oxidant is 25:20-25:5-6:10-20.

Preferably, the sample includes soil or a body of water.

The invention provides a method for determining ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample. In the present invention, oxidizing agent (sodium hypochlorite and sodium chloroisocyanurate) and color developing agent (salicylic acid) system are used in the determination of ammonium nitrogen, and ammonium nitrogen is oxidized to monochloramine under the action of the oxidizing agent, and the formed monochloride The amine and the developer form indophenol, which is measured by measuring the UV absorbance at 660 nm. The color reaction speed is stable and uniform, and can be carried out on a 96-well plate. For nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen measurements, the first Griess reagent (N-(1-naphthyl)ethylenediamine dihydrochloride) and the second Griess reagent system (sulfonamide and chlorine Vanadium), the detection limit is low, the sensitivity is high, and the reaction process is stable and uniform, and can be carried out on a 96-well plate. The data of the examples show that the assay method provided by the present invention has high precision and accuracy, and has a lower detection limit for various nitrogen forms in the sample.

Description of drawings

Fig. 1 is ammonium nitrogen concentration-absorbance standard curve diagram;

Fig. 2 is nitrate nitrogen concentration-absorbance standard curve figure;

Fig. 3 is nitrite nitrogen concentration-absorbance standard curve figure;

Figure 4 is a graph of total dissolved nitrogen concentration-absorbance standard curve.

Detailed ways

The invention provides a method for determining ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample. First, a standard curve is established; the standard curve includes ammonium nitrogen concentration-absorbance standard curve, nitric nitrogen concentration Nitrogen concentration-absorbance standard curve, nitrite nitrogen concentration-absorbance standard curve and total dissolved nitrogen concentration-absorbance standard curve.

In the present invention, the sample is preferably soil or water body. In the present invention, when measuring ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in the soil, preferably the soil is prepared into a soil solution; the preparation method of the soil solution preferably includes the following steps :

Take fresh soil and pass it through a 2mm sieve, weigh 0.5g of the sieved soil (fresh weight), add 5mL of K ₂ SO ₄ aqueous solution with a concentration of 0.5mol/L, shake and extract at room temperature for 1h, pass through a 0.45mm filter membrane, and take the filtrate As a soil solution; the soil solution is divided into four groups for the determination of ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen; in the determination, the volume of each soil solution is preferably 1 mL.

In the present invention, the establishment of the ammonium nitrogen concentration-absorbance standard curve comprises the following steps:

Prepare an ammonium nitrogen standard solution, mix the ammonium nitrogen standard solution, an oxidant and a chromogenic agent, develop color, measure the absorbance at 660 nm, and obtain an ammonium nitrogen concentration-absorbance standard curve; the oxidant is sodium hypochlorite and chlorine An aqueous solution of sodium isocyanurate, and the color developer is an aqueous sodium hydroxide solution of salicylic acid.

In the present invention, the solute of the ammonium nitrogen standard solution is preferably ammonium chloride; in a specific embodiment of the present invention, the preparation method of the ammonium nitrogen standard solution preferably includes the following steps: weighing 3.819 g of chlorinated ammonium chloride Dissolve ammonium in water and dilute to 1000mL to obtain an ammonium nitrogen standard stock solution with a concentration (calculated as nitrogen) of 1000mg/L; take an appropriate amount of ammonium nitrogen standard stock solution and dilute to 100mL, and prepare the concentrations (calculated as nitrogen) 0.1mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.5m/L and 5mg/L ammonium nitrogen standard solution, respectively.

In the present invention, the mass ratio of sodium hypochlorite and sodium chloroisocyanurate in the oxidant is preferably 6.5-7.2:0.025-0.035. In a specific embodiment of the present invention, the preparation method of the oxidizing agent preferably includes the following steps: measuring 65 mL of sodium hypochlorite solution (available chlorine content>5.3%), dissolving it in 1000 mL water; under stirring conditions, slowly adding 0.025 g of chlorinated Sodium isocyanurate, standing overnight to obtain an oxidizing agent.

In a specific embodiment of the present invention, the preparation method of the color developer preferably includes the following steps: weighing 144 g of sodium salicylate, and dissolving it into 1000 mL of NaOH solution with a concentration of 0.3 mol/L to obtain the color developer.

In the present invention, the order of mixing the ammonium nitrogen standard solution, the oxidant and the color developer is preferably that the ammonium nitrogen standard solution and the oxidant are mixed first, and then mixed with the color developer. In the present invention, the addition amount of the oxidizing agent and the color developer is preferably excessive relative to the ammonium nitrogen content in the ammonium nitrogen standard solution, so that the ammonium nitrogen can be completely oxidized to monochloramine, and then combined with the subsequent salicylic acid Indophenol was formed and all characterized by color development.

In the present invention, the temperature of the color development is preferably room temperature, and the time is preferably 30 min.

In a specific embodiment of the present invention, the specific process of mixing and developing the ammonium nitrogen standard solution, the oxidant and the chromogenic agent is as follows: mixing 100 μL of the ammonium nitrogen standard solution, 20 μL of the oxidant and 50 μL of the chromogenic agent, at room temperature Under color development for 30min.

In the present invention, the establishment of the nitrate nitrogen concentration-absorbance standard curve includes the following steps:

Prepare a nitrate nitrogen standard solution, mix the nitrate nitrogen standard solution, nitrate reductase, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain a nitrate nitrogen concentration-absorbance standard curve ; The first Griess reagent is an aqueous solution of N-(1-naphthyl)ethylenediamine dihydrochloride, and the second Griess reagent is an aqueous hydrochloric acid solution of sulfonamide and vanadium chloride.

In the present invention, the solute of the nitrate nitrogen standard solution is preferably potassium nitrate; in a specific embodiment of the present invention, the preparation method of the nitrate nitrogen standard solution preferably includes the following steps: weighing 7.221g of potassium nitrate dissolved in In water, dilute to 1000mL to obtain a standard stock solution of nitrate nitrogen with a concentration (in nitrogen) of 1000mg/L; take an appropriate amount of standard stock solution of nitrate nitrogen and dilute to 100mL, and the preparation concentration (in terms of nitrogen) is 0.1 mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.5mg/L and 5mg/L nitrate nitrogen standard solution.

In the present invention, the nitrate reductase is preferably added in the form of an aqueous solution. In a specific embodiment of the present invention, the preparation method of the nitrate reductase solution preferably includes the following steps: adding 3 units of nitrate to Add 1 mL of a phosphate buffer solution with a pH of 7.5 to the ampoule of the reductase powder preparation, shake and mix for 30 min, and store it at -20°C for later use to obtain a nitrate reductase stock solution, which can be Stable storage for 3 to 4 months; carefully transfer all the nitrate reductase stock solution in the ampoule into a 50mL test tube with a pipette, rinse the original ampoule repeatedly with a phosphate buffer solution with a pH of 7.5, and moisten the phosphate. The whole lotion was transferred into the above 50mL test tube, and the volume was adjusted to 20mL with a phosphate buffer solution with a pH of 7.5 to obtain a nitrate reductase solution with a concentration of 0.15U/mL. The nitrate reductase solution can be stably stored at 4 °C 18h.

In a specific embodiment of the present invention, the preparation method of the first Griess reagent is preferably as follows: weighing 50 mg of N-(1-naphthyl)ethylenediamine dihydrochloride and mixing it with 250 mL of water to obtain the first Griess reagent.

In the present invention, the mass ratio of sulfonamide and vanadium chloride in the second Griess reagent is preferably 4.5-5.5:0.10-0.20, more preferably 4.8-5.3:0.12-0.20, more preferably 5.0:0.14. In a specific embodiment of the present invention, the preparation method of the second Griess reagent preferably includes the following steps: mixing 5.0 g of sulfonamide, 0.14 g of vanadium chloride and 500 mL of HCl solution with a concentration of 3 mol/L to obtain the second Griess reagent, the second Griess reagent is stable for several months.

In the present invention, the order in which the nitrate nitrogen standard solution, nitrate reductase, the first Griess reagent and the second Griess reagent are mixed is preferably firstly mixing the nitrate nitrogen standard solution and nitrate reductase, and then rapidly Add the first Griess reagent and the second Griess reagent. In the present invention, the nitrate reductase, the first Griess reagent and the second Griess reagent are preferably added in excess relative to the nitrate nitrogen content in the nitrate nitrogen standard solution, so that the nitrate nitrogen can be completely converted into nitrite nitrogen Nitrogen, and all are characterized by color development.

In the present invention, the temperature of the color development is preferably 37° C., and the time is preferably 60 min.

In a specific embodiment of the present invention, the specific process of mixing the nitrate nitrogen standard solution, nitrate reductase, the first Griess reagent and the second Griess reagent, and developing the color is preferably:

After mixing 100 μL of nitrate nitrogen standard solution and 100 μL of nitrate reductase solution, 50 μL of the first Griess reagent and 50 μL of the second Griess reagent were quickly added in sequence, and the color was developed at 37° C. for 60 min.

In the present invention, the establishment of the nitrite nitrogen concentration-absorbance standard curve comprises the following steps:

Prepare a nitrite nitrogen standard solution, mix the nitrite nitrogen standard solution, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain a nitrite nitrogen concentration-absorbance standard curve;

In the present invention, the solute of the nitrite nitrogen standard solution is preferably sodium nitrite; in a specific embodiment of the present invention, the preparation method of the nitrite nitrogen standard solution preferably includes the following steps: weighing 4.928g Sodium nitrite is dissolved in water, and the volume is adjusted to 1000mL to obtain a nitrite nitrogen standard stock solution with a concentration (calculated as nitrogen) of 1000mg/L; Nitrous nitrogen standard solution of 0.1 mg/L, 0.2 mg/L, 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.5 mg/L and 5 mg/L in nitrogen terms.

In the present invention, the first Griess reagent and the second Griess reagent are preferably the same as the first Griess reagent and the second Griess reagent involved in the establishment of the nitrate nitrogen concentration-absorbance standard curve, which will not be repeated here.

In the present invention, the order in which the nitrite nitrogen standard solution, the first Griess reagent and the second Griess reagent are mixed is preferably as follows: the first Griess reagent and the second Griess reagent are rapidly added to the nitrite nitrogen standard solution in sequence. . In the present invention, the added amount of the first Griess reagent and the second Griess reagent is preferably an excess relative to the nitrite nitrogen content in the nitrite nitrogen standard solution, so that all the nitrite nitrogen develops color.

In the present invention, the temperature of the color development is preferably 37° C., and the time is preferably 60 min.

In a specific embodiment of the present invention, the specific process of mixing the nitrite nitrogen standard solution, the first Griess reagent and the second Griess reagent, and developing the color is preferably as follows: quickly add 50 μL of nitrite nitrogen standard solution to 100 μL of the nitrite nitrogen standard solution. The first Griess reagent and 50 μL of the second Griess reagent were developed for 60 min at 37°C.

In the present invention, the establishment of the total dissolved nitrogen concentration-absorbance standard curve comprises the following steps:

Prepare a total dissolved nitrogen standard solution, mix the total dissolved nitrogen standard solution, persulfate oxidant and water to obtain a digestion solution; combine the digestion solution, nitrate reductase, the first Griess reagent and the second Griess reagent Mix and develop color, measure the absorbance at 540nm, and obtain the standard curve of total dissolved nitrogen concentration-absorbance.

In the present invention, the solute of the total dissolved nitrogen standard solution is preferably glycine. In a specific embodiment of the present invention, the preparation method of the total dissolved nitrogen standard solution preferably includes the following steps:

Weigh 5.362g of glycine and dissolve it in water, and dilute to 1000mL to obtain a total dissolved nitrogen standard stock solution with a concentration (calculated as nitrogen) of 1000mg/L; take an appropriate amount of total dissolved nitrogen standard stock solution to 100mL, and prepare The concentration (calculated as nitrogen) is 0.1mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.5mg/L, 5mg/L total dissolved nitrogen standard solution.

In the present invention, the persulfate oxidant is preferably a mixture of potassium persulfate, potassium permanganate, sodium hydroxide and boric acid; among the persulfate oxidants, potassium persulfate, potassium permanganate, sodium hydroxide and The mass ratio of boric acid is preferably 25:20 to 25:5 to 6:10 to 20, and more preferably 25:25:6.2:15.

In the present invention, the mass ratio of the glycine to the persulfate oxidant is preferably 0.02-1.00: 2500-3500, and the present invention controls the mass ratio of the glycine to the persulfate oxidant to be 0.02-1.00: 2500-3500, It can ensure that glycine is completely oxidized to obtain nitrate nitrogen.

In the present invention, the mixing sequence of the total dissolved nitrogen standard solution, the persulfate oxidant and water is preferably as follows: mixing the persulfate oxidant with water, an aqueous persulfate oxidant solution; then mixing the total dissolved nitrogen standard The solution is mixed with the aqueous persulfate oxidant solution.

In the present invention, the temperature of the digestion is preferably 120° C., and the time is preferably 40 min.

In a specific embodiment of the present invention, the total dissolved nitrogen standard solution is prepared, and the total dissolved nitrogen standard solution, the persulfate oxidant and water are mixed, and the specific process of obtaining the digestion solution preferably includes the following steps:

Weigh 5.362g of glycine and dissolve it in water, dilute to 1000mL, and prepare a total dissolved nitrogen stock solution with a concentration (calculated as nitrogen) of 1000mg/L; In terms of nitrogen), the total dissolved nitrogen standard solution of 0.1 mg/L, 0.2 mg/L, 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.5 mg/L and 5 mg/L;

Weigh 25g potassium persulfate, 25g potassium permanganate, 6.2g sodium hydroxide and 15g boric acid, and dilute to 1L with deionized water to obtain an aqueous persulfate oxidant solution;

Measure 2.5 mL of total dissolved nitrogen standard solution of different concentrations and 2.5 mL of persulfate oxidant aqueous solution, and digest at 120 °C for 40 min to obtain a digested solution.

In the present invention, the nitrate reductase, the first Griess reagent and the second Griess reagent are preferably the nitrate reductase, the first Griess reagent and the second Griess reagent involved in the establishment of the nitrate nitrogen concentration-absorbance standard curve The reagents are the same and will not be repeated here.

In the present invention, the order of mixing the digestion solution, nitrate reductase, the first Griess reagent and the second Griess reagent is preferably: first mix the digestion solution and nitrate reductase, and then quickly (30 seconds) inside) add the first Griess reagent and the second Griess reagent. In the present invention, the nitrate reductase, the first Griess reagent and the second Griess reagent are preferably added in excess relative to the nitrate nitrogen content in the digestion solution, so that all nitrate nitrogen is converted into nitrite, and the nitrogen Nitrate nitrogen is all colored.

In the present invention, the temperature of the color development is preferably 37° C., and the time is preferably 60 min.

In a specific embodiment of the present invention, the specific process of mixing the digestion solution, nitrate reductase, the first Griess reagent and the second Griess reagent, and developing the color is preferably as follows: mixing 100 μL of the digestion solution and 100 μL of the nitrate reductase solution Then, 50 μL of the first Griess reagent and 50 μL of the second Griess reagent were quickly added in sequence, and the color was developed at 37° C. for 60 min.

In the present invention, the total dissolved nitrogen includes nitrate nitrogen, nitrite nitrogen, ammonium nitrogen and dissolved organic nitrogen.

In the present invention, the concentration of corresponding nitrogen in the ammonium nitrogen standard solution, nitrate standard solution, nitrite standard solution and total dissolved nitrogen standard solution is preferably less than 5 mg/L; The concentration of various forms of nitrogen in ) generally does not exceed 5mg/L, so when the corresponding standard solution is prepared, 5mg/L is also used as the upper limit of concentration.

In the present invention, the absorbance values of the ammonium nitrogen standard solution, the nitric acid standard solution, the nitrite standard solution and the total dissolved nitrogen standard solution are all preferably in the range of 0.5 to 2.5; If the absorbance of the standard solution exceeds 3.0, it is preferable to dilute before measuring. The invention controls the absorbance value of each form of nitrogen standard solution or the sample solution to be tested to 0.5-2.5, ensures the accuracy of the absorbance value, and further improves the standard curve and the final sample to be tested. Accuracy of nitrate nitrogen and total dissolved nitrogen concentrations.

After obtaining the ammonium nitrogen concentration-absorbance standard curve, the nitrate nitrogen concentration-absorbance standard curve, the nitrite nitrogen concentration-absorbance standard curve and the total dissolved nitrogen concentration-absorbance standard curve, the present invention measures the ammonium nitrogen and nitric acid in the sample. nitrogen, nitrite, and total dissolved nitrogen.

In the present invention, the steps of measuring ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in the sample are preferably:

The sample to be tested is prepared into a sample solution to be tested, and the sample solution to be tested is subjected to color development according to the color reaction when each standard curve is established to obtain the absorbance of the corresponding nitrogen form, and is brought into the corresponding standard curve to obtain the standard The ammonium nitrogen content was measured by the curve, the nitrate nitrogen content was measured by the standard curve, the nitrite nitrogen content was measured by the standard curve, and the total dissolved nitrogen content was measured by the standard curve.

In the present invention, the step of determining the content of ammonium nitrogen in the sample to be tested is preferably as follows: mixing 100 μL of the sample solution to be tested, 20 μL of oxidant and 50 μL of color developer, developing color at room temperature for 30 min, and measuring the absorbance at 660 nm, Bring the obtained absorbance value into the standard curve of ammonium nitrogen content to obtain the standard curve to measure the ammonium nitrogen content, and the obtained standard curve to measure the ammonium nitrogen content is the ammonium nitrogen content in the sample to be tested.

In the present invention, the step of determining the content of nitrite nitrogen in the sample to be tested is preferably as follows: rapidly add 50 μL of the first Griess reagent and 50 μL of the second Griess reagent to 100 μL of the sample solution to be tested, and develop color at 37° C. for 60 min , measure the absorbance at 540nm, bring the obtained absorbance value into the nitrite nitrogen standard curve, obtain the standard curve to measure the nitrite nitrogen content, and the obtained standard curve to measure the nitrite nitrogen content is the sample to be tested Nitrite nitrogen content.

In the present invention, the step of determining the nitrate nitrogen content in the sample to be tested is preferably as follows: after mixing 100 μL of the sample solution to be tested and 100 μL of nitrate reductase solution, 50 μL of the first Griess reagent and 50 μL of the second Griess reagent are quickly added in sequence. Griess reagent, develop color at 37°C for 60min, measure the absorbance at 540nm, and insert the obtained absorbance value into the nitrate nitrogen standard curve to obtain the apparent nitrate nitrogen content of the sample, the sample apparent nitrate nitrogen The difference between the content and the nitrite nitrogen content measured by the standard curve is the nitrate nitrogen content in the sample to be tested.

In the present invention, the apparent nitrate nitrogen content of the sample is equal to the content of total nitrite nitrogen in the sample to be tested after reduction, and the content of total nitrite nitrogen in the sample to be tested after reduction is the nitrate content in the sample before reduction The sum of the nitrogen content and the nitrite nitrogen content, therefore, the difference between the apparent nitrate nitrogen content of the sample and the nitrite nitrogen content in the sample to be tested is the nitrate nitrogen content in the sample to be tested.

In the present invention, the determination step of the total dissolved nitrogen content in the sample to be tested is preferably as follows: 2.5 mL of the sample to be tested and 2.5 mL of an aqueous solution of persulfate oxidant (weigh 25 g of potassium persulfate, 25 g of potassium permanganate, 6.2g of sodium hydroxide and 15g of boric acid, and dilute to 1L with deionized water to obtain an aqueous solution of persulfate oxidant; ) Mix and digest at 120°C for 40min to obtain a digested solution; 100 μL of the digested solution and 100 μL of nitrate reductase solution After mixing, then quickly add 50 μL of the first Griess reagent and 50 μL of the second Griess reagent in turn, develop color at 37 ° C for 60 min, measure the absorbance at 540 nm, and bring the obtained absorbance value into the total dissolved nitrogen standard curve, A standard curve is obtained to measure the total dissolved nitrogen content, and the obtained standard curve to measure the total dissolved nitrogen content is the total dissolved nitrogen content in the sample to be tested.

The methods for determining ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in the samples provided by the present invention are described in detail below with reference to the examples, but they cannot be understood as limiting the protection scope of the present invention.

Example 1

Preparation of soil solution: take fresh soil and pass it through a 2mm sieve, weigh 0.5g of the soil (fresh weight) from the sieve, add 5mL of K ₂ SO ₄ aqueous solution with a concentration of 0.5mol/L, shake and extract at room temperature for 1h, pass 0.45μm filter membrane, take the filtrate as soil solution; divide the soil solution into four groups for the determination of ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen; during measurement, the volume of each described soil solution to 1mL.

(1) Establishment of ammonium nitrogen concentration-absorbance standard curve

Preparation of oxidant: measure 65mL of sodium hypochlorite solution (available chlorine content>5.3%), dissolve in 1000mL of water; under stirring conditions, slowly add 0.025g of sodium chloroisocyanurate, and let stand overnight to obtain the oxidant.

Preparation of color developer: Weigh 144 g of sodium salicylate and dissolve it into 1000 mL of NaOH solution with a concentration of 0.3 mol/L to obtain a color developer.

Preparation of ammonium nitrogen standard solution: Weigh 3.819g of ammonium chloride and dissolve it in water, and dilute to 1000mL to obtain an ammonium nitrogen standard stock solution with a concentration (calculated as nitrogen) of 1000mg/L; take an appropriate amount of ammonium nitrogen standard stock solution Dilute the volume to 100mL, and prepare the ammonium state of 0.1mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.5mg/L and 5mg/L respectively. Nitrogen standard solution.

Color development reaction: 100 μL of ammonium nitrogen standard solution + 20 μL of oxidant + 50 μL of chromogenic reagent; develop color at room temperature for 30 min, measure absorbance at 660 nm, and establish ammonium nitrogen concentration-absorbance standard curve, the results are shown in Figure 1, The parameters of the obtained standard curve are shown in Table 1. The detection limit of ammonium nitrogen in the obtained soil solution was determined by the external standard method, and the results are shown in Table 1. It can be seen from Table 1 that the ammonium nitrogen concentration-absorbance standard curve is in the range of ammonium nitrogen concentration (calculated as nitrogen) from 0.1 to 5 mg/L, the slope is 0.517, the intercept is 0.064, and the correlation coefficient is 0.95; The limit of detection (LOD) of this standard curve for ammonium nitrogen in soil solution is 0.021mg/L, which is 0.21mg/kg when converted to soil samples.

Table 1 Standard curve and detection limit for determination of ammonium nitrogen by external standard method

Four different soil samples were selected, and soil solutions were prepared according to the preparation method of soil samples; the same color reaction was used to determine the concentration of ammonium nitrogen in the soil solution; and the external standard method was used to verify the ammonium nitrogen concentration-absorbance standard curve The precision and accuracy of the determination of the four soil samples are shown in Table 2.

Table 2 The precision and accuracy of the standard curve for the determination of ammonium nitrogen by external standard method

It can be seen from Table 2 that the coefficient of variation of the ammonium nitrogen standard curve provided by the present invention is 2.57-5.21%, indicating that the method has high precision; the recovery rate of standard addition is 81-102%, indicating that the accuracy is high.

(2) Establishment of the standard curve of nitrate nitrogen concentration-absorbance

Preparation of nitrate reductase solution: add 1 mL of pH 7.5 phosphate buffer solution to an ampoule containing 3 units of nitrate reductase powder preparation (AtNaR2, EC# 1.7.1.1.), shake and mix for 30 min , stored at -20°C for later use, to obtain nitrate reductase stock solution;

Carefully transfer all the nitrate reductase stock solution in the ampoule into a 50mL test tube with a pipette, rinse the original ampoule repeatedly with a phosphate buffer solution with a pH of 7.5, and transfer the full amount of the phosphate rinse solution into the above 50mL test tube, Dilute to 20 mL with a phosphate buffer solution with a pH of 7.5 to obtain a nitrate reductase solution with a concentration of 0.15 U/mL; this solution can be stored stably at 4 °C for 18 h.

To prepare the first Griess reagent: Weigh 50 mg of N-(1-naphthyl)ethylenediamine dihydrochloride and mix with 250 mL of water to obtain the first Griess reagent.

Preparation of the second Griess reagent: 5 g of sulfonamide, 0.14 g of vanadium chloride and 500 mL of HCl solution with a concentration of 3 mol/L were mixed to obtain the second Griess reagent.

Preparation of nitrate nitrogen standard solution: Weigh 7.221g potassium nitrate and dissolve it in water, and dilute to 1000mL to obtain a nitrate nitrogen standard stock solution with a concentration (calculated as nitrogen) of 1000mg/L; take an appropriate amount of nitrate nitrogen standard stock solution to determine volume to 100mL, and obtain the nitrate standard with concentrations (calculated as nitrogen) of 0.1mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.5mg/L and 5mg/L respectively solution.

Color reaction: 100 μL nitrate nitrogen standard solution + 100 μL nitrate reductase solution + 50 μL first Griess reagent (add quickly) + 50 μL second Griess reagent (add quickly), a total of 300 μL reaction system; the reaction system was displayed at 37°C. Color for 60min, absorbance was measured at 540nm, and a standard curve of nitrate nitrogen concentration-absorbance was established. The results are shown in Figure 2, and the parameters of the obtained standard curve are shown in Table 3. The detection limit of nitrate nitrogen in the obtained soil solution was determined by the external standard method, and the results are shown in Table 3. It can be seen from Table 3 that the standard curve of nitrate nitrogen concentration-absorbance is in the range of nitrate nitrogen concentration (calculated as nitrogen) from 0.1 to 5 mg/L, the slope is 0.197, the intercept is 0.076, and the correlation coefficient is 0.97; The limit of detection (LOD) of this standard curve for nitrate nitrogen in soil solution was 0.024mg/L, and when converted to soil samples, the limit of detection was 0.24mg/kg.

Table 3 Determination of nitrate nitrogen standard curve and detection limit by external standard method

Four different soil samples were selected, and the soil solution was prepared according to the preparation method of soil samples; the same color reaction was used to determine the concentration of nitrate nitrogen in the soil solution; and the external standard method was used to verify the nitrate nitrogen concentration-absorbance standard curve The precision and accuracy of the determination of the four soil samples are shown in Table 4.

Table 4. The precision and accuracy of the standard curve for the determination of nitrate nitrogen by external standard method

As can be seen from Table 4: the coefficient of variation of the nitrate nitrogen standard curve provided by the present invention is 2.66-4.97%, indicating that the method has high precision; the standard addition recovery rate is 86-110%, indicating that the accuracy is high.

(3) Establishment of nitrite nitrogen concentration-absorbance standard curve

Refer to (2) for the preparation of the first Griess reagent and the second Griess reagent.

Preparation of nitrite nitrogen standard solution: Weigh 4.928g sodium nitrite and dissolve it in water, and dilute to 1000mL to obtain a nitrite nitrogen standard stock solution with a concentration (calculated as nitrogen) of 1000mg/L; take an appropriate amount of the standard stock solution to determine The volume is up to 100mL, and the concentration (calculated as nitrogen) is 0.1mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.5mg/L and 5mg/L nitrite state respectively. Nitrogen standard solution.

Color reaction: 100 μL nitrite nitrogen standard solution + 50 μL first Griess reagent (add quickly) + 50 μL second Griess reagent (add quickly), a total of 200 μL reaction system; the reaction system develops color at 37°C for 60 min, at 540 nm The absorbance was measured, and a nitrite nitrogen concentration-absorbance standard curve was established. The results are shown in Figure 3, and the parameters of the obtained standard curve are shown in Table 5. The detection limit of nitrite nitrogen in the obtained soil solution was determined by the external standard method, and the results are shown in Table 3. It can be seen from Table 3 that the nitrite nitrogen concentration-absorbance standard curve is in the range of nitrite nitrogen concentration (calculated as nitrogen) from 0.1 to 5 mg/L, the slope is 0.289, the intercept is 0.081, and the correlation coefficient is 0.97; the limit of detection (LOD) of this standard curve for nitrate nitrogen in soil solution is 0.021mg/L, which is 0.21mg/kg when converted to soil samples.

Table 5 Determination of nitrite nitrogen standard curve and detection limit by external standard method

Four different soil samples were selected, and the soil solution was prepared according to the preparation method of the soil samples; the same color reaction was used to determine the concentration of nitrite nitrogen in the soil solution; and the external standard method was used to verify the concentration of nitrite nitrogen - absorbance The precision and accuracy of the standard curve for the determination of the four soil samples are shown in Table 6.

Table 6. The precision and accuracy of the standard curve for the determination of nitrite nitrogen by external standard method

It can be seen from Table 6 that the coefficient of variation of the nitrite nitrogen standard curve provided by the present invention is 3.17-4.35%, indicating that the method has high precision; the standard addition recovery rate is 89-104%, indicating that the accuracy is high.

(4) Establishment of the standard curve of total dissolved nitrogen (TDN) concentration-absorbance

Refer to (2) for the preparation of nitrate reductase solution, first Griess reagent and second Griess reagent.

Prepare an aqueous solution of persulfate oxidant: weigh 25g potassium persulfate (K ₂ S ₂ O ₈ ), 25g potassium permanganate, 6.2g sodium hydroxide (NaOH) and 15g boric acid (H ₃ BO ₃ ) with deionized water Dilute to 1 L to obtain an aqueous solution of persulfate oxidant.

To prepare a standard solution of total dissolved nitrogen (TDN): Weigh 5.362 g of glycine and dissolve it in water, dilute to 1000 mL, and prepare a total dissolved nitrogen stock solution with a concentration (calculated as nitrogen) of 1000 mg/L; take an appropriate amount of total dissolved nitrogen Dilute the nitrogen stock solution to 100mL, and prepare the concentrations (calculated in nitrogen) as 0.1mg/L, 0.2mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.5mg/L and 5mg/L respectively total dissolved nitrogen standard solution.

Color reaction: respectively take a series of 2.5mL total dissolved nitrogen standard solution and mix with 2.5mL persulfate oxidant aqueous solution, digest at 120 ℃ for 40min to obtain a digestion solution.

100 μL digestion solution + 100 μL nitrate reductase solution + 50 μL first Griess reagent (add quickly) + 50 μL second Griess reagent (add quickly), a total of 300 μL reaction system; the reaction system was developed at 37°C for 60 min at 540 nm The absorbance was measured, and a total dissolved nitrogen (TDN) concentration-absorbance standard curve was established. The results are shown in FIG. 4 , and the parameters of the obtained standard curve are shown in Table 7. The detection limit of total dissolved nitrogen in the obtained soil solution was determined by the external standard method, and the results are shown in Table 7. It can be seen from Table 7: the total dissolved nitrogen concentration-absorbance standard curve is in the range of the total dissolved nitrogen concentration (calculated as nitrogen) from 0.1 to 5 mg/L, the slope is 0.379, the intercept is 0.093, and the correlation coefficient is 0.97 ; The limit of detection (LOD) of this standard curve for total dissolved nitrogen in soil solution is 0.24mg/L, which is 2.4mg/kg when converted to soil samples.

Table 7 Standard curve and detection limit for the determination of total dissolved nitrogen by external standard method

Four different soil samples were selected, and soil solutions were prepared according to the preparation method of soil samples; the same color reaction was used to determine the concentration of total dissolved nitrogen in the soil solution; and the external standard method was used to verify the concentration of total dissolved nitrogen-absorbance The precision and accuracy of the standard curve for the determination of the four soil samples are shown in Table 8.

Table 8 Precision and accuracy of the standard curve for the determination of total dissolved nitrogen by external standard method

It can be seen from Table 8 that the coefficient of variation of the total dissolved nitrogen standard curve provided by the present invention is 3.86-5.35%, indicating that the method has high precision; the standard addition recovery rate is 89-110%, indicating that the accuracy is high.

It can be seen from the examples that the assay method provided by the present invention has high precision and accuracy, and has a lower detection limit for various nitrogen forms in the sample.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (4)

1. the assay method of ammonium nitrogen, nitrate nitrogen, nitrite nitrogen and total dissolved nitrogen in a sample, is characterized in that, comprises the following steps: The sample to be tested is prepared into a sample solution to be tested, the sample solution to be tested is mixed with an oxidant and a chromogenic agent, and the color is developed, the absorbance at 660 nm is measured, and the absorbance of the sample solution to be tested at 660 nm obtained according to the measurement and the predetermined ammonium nitrogen concentration-absorbance standard curve to obtain the ammonium nitrogen content in the sample to be tested; Described oxidizing agent is the aqueous solution of sodium hypochlorite and chloroacetonitrile sodium urate, and described color developing agent is the sodium hydroxide solution of salicylic acid; The mass ratio of sodium hypochlorite to sodium chloroisocyanurate in the oxidant is 6.5-7.2:0.025-0.035; Mix the sample solution to be tested with the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, according to the measured absorbance at 540 nm of the sample solution to be tested and the predetermined nitrite nitrogen concentration -Absorbance standard curve to obtain the nitrite nitrogen content in the sample to be tested; Mix the sample solution to be tested with nitrate reductase, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain the absorbance at 540 nm of the sample solution to be tested according to the measurement and the predetermined value. The nitrate nitrogen concentration-absorbance standard curve was obtained to obtain the apparent nitrate nitrogen content of the sample; The first Griess reagent is an aqueous solution of N-(1-naphthyl)ethylenediamine dihydrochloride, and the second Griess reagent is an aqueous hydrochloric acid solution of sulfonamide and vanadium chloride; The mass ratio of sulfonamide and vanadium chloride in the second Griess reagent is 4.5-5.5:0.10-0.20; The solute of described nitrate nitrogen standard solution is potassium nitrate; Mix the sample solution to be tested with persulfate oxidant and water to obtain a digestion solution; mix the digestion solution, nitrate reductase, the first Griess reagent and the second Griess reagent, develop color, and measure the absorbance at 540 nm , according to the measured absorbance of the sample solution to be tested at 540 nm and the predetermined total dissolved nitrogen concentration-absorbance standard curve, obtain the total dissolved nitrogen content in the sample to be tested; The persulfate oxidant is a mixture of potassium persulfate, potassium permanganate, sodium hydroxide and boric acid; The solute of the total dissolved nitrogen standard solution is glycine; The difference between the apparent nitrate nitrogen content of the sample and the nitrite nitrogen content in the sample to be tested is the nitrate nitrogen content in the sample to be tested; Establishment of standard curve: the standard curve includes ammonium nitrogen concentration-absorbance standard curve, nitrate nitrogen concentration-absorbance standard curve, nitrite nitrogen concentration-absorbance standard curve and total dissolved nitrogen concentration-absorbance standard curve; The establishment of the ammonium nitrogen concentration-absorbance standard curve includes the following steps: Prepare an ammonium nitrogen standard solution, mix the ammonium nitrogen standard solution, an oxidant and a color developing agent, develop the color, measure the absorbance at 660 nm, and obtain an ammonium nitrogen concentration-absorbance standard curve; The solute of the ammonium nitrogen standard solution is ammonium chloride; The establishment of the nitrate nitrogen concentration-absorbance standard curve includes the following steps: Prepare a nitrate nitrogen standard solution, mix the nitrate nitrogen standard solution, nitrate reductase, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain a nitrate nitrogen concentration-absorbance standard curve ; The establishment of the nitrite nitrogen concentration-absorbance standard curve includes the following steps: Prepare a nitrite nitrogen standard solution, mix the nitrite nitrogen standard solution, the first Griess reagent and the second Griess reagent, develop color, measure the absorbance at 540 nm, and obtain a nitrite nitrogen concentration-absorbance standard curve; The solute of the nitrite nitrogen standard solution is sodium nitrite; The establishment of the total dissolved nitrogen concentration-absorbance standard curve includes the following steps: Prepare a total dissolved nitrogen standard solution, mix the total dissolved nitrogen standard solution, persulfate oxidant and water to obtain a digestion solution; combine the digestion solution, nitrate reductase, the first Griess reagent and the second Griess reagent Mix and develop color, measure the absorbance at 540nm, and obtain the standard curve of total dissolved nitrogen concentration-absorbance. 2 . The assay method according to claim 1 , wherein the mass ratio of the glycine to the persulfate oxidant is 0.02-1.00: 2500-3500. 3 . 3. assay method according to claim 1, is characterized in that, the mass ratio of potassium persulfate, potassium permanganate, sodium hydroxide and boric acid in described persulfate oxidant is 25:20～25:5～6 : 10 to 20. 4. The assay method according to claim 1, wherein the sample comprises soil or water.

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