Nitrite and nitrate separation system in water samples and nitrogen and oxygen isotope determination method

Abstract

The present application belongs to the technical field of material testing and analysis, and specifically relates to a system for separating nitrite and nitrate in water samples and a method for determining nitrogen and oxygen isotopes. The separation system includes a connected hanging bottle, a three-way head, a flow rate controller, a resin glass column, a peristaltic tube, a bottle stopper piercer, a headspace bottle, and a water ring vacuum pump, wherein an ion exchange resin is precipitated inside the resin glass column, and after the water sample to be tested flows through the resin glass column, the nitrite and nitrate therein are completely exchanged with the chloride ions in the resin glass column, and then, the KCl solution flows through the resin glass column to achieve elution and separation of nitrite and nitrate. The system for separating nitrite and nitrate in water samples of the present application can completely separate nitrate and nitrite in water samples, and its separation degree, full process blank and recovery rate meet the accuracy requirements of isotope analysis, thereby improving the accuracy of the nitrogen and oxygen isotope values ​​of nitrate and nitrite determined by bacterial denitrification.

Description

Technical Field

[0001] The present application belongs to the technical field of material testing and analysis, and specifically relates to a nitrite and nitrate separation system in a water sample and a nitrogen and oxygen isotope determination method. Background Art

[0002] Nitrogen (N) is not only a basic element and an important component of organisms, but also a key pollution factor in the environment. Excessive N will lead to increased pollution of the atmosphere, rivers, lakes, oceans and groundwater, and will also threaten human health. N is mainly present in nature in the form of N2, N2O, NH4 + 、NO2 - 、NO3 - It exists in the form of organic nitrogen. Different forms of N are mutually transformed through the nitrogen cycle. Among them, nitrate is the most important and stable form of nitrogen in aquatic ecosystems. Excessive nitrate pollution will not only lead to eutrophication of water bodies, but also cause the death of fish and shrimp in the water, seriously destroying the ecological balance. Secondly, excessive nitrate poses a great threat to human health. In an oxygen-deficient environment, nitrate can be reduced to toxic nitrite in the digestive tract. Nitrite can oxidize low-iron hemoglobin to high-iron hemoglobin in the human body, causing it to lose the ability to transport oxygen. In addition, nitrite can also react with secondary amine compounds to generate carcinogenic nitrosamines, which will increase the risk of various cancer cell lesions. Therefore, long-term drinking of water containing high concentrations of nitrate will cause poisoning in humans and animals. Therefore, it is crucial to explore the source and transformation of nitrogen in nitrate in the aquatic environment.

[0003] At present, stable isotope technology has become a recognized effective technical means to identify the source of nitrate pollution in water bodies. Commonly used pretreatment methods for nitrate nitrogen and oxygen isotopes include distillation, diffusion, silver nitrate method (ion exchange method), bacterial denitrification method and hydrazoic acid method. The distillation method is to uniformly digest the organic nitrogen and inorganic nitrogen in the sample to obtain NO3 - , reduced to NH4 by distillation reaction + However, this method requires special equipment and is difficult to carry out in the field and other places with poor equipment. In addition, the glassware used in the experiment is easy to absorb NH4 + The method has a large demand for samples and a high detection limit, which cannot meet the requirements of low-concentration, large-scale and large-scale investigations in marine sites. The diffusion method was first used to determine NO3 - and NH4 + The content of δ 15The N value still has a large error. The sample preparation process of the silver nitrate method is relatively labor-intensive (sample preparation takes 3-5 days) and costly. - Will interfere with the anion exchange resin to NO3 - adsorption, and for low concentrations of NO3 - The sample needs a large sample volume. The hydrazo acid method is generally not used because the reagents used are too toxic.

[0004] Among them, the principle of bacterial denitrification to pretreat nitrate nitrogen and oxygen isotopes is that the Pseudomonas chlororaphis strain naturally lacks gaseous nitrous oxide (N2O) reduction activity and first reduces all nitrate to nitrite, and finally releases nitrous oxide (N2O). The advantages of bacterial denitrification are: (1) This technology requires a smaller sample amount (1-100nmol) than the ion exchange method (100-200mmol) by three orders of magnitude, and allows the analysis of lower (1μmol / L) NO3 - concentration sample. (2) The preparation process is less labor-intensive (2-3 days of sample preparation), while the ion exchange method requires 3-5 days of sample preparation. (3) The method achieves a high level of sensitivity (δ 15 The error of N is 0.1-0.5‰, δ 18 O error is 0.1-1‰). (4) The cost is relatively low, about 5 euros per sample, compared with ion exchange method (60 euros per sample). However, bacterial denitrification also has the following disadvantages: denitrifying bacteria will convert NO2 - and NO3 - All of them are converted into N2O, which makes it impossible to distinguish NO2 from N2O. - and NO3 - Nitrogen and oxygen isotope values.

[0005] In order to solve the shortcomings of bacterial denitrification, Grange et al. used anti-blood acid to destroy NO2 - , pH must be below 3.5 to be completely removed, but in this process NO2 - Will react to produce NO3 - , NO2 - When the concentration is above 50μmol / L, the isotopic difference is 1‰; when NO3 - When the concentration exceeds 500μmol / L, the bubbling time should be more than 5h, NO2 - Will react to produce NO3 - ; and the NO2 in the acidified sample -Böhlke et al. first used two denitrifying bacteria, Stenotrophomonas nitritireducens and Pseudomonas aureofaciens, to specifically reduce NO2 - To N2O, and then to NO3 - For N2O, low concentration of NO2 in the same water sample was achieved - and NO3 - The method can accurately determine the isotopic composition, but it is cumbersome to operate and has a long culture cycle. Due to the harsh survival conditions of bacteria, its scope of application is limited. Summary of the invention

[0006] In order to solve at least one technical problem existing in the prior art, the present application provides a nitrite and nitrate separation system in a water sample and a nitrogen and oxygen isotope determination method.

[0007] In the first aspect, the present application discloses a system for separating nitrite and nitrate in a water sample, comprising a hanging bottle, a resin glass column, a first peristaltic tube, a first bottle stopper piercer, a headspace bottle, a water ring vacuum pump, a second peristaltic tube, and a second bottle stopper piercer, wherein the liquid outlet of the hanging bottle is connected to the liquid inlet of the resin glass column through a flow rate controller, the liquid outlet of the resin glass column is connected to the first bottle stopper piercer piercing the bottle cap of the headspace bottle through the first peristaltic tube, and the first air inlet of the water ring vacuum pump is connected to the second bottle stopper piercer piercing the bottle cap of the headspace bottle through the second peristaltic tube;

[0008] Wherein, a pre-treated ion exchange resin is deposited below the liquid surface of the deionized water inside the resin glass column;

[0009] In addition, the hanging bottle can successively contain a standard solution, a pretreated water sample to be tested, and a KCl solution of a predetermined concentration. The standard solution and the water sample to be tested can flow through the resin glass column at a predetermined flow rate under the control of the flow rate controller, so that the nitrite and nitrate in the standard solution and the water sample to be tested are completely exchanged with the chloride ions in the resin glass column, thereby achieving pre-concentration of nitrite and nitrate. Subsequently, the KCl solution can also flow through the resin glass column at a predetermined flow rate under the control of the flow rate controller, thereby eluting and separating nitrite and nitrate.

[0010] In an optional embodiment, the pretreated water sample to be tested refers to the water sample to be tested after being filtered through a filter membrane.

[0011] In an optional embodiment, the ion exchange resin in the resin glass column is AG1-X8 anion exchange resin, and the pretreatment of the ion exchange resin refers to soaking the AG1-X8 anion exchange resin in 6% HCl, then rinsing it with deionized water until the pH of the rinsed solution is 5-6, and finally soaking it in deionized water.

[0012] In an optional embodiment, the resin glass column has a quartz wool bottom, the inner diameter of the column ranges from 0.56 to 0.6 cm, the column height ranges from 35 to 40 cm, and in addition, the height of the AG1-X8 anion exchange resin settled below the deionized water surface ranges from 26.5 to 27.5 cm.

[0013] In an optional embodiment, the system for separating nitrite and nitrate in water sample further comprises a three-way head and a third peristaltic tube, wherein the liquid outlet of the hanging bottle is connected to the liquid inlet of the three-way head, one of the liquid outlets of the three-way head is connected to the liquid inlet of the resin glass column through a flow rate controller, and the other liquid outlet of the three-way head is connected to the second air inlet of the water ring vacuum pump through the third peristaltic tube;

[0014] In addition, the flow rate controller is a peristaltic pump.

[0015] In a second aspect, the present application also discloses a method for determining nitrogen and oxygen isotopes, comprising the following steps:

[0016] Step 1: Perform a separation experiment on nitrite and nitrate in a standard solution by using the nitrite and nitrate separation system in a water sample as described in any one of the first aspects. The separation experiment is specifically divided into the following sub-steps:

[0017] Sub-step 1.1, adding a predetermined amount of standard solution into a hanging bottle, and allowing the standard solution in the hanging bottle to flow through the resin glass column at a predetermined flow rate under the control of a flow rate controller;

[0018] Sub-step 1.2, collecting the liquid flowing out of the resin glass column, and measuring the nitrite and nitrate in the liquid in real time, and when it is measured that the liquid does not contain nitrite and nitrate, proceeding to sub-step 1.3;

[0019] Sub-step 1.3, adding a predetermined amount and a predetermined concentration of KCl eluent into a hanging bottle, and the KCl eluent in the hanging bottle flows through the resin glass column at a predetermined flow rate under the control of a flow rate controller;

[0020] Sub-step 1.4, collecting the KCl eluate flowing out from the bottom of the resin glass column in sub-step 1.3, and dividing the KCl eluate flowing out successively into multiple groups of KCl eluates of equal volume during the collection;

[0021] Sub-step 1.5, respectively determining the nitrite and nitrate contents in the multiple groups of KCl eluents, and finally drawing an elution curve diagram of the KCl eluent according to the changes in the contents;

[0022] Step 2: Separate the nitrite and nitrate in another standard solution by the nitrite and nitrate separation system in water sample described in any one of the first aspects, wherein the separation process is specifically divided into the following sub-steps:

[0023] Sub-step 2.1, treating the standard solution with reference to the above sub-steps 1.1 to 1.3;

[0024] Sub-step 2.2, using the elution curve of the KCl eluate obtained in sub-step 1.5 as a reference, collecting a predetermined number of groups of the KCl eluate flowing out of the bottom of the resin glass column in sub-step 2.1, wherein the collected components include components that concentrate on nitrite and components that concentrate on nitrate;

[0025] Step 3: Determine the nitrogen and oxygen isotope values ​​of the concentrated nitrite-containing component and the concentrated nitrate-containing component obtained in sub-step 2.2, respectively, wherein the deviation of the nitrogen and oxygen isotope values ​​obtained by the determination from the nitrogen and oxygen isotope values ​​of nitrite and nitrate in the standard solution of step 1 is within a predetermined range.

[0026] Step 4, referring to step 2, separating the nitrite and nitrate in the water sample to be tested, wherein the water sample to be tested has the same volume as the standard solution and contains nitrite and nitrate;

[0027] Step 5: respectively measure the nitrogen and oxygen isotope values ​​of the components concentrated containing nitrite and the components concentrated containing nitrate obtained in step 4.

[0028] In an optional embodiment, in the step 4, before adding the water sample to be tested into the hanging bottle, the step further includes:

[0029] The water sample to be tested is filtered through a filter membrane.

[0030] In an optional embodiment, the ion exchange resin in the resin glass column is AG1-X8 anion exchange resin, and before the sub-step 1.1 and the sub-step 2.1, the following steps are also included:

[0031] The AG1-X8 anion exchange resin was soaked in 6% HCl, then rinsed with deionized water until the pH of the rinsed solution was 5-6, and then soaked in deionized water; and

[0032] Fill the resin glass column with quartz wool bottom with deionized water, and then use a rubber-tipped dropper to add an appropriate amount of AG1-X8 anion exchange resin soaked in deionized water into the resin glass column in a natural sedimentation manner; and

[0033] The ion exchange resin in the resin glass column is pre-cleaned with a KCl solution of a predetermined amount and a predetermined concentration. In an optional embodiment, in the sub-steps 1.2 and 1.5, the nitrite and nitrate in the column fluid are measured in real time by an ion chromatograph.

[0034] In an optional embodiment, in step three and step five, the component containing nitrite and the component containing nitrate are respectively added to denitrifying bacteria, and then the nitrogen and oxygen isotope values ​​are determined using an isotope mass spectrometer.

[0035] This application has at least the following beneficial technical effects:

[0036] 1) The nitrite and nitrate separation system in the water sample of the present application can completely separate the nitrate and nitrite in the water sample, and its separation degree, full process blank and recovery rate meet the accuracy requirements of isotope analysis, thereby improving the accuracy of the nitrogen and oxygen isotope values ​​of nitrate and nitrite determined by bacterial denitrification method;

[0037] 2) The nitrite and nitrate separation system in the water sample of the present application can simultaneously analyze the nitrogen and oxygen isotope ratios of nitrite and nitrate;

[0038] 3) The nitrite and nitrate separation system in the water sample of the present application belongs to an automatic column passing system. When multiple separation systems are used together, multiple samples can be passed through the column at the same time, thereby improving the pretreatment efficiency;

[0039] 4) The nitrite and nitrate separation system in the water sample of the present application can complete the pretreatment of nitrate and nitrite in the water sample in an environment isolated from air, which can avoid the contamination of the sample by nitrogen in the air during the sample pretreatment process of the ion exchange resin, thereby further improving the determination accuracy of nitrogen and oxygen isotope values. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a simplified structural diagram of the system for separating nitrite and nitrate in water samples of the present application;

[0041] Figure 2 The diagram shows the composition of a fully automatic closed column system composed of multiple sets of nitrite and nitrate separation systems in water samples connected in parallel in a preferred embodiment;

[0042] Figure 3 It is a flow chart of each sub-step in step 1 of the nitrogen and oxygen isotope determination method of the present application;

[0043] Figure 4 This is a flowchart of steps 2 to 5 in the nitrogen and oxygen isotope determination method of this application.

[0044] Figure 5 This is the elution curve obtained in sub-step 1.5 in a preferred embodiment of the nitrogen and oxygen isotope determination method of the present application. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the implementation of this application clearer, the technical scheme in the embodiment of this application will be described in more detail below in conjunction with the drawings in the embodiment of this application. The described embodiment is a part of the embodiment of this application, not all of the embodiments. The embodiments described below with reference to the drawings are exemplary and are intended to be used to explain this application, and should not be understood as limiting this application.

[0046] In a first aspect, the present application discloses a system for separating nitrite and nitrate in a water sample; Figure 1 As shown, the separation system may include a connected hanging bottle 1, a resin glass column 3, a first peristaltic tube 4, a first bottle stopper piercer 5, a headspace bottle 6, a water ring vacuum pump 8, a second peristaltic tube 9, and a second bottle stopper piercer 10.

[0047] Specifically, the liquid outlet of the hanging bottle 1 is connected to the inlet of the flow rate controller through a pipeline, and the outlet of the flow rate controller is connected to the liquid inlet of the resin glass column 3. The liquid outlet of the resin glass column 3 is connected to the first bottle stopper piercer 5 set on the bottle cap of the head space bottle 6 through the first peristaltic tube 4.

[0048] In addition, the first air inlet of the water ring vacuum pump 8 is connected to the second bottle stopper piercer 10 arranged on the bottle cap of the head space bottle 6 through the second peristaltic tube 9.

[0049] The pre-treated ion exchange resin is deposited below the liquid surface of the deionized water in the resin glass column 3 .

[0050] It should be noted that the ion exchange resin can also be selected as needed. In the present embodiment, the ion exchange resin in the resin glass column 3 is AG1-X8 anion exchange resin. The pretreatment of the above-mentioned ion exchange resin refers to soaking the AG1-X8 anion exchange resin with 6% HCl and then rinsing it with deionized water until the pH of the solution after rinsing is approximately 5-6. Finally, soak the AG1-X8 anion exchange resin in deionized water for standby use.

[0051] Similarly, the size of the resin glass column 3 and its internal structure can be appropriately selected as needed. For example, a small amount of quartz wool can be added to the bottom of the resin glass column, and the inner diameter range of the resin glass column 3 can be selected from 0.56-0.60cm, and the column height range can be selected from 35~40cm.

[0052] In this embodiment, first, a small amount of quartz wool is added to the bottom of the resin glass column, and then the entire resin column is filled with deionized water. If there are bubbles in the filled deionized water, the bubbles in the deionized water are discharged by knocking on the glass column, and then the spare resin soaked in the deionized water is sucked with a rubber-tipped dropper, and then the rubber-tipped dropper is extended below the deionized water surface, and the resin is slowly added, and it is waited for it to settle naturally until it reaches a suitable height. Finally, the ion exchange resin in the resin glass column 3 is pre-cleaned with a predetermined amount and a predetermined concentration of KCl solution; in this embodiment, the height range of the AG1-X8 anion exchange resin preferably settled below the deionized water surface is 26.5-27.5cm, and the volume of the preferred KCl solution is 50mL, and the concentration is 2mol / L.

[0053] In addition, the hanging bottle 1 can contain the standard solution, the pre-treated water sample to be tested and the KCl solution of predetermined concentration respectively according to the sequence of the processing steps.

[0054] The pretreated water sample to be tested refers to the water sample to be tested that has been filtered through a filter membrane (e.g., a 0.22 μm Whatman glass fiber membrane); the standard solution and the pretreated water sample to be tested can flow through the resin glass column 3 at a predetermined flow rate under the control of a flow rate controller, so that the nitrite and nitrate in the standard solution and the water sample to be tested are completely exchanged with the chloride ions (Cl-) in the resin glass column 3 to achieve pre-concentration of nitrite and nitrate. Subsequently, the KCl solution can also flow through the resin glass column 3 at a predetermined flow rate under the control of the flow rate controller to elute and separate nitrite and nitrate.

[0055] Furthermore, bubbles may be generated in the process of the liquid in the hanging bottle 1 flowing into the resin glass column 3 through the flow rate controller. In order to prevent or reduce the influence of the bubbles on the pre-concentration treatment of nitrite and nitrate in the resin glass column 3, the nitrite and nitrate separation system in the water sample of the present application can also include a three-way head 2 and a third peristaltic tube 7.

[0056] Specifically, the liquid outlet of the hanging bottle 1 is connected to the liquid inlet of the tee 2 through a pipeline, one of the liquid outlets of the tee 2 is connected to the inlet of the flow rate controller through a pipeline, and the outlet of the flow rate controller is connected to the liquid inlet of the resin glass column 3; in addition, the other liquid outlet of the tee 2 is connected to the second air inlet of the water ring vacuum pump 8 through the third peristaltic tube 7.

[0057] Furthermore, the above-mentioned flow rate controller of the present application can be selected as a variety of process control devices according to needs, such as a control valve, a peristaltic pump 11, etc. This embodiment preferably controls the peristaltic pump 11 which is more convenient and more accurate.

[0058] It should also be noted that in this application, two or more water sample nitrite and nitrate separation systems can be selected and used in combination as needed, such as Figure 2 As shown, in one embodiment, four separation systems are fixed together for use by fixing a mounting plate (each separation system is used independently), thereby enabling multiple samples to pass through the column at the same time, thereby improving the pretreatment efficiency.

[0059] In a second aspect, the present application also discloses a method for determining nitrogen and oxygen isotopes, such as Figure 3-Figure 4 As shown, the determination method may include the following steps:

[0060] Step 1: Perform a separation experiment on nitrite and nitrate in a standard solution by using the nitrite and nitrate separation system in a water sample according to any one of the first aspects. The separation experiment is specifically divided into the following sub-steps:

[0061] Sub-step 1.1, adding a predetermined amount of standard solution into the hanging bottle 1, and the standard solution in the hanging bottle 1 flows through the resin glass column 3 at a predetermined flow rate under the control of the flow rate controller;

[0062] Sub-step 1.2, collecting the liquid flowing out of the resin glass column 3, and measuring the nitrite and nitrate in the liquid in real time. When it is measured that the liquid does not contain nitrite and nitrate, proceed to sub-step 1.3;

[0063] Sub-step 1.3, adding a predetermined amount and a predetermined concentration of KCl eluent into the hanging bottle 1, and the KCl eluent in the hanging bottle 1 flows through the resin glass column 3 at a predetermined flow rate under the control of a flow rate controller;

[0064] Sub-step 1.4, collecting the KCl eluate flowing out from the bottom of the resin glass column 3 in sub-step 1.3, and dividing the KCl eluate flowing out successively into multiple groups of KCl eluates of equal volume during the collection;

[0065] Sub-step 1.5, respectively determine the nitrite and nitrate contents in multiple groups of KCl eluents, and finally draw an elution curve diagram of the KCl eluent according to the changes in the contents.

[0066] Step 2: Separate the nitrite and nitrate in another standard solution (with the same composition and volume as the standard solution in step 1) by the nitrite and nitrate separation system in water sample described in any one of the first aspects above, and the separation process is specifically divided into the following sub-steps:

[0067] Sub-step 2.1, treating the standard solution with reference to the above sub-steps 1.1 to 1.3;

[0068] Sub-step 2.2, using the elution curve of the KCl eluate obtained in sub-step 1.5 as a reference, collecting a predetermined number of groups of the KCl eluate flowing out of the bottom of the resin glass column 3 in sub-step 2.1, wherein the collected components include components that concentrate on nitrite and components that concentrate on nitrate;

[0069] Step 3, respectively measuring the nitrogen oxygen isotope values ​​of the concentrated nitrite-containing components and the concentrated nitrate-containing components obtained in sub-step 2.3, wherein the deviation of the nitrogen oxygen isotope values ​​obtained by the measurement from the nitrogen oxygen isotope values ​​of nitrite and nitrate in the standard solution of step 1 is within a predetermined range; that is, step 2 and step 3 can verify that, in step 1, the separation of nitrite and nitrate in the standard solution is very thorough, and the elution curve diagram obtained in step 1 can be fully applied to the subsequent separation treatment of the water sample to be tested having the same components;

[0070] Step 4: Separating the nitrite and nitrate in the water sample to be tested by the nitrite and nitrate separation system in water sample described in any one of the first aspects above, wherein the water sample to be tested has the same volume as the standard solution in step 1 and step 2 and contains nitrite and nitrate, and the separation process is specifically divided into the following sub-steps:

[0071] Sub-step 4.1, processing the water sample to be tested with reference to the above sub-steps 2.1-2.2;

[0072] Step 5: respectively measure the nitrogen and oxygen isotope values ​​of the component containing nitrite and the component containing nitrate obtained in step 4.

[0073] Furthermore, in step 4, before adding the water sample to be tested into the hanging bottle 1, the water sample to be tested is filtered through a filter membrane.

[0074] Furthermore, when the ion exchange resin in the resin glass column 3 is AG1-X8 anion exchange resin, before sub-step 1.1 and sub-step 2.1 (including sub-step 4.1), the following steps are also included:

[0075] The AG1-X8 anion exchange resin was soaked in 6% HCl, then rinsed with deionized water until the pH of the rinsed solution was 5-6, and then soaked in deionized water; and

[0076] Fill the resin glass column 3 with quartz wool bottom with deionized water, and then use a rubber-tipped dropper to add a proper amount of AG1-X8 anion exchange resin soaked in deionized water into the resin glass column 3 by natural sedimentation until it reaches a suitable height; and

[0077] The ion exchange resin in the resin glass column 3 is pre-cleaned with a KCl solution of a predetermined amount and a predetermined concentration.

[0078] Furthermore, in sub-steps 1.2 and 1.5, the nitrite and nitrate in the column liquid are measured in real time by an ion chromatograph. In steps 3 and 5, the components containing nitrite and the components containing nitrate are added to denitrifying bacteria respectively, and then the nitrogen and oxygen isotope values ​​are determined by an isotope mass spectrometer.

[0079] The nitrogen and oxygen isotope determination method of the present application will be further described below through a specific example:

[0080] A1. Selection of standard solution and water sample to be tested:

[0081] In this embodiment, we select the known 15 N and δ 18 O mixed solution of KNO3 and NaNO2 (including 3.2 mg / L NO2 - and 3.2mg / L NO3 - ) as the standard solution and the water sample to be tested.

[0082] A2. Pretreatment steps of the water sample to be tested and AG1-X8 anion exchange resin:

[0083] The water samples to be tested were filtered with 0.22 μm filter membrane (Whatman) for later use. In addition, the pretreatment of AG1-X8 anion exchange resin refers to the description of the first and second aspects above, which will not be repeated here.

[0084] A3. Filling of resin glass column 3:

[0085] A resin glass column with an inner diameter of 0.56 cm and a column height of 37.5 cm is selected, and then a resin glass column with a suitable resin height (preferably 26.5-27.5 cm) is obtained according to the filling method described in the first and second aspects above;

[0086] A4, resin glass column 3 pre-concentrates NO3 in the standard solution - and NO2 - :

[0087] Take 200ml of the standard solution in step A1 and slowly add it to the hanging bottle 1. The water sample in the hanging bottle 1 enters the resin glass column 3 through the peristaltic tube and is pumped by the peristaltic pump 11. The pump speed is adjusted to 0.9rpm and the flow rate is 100ml / h. After the water sample passes through the column, the liquid under the column is collected. When the ion chromatograph measures that there is no NO3 in the liquid under the column, - and NO2 - Explain that NO3 in water samples - and NO2 - After the Cl- in the ion exchange resin column is completely exchanged, the pre-concentration step is completed and the next step of elution can be carried out;

[0088] A5. KCl solution elution to separate NO3 in standard solution - and NO2 - :

[0089] Add 192 ml of 0.25 mol / L KCl solution into hanging bottle 1, which is connected to resin glass column 3 (i.e., the pre-concentrated NO3 in step A4) through a peristaltic tube. - and NO2 - The subsequent AG1-X8 anion exchange resin column was connected and pumped using a peristaltic pump with a pump speed of 0.3 rpm and a flow rate of 30 ml / h.

[0090] Furthermore, each 3 ml of KCl eluate after column passage was collected as a group, and Ag2O was added and stirred with a glass rod to neutralize excess Cl-. The eluate was then filtered with a 0.45 μm filter head and collected in a 15 ml centrifuge tube. A total of 192 ml was eluted, with a total of 64 groups.

[0091] In addition, the collected 64 groups of KCL eluates were used to determine the NO2 - and NO3 - , draw as Figure 5 The elution curve is shown in the figure. It can be seen from the elution curve that NO2 appears in groups 17-22. - The concentration peak of NO3 - The concentration peak appeared in groups 44-64.

[0092] Finally, the NO2 in 64 eluates was determined by ion chromatography. - and NO3 - Content, and calculate NO2 - and NO3 - Whether the recovery rate reaches 100%. This proves that the experimental scheme can completely separate NO2 - and NO3 - , and the recovery yield can reach 100%. Figure 5 The results show that in this embodiment, NO2- and NO3 - Separate out, and the separation degree is above 2, followed by NO2 - The nitrogen recovery rate can reach 99-104%, NO3 - The nitrogen recovery rate can reach 100-117%.

[0093] A6, resin glass column 3 pre-concentrates the NO3 in another standard solution - and NO2 - :

[0094] Take another 200 ml portion of the standard solution in step A1 and perform pre-concentration treatment according to step A4 (a new resin glass column 3 needs to be selected for this step, and the treatment time is preferably 2 h).

[0095] A7, KCl solution to separate the NO3 in another standard solution - and NO2 - :

[0096] Referring to step A5, use 192 ml of 0.25 mol / L KCL to treat the resin glass column 3 (i.e., the pre-concentrated NO3 in step A6). - and NO2 - The AG1-X8 anion exchange resin column was then used for elution.

[0097] Further, the 192 ml of eluate was divided into four groups. The first group was 45 ml, the second group was 21 ml (the eluted NO2 - The concentrated area, that is, the concentrated components containing nitrite), the third group 60ml, the fourth group 66ml (eluted NO3 - Concentrated areas, i.e., components containing nitrates are concentrated).

[0098] A8. Determination of NO2 by bacterial denitrification - and NO3 - N and O isotope values:

[0099] The second group (NO2 - ) and the fourth group (NO3 - ) were added to the denitrifying bacteria and then the nitrogen and oxygen isotope values ​​were determined using an isotope mass spectrometer.

[0100] In addition, potassium nitrate solution and sodium nitrite solution (3.2 mg / L NO2 - and 3.2mg / L NO3 - ) and the nitrogen and oxygen isotope values ​​of, as well as the nitrogen isotope values ​​of solid potassium nitrate and solid sodium nitrite.

[0101] After measurement and comparison, the second group of eluent (NO2 - The nitrogen isotope value of the concentrated area (-10.5‰) and the sodium nitrite solution (3.2 mg / L NO2 - The deviation of the nitrogen isotope value of sodium nitrite solid (-11‰) is within 0.5‰.

[0102] The second eluent (NO2 - concentration area) (-25.4‰) and sodium nitrite solution (3.2 mg / L NO2 - )’s oxygen isotope (-24.2‰) is within 1.2‰.

[0103] The fourth eluent (NO3 - concentration area) (-0.6‰) and potassium nitrate solution (3.2 mg / L NO3 - The deviation of the nitrogen isotope value of ) (-0.8‰) and the nitrogen isotope value of potassium nitrate solid (-0.8‰) is within 0.2‰.

[0104] The fourth eluent (NO3 - concentration area) oxygen isotope value (22.1‰) and potassium nitrate solution (3.2 mg / L NO3 - )’s oxygen isotope (21.7‰) is within 0.4‰.

[0105] The above specific data can be found in Table 1 below:

[0106] Table 1: Nitrogen and oxygen isotope values ​​and errors determined by bacterial denitrification

[0107]

[0108] This proves that NO2 separated by ion exchange resin column - The NaNO2 in the mixed solution of KNO3 and NaNO2 comes from the nitrate, while the KNO3 comes from the nitrate. Then the four groups of solutions are tested by ion chromatography to determine the ion concentration. - The nitrogen recovery rate can reach 99-104%, NO3 - The nitrogen recovery rate can reach 100-107%.

[0109] A9, resin glass column 3 pre-concentrates NO3 in the water sample to be tested - and NO2 - :

[0110] Take 200 ml of the water sample to be tested in step A2 and perform pre-concentration treatment according to step A4 (a new resin glass column 3 needs to be selected for this step).

[0111] A10, refer to step A7, and use KCl solution to elute and separate the NO3 in the water sample to be tested - and NO2 - , and the eluates were grouped.

[0112] A11, refer to step A8, and determine the NO2 in each eluate in step A10 by bacterial denitrification method - and NO3 - N and O isotope values.

[0113] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A method for determining nitrogen and oxygen isotopes, characterized in that: The steps include: Step 1: A separation experiment of nitrite and nitrate in a standard solution is performed using a nitrite and nitrate separation system in a water sample, wherein the nitrite and nitrate separation system in a water sample comprises a hanging bottle (1), a resin glass column (3), a first peristaltic tube (4), a first bottle stopper piercer (5), a headspace bottle (6), a water ring vacuum pump (8), a second peristaltic tube (9), and a second bottle stopper piercer (10), wherein the liquid outlet of the hanging bottle (1) is connected to the liquid inlet of the resin glass column (3) through a flow rate controller, and the outlet of the resin glass column (3) is connected to the liquid outlet of the hanging bottle (1). The liquid inlet is connected to the first bottle stopper piercer (5) disposed on the bottle cap of the headspace bottle (6) through the first peristaltic tube (4), and the first air inlet of the water ring vacuum pump (8) is connected to the second bottle stopper piercer (10) disposed on the bottle cap of the headspace bottle (6) through the second peristaltic tube (9), wherein a pretreated ion exchange resin is precipitated below the liquid surface of the deionized water inside the resin glass column (3), and the ion exchange resin is an AG1-X8 anion exchange resin. The separation experiment is specifically divided into the following sub-steps: Sub-step 1.1, adding a predetermined amount of standard solution into the hanging bottle (1), and the standard solution in the hanging bottle (1) flows through the resin glass column (3) at a predetermined flow rate under the control of a flow rate controller; Sub-step 1.2, collecting the liquid flowing out of the resin glass column (3), and measuring the nitrite and nitrate in the liquid in real time. When it is measured that the liquid does not contain nitrite and nitrate, proceed to sub-step 1.3; Sub-step 1.3, adding a predetermined amount and a predetermined concentration of KCl eluent into the hanging bottle (1), and the KCl eluent in the hanging bottle (1) flows through the resin glass column (3) at a predetermined flow rate under the control of a flow rate controller; Sub-step 1.4, collecting the KCl eluate flowing out from the bottom of the resin glass column (3) in sub-step 1.3, and dividing the KCl eluate flowing out successively into multiple groups of KCl eluates of equal volume during the collection; Sub-step 1.5, respectively determining the nitrite and nitrate contents in the multiple groups of KCl eluents, and finally drawing an elution curve diagram of the KCl eluent according to the changes in the contents; Step 2: Separate the nitrite and nitrate in another standard solution by the nitrite and nitrate separation system in the water sample. The separation process is specifically divided into the following sub-steps: Sub-step 2.1, treating the standard solution with reference to the above sub-steps 1.1 to 1.3; Sub-step 2.2, using the elution curve of the KCl eluate obtained in sub-step 1.5 as a reference, collecting a predetermined number of groups of the KCl eluate flowing out of the bottom of the resin glass column (3) in sub-step 2.1, wherein the collected components include components that concentrate on nitrite and components that concentrate on nitrate; Step 3, adding the concentrated nitrite-containing component and the concentrated nitrate-containing component obtained in sub-step 2.2 to denitrifying bacteria respectively, and then determining the nitrogen and oxygen isotope values ​​using an isotope mass spectrometer, wherein the deviation of the nitrogen and oxygen isotope values ​​obtained by the determination and the nitrogen and oxygen isotope values ​​of nitrite and nitrate in the standard solution of step 1 is within a predetermined range; Step 4, referring to step 2, separating the nitrite and nitrate in the water sample to be tested, wherein the water sample to be tested has the same volume as the standard solution and contains nitrite and nitrate; Step 5: Add the concentrated nitrite-containing component and the concentrated nitrate-containing component obtained in step 4 to denitrifying bacteria respectively, and then determine the nitrogen and oxygen isotope values ​​using an isotope mass spectrometer.

2. The method for determining nitrogen and oxygen isotopes according to claim 1, characterized in that: In the step 4, before adding the water sample to be tested into the hanging bottle (1), the step further includes: The water sample to be tested is filtered through a filter membrane.

3. The method for determining nitrogen and oxygen isotopes according to claim 1, wherein: Before the sub-step 1.1 and the sub-step 2.1, the following steps are also included: AG1-X8 anion exchange resin was soaked in 6% HCl, then rinsed with deionized water until the pH of the rinsed solution was 5-6, and then soaked in deionized water; as well as Fill the resin glass column (3) with quartz wool bottom with deionized water, and then use a rubber-tipped dropper to add an appropriate amount of AG1-X8 anion exchange resin soaked in deionized water into the resin glass column (3) by natural sedimentation; as well as The ion exchange resin in the resin glass column (3) is pre-cleaned using a KCl solution of a predetermined amount and a predetermined concentration.

4. The method for determining nitrogen and oxygen isotopes according to claim 1, characterized in that: In the sub-step 1.2 and the sub-step 1.5, the nitrite and nitrate in the column liquid are measured in real time by an ion chromatograph.

5. The method for determining nitrogen and oxygen isotopes according to claim 3, characterized in that: The resin glass column (3) has a quartz wool bottom, an inner diameter of the column ranging from 0.56 to 0.6 cm, a column height ranging from 35 to 40 cm, and in addition, a height of the AG1-X8 anion exchange resin settled below the deionized water surface ranging from 26.5 to 27.5 cm.

6. The method for determining nitrogen and oxygen isotopes according to claim 1, characterized in that: The system for separating nitrite and nitrate in water samples further comprises a three-way head (2) and a third peristaltic tube (7), wherein the liquid outlet of the hanging bottle (1) is connected to the liquid inlet of the three-way head (2), one of the liquid outlets of the three-way head (2) is connected to the liquid inlet of the resin glass column (3) through a flow rate controller, and the other liquid outlet of the three-way head (2) is connected to the second air inlet of the water ring vacuum pump (8) through the third peristaltic tube (7); In addition, the flow rate controller is a peristaltic pump (11).

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