A water-soluble double-labeled nitrogen gas ( 15 Preparation and Quantitative Methods of N2 Standard Samples

Abstract

This invention discloses a method for dissolving dual-labeled nitrogen gas in water ( 15 The preparation and quantification method of N2 standard samples belongs to the field of stable isotope analysis technology. This method will... 15 N-labeled ammonium chloride solution reacts with sodium hypochlorite in a completely sealed reaction flask filled with liquid and without a headspace, resulting in in-situ formation and dissolution. 15 N 15 Nitrogen gas was directly measured in its dissolved state in the liquid phase using membrane sample mass spectrometry (MIMS). 15 N 15 The m / z 30 characteristic signal of N was obtained by simultaneously monitoring the concentrations of residual monochloramine, dichloramine, and trichloramine in the reaction solution, and corrected based on the principle of nitrogen conservation. 15 N 15 The true concentration of nitrogen (N). This invention eliminates the need for gas collection, transfer, and cold trap purification throughout the entire process, avoiding nitrogen escape losses; chloramine correction significantly improves quantitative accuracy. It is simple to operate and allows for the flexible preparation of a series of concentration standards ranging from 10 to 1000 μmol / L. The signal drift of the standards is less than 3% within 30 days, making it suitable for isotope ratio mass spectrometry calibration, denitrification tracing, and isotope analysis of dissolved nitrogen in environmental water samples.

Description

Technical Field

[0001] This invention belongs to the field of stable isotope standard material preparation and analytical chemistry technology, specifically relating to a method for dissolving double-labeled nitrogen gas in water (…). 15 Preparation and quantification methods of N2 standard samples. Background Technology

[0002] Stable isotope standards are fundamental to standardized isotope measurements, used for calibrating mass spectrometry instruments, evaluating the accuracy of measurement methods, and ensuring laboratory quality control. In nitrogen cycle studies, dual labeling is crucial. 15 N2 gas can be used in key applications such as denitrification measurement and nitrogen emission flux calculation.

[0003] Currently, commercially available nitrogen stable isotope standard materials are mainly based on 14 N 14 N and 14 N 15 N is the primary focus, targeting 15 N 15 Liquid standards with N (mass number 30) are scarce. Existing preparation methods have the following problems:

[0004] (1) Traditional methods require multiple steps such as gas collection, transfer, and pressurized dissolution, which can easily lead to nitrogen escape and inaccurate quantification.

[0005] (2) The gas collection device is complex and requires steps such as cold trap purification and gas transfer, which is cumbersome to operate;

[0006] (3) There is a lack of in-situ, real-time and online quantitative methods, making it impossible to quickly prepare a series of concentration standards.

[0007] To address the above-mentioned problems, this invention provides a simple, accurate, and gas-free in-situ quantitative method. Summary of the Invention

[0008] The purpose of this invention is to provide a method for dissolving dual-labeled nitrogen gas in water (… 15 The preparation and quantification methods for N2 standard samples enable in-situ, real-time, and online measurement, avoiding escape losses during gas transfer.

[0009] The technical solution adopted in this invention is as follows:

[0010] A water-soluble double-labeled nitrogen gas ( 15 The preparation and quantification method of N2 standard samples includes the following steps:

[0011] (1) Preparation of degassed water: High-purity water was boiled and degassed for 30 minutes under vacuum (≤0.1 Pa), or high-purity helium was bubbled and degassed for 2 hours to reduce the background dissolved N2 concentration to below 0.01 μmol / L. The background was confirmed to be acceptable by measuring the m / z 28 signal using MIMS.

[0012] (2) 15 Preparation of N-labeled ammonium chloride mother liquor: Weigh out 15 N abundance ≥ 98 atom% 15 N-labeled ammonium chloride was prepared with degassed water to a concentration of 100 mmol / L. 15 The NH4Cl mother liquor was sealed and stored at 4°C.

[0013] (3) Preparation of sodium hypochlorite working solution: Standardize the effective chlorine concentration of commercially available sodium hypochlorite solution, dilute with degassed water to 0.5 mol / L, prepare fresh before use, and store away from light.

[0014] (4) Preparation of reaction flask and start-up of reaction: Take a clean, sealed reaction flask (50 mL), completely fill it with degassed water or phosphate buffer (pH=8.0~8.5), and inject a certain volume of solution through the diaphragm using a microsyringe. 15 Add the NH4Cl mother liquor, then inject the calculated amount of sodium hypochlorite working solution (Cl:N molar ratio = 1.5:1), ensuring no air bubbles remain in the flask. Shake for 30 minutes, maintaining the temperature at 25°C. The reaction formula is as follows:

[0015]

[0016] (5) In-situ MIMS detection: After the reaction is complete, MIMS continuously samples and records m / z 30 ( 15 The N2 signal changes over time. After the signal stabilizes (approximately 5 minutes), the steady-state signal intensity is recorded. Simultaneously, the m / z 53 (NH2Cl), m / z 85 (NHCl2), and m / z 88 (NCl3) signals are monitored.

[0017] (6) Calculate according to the following formula 15 The actual concentration of N2:

[0018]

[0019] in, For the initial 15 NH4Cl concentration, , , The concentrations of monochloramine, dichloramine, and trichloramine, as measured by MIMS, are shown in μmol / L.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) No gas collection required: The gas is dissolved in the liquid phase in situ and directly measured by MIMS, avoiding escape loss during the transfer process;

[0022] (2) Easy to operate: closed reaction bottle, single gas path design, no gas transfer device required;

[0023] (3) Flexible preparation of a series of standards: by adjusting 15 The volume of NH4Cl mother liquor added allows for the preparation of multiple concentration points in a single step.

[0024] (4) High accuracy: Quantitative accuracy is ensured through chloramine residue correction. Detailed Implementation

[0025] Preparation of degassed water: Degassed high-purity water (18.2 MΩ·cm) by boiling for 30 minutes under vacuum (≤0.1 Pa), or by bubbling with high-purity helium for 2 hours, and then cooling for later use. The m / z 28 signal was measured by membrane mass spectrometry, requiring a background dissolved nitrogen concentration below 0.01 μmol / L.

[0026] 15 N-labeled ammonium chloride mother liquor: Weigh out 15 N abundance not less than 98% 15 N-labeled ammonium chloride was used to prepare a high-concentration stock solution with degassed water. Approximately 53.5 mg was weighed out. 15 NH4Cl was dissolved in 10 mL of degassed water to obtain a concentration of 100 mmol / L. 15 NH4Cl mother liquor (equivalent to) 15 NH4 + The concentration is 100,000 μmol / L. The stock solution is sealed and stored at 4°C. When using, it should be drawn up with a microsyringe.

[0027] Sodium hypochlorite working solution: Before use, standardize the effective chlorine concentration of a commercially available sodium hypochlorite solution and dilute it with degassed water to a fixed concentration. Prepare a 0.5 mol / L (500,000 μmol / L) sodium hypochlorite working solution, prepare fresh before use, and store protected from light. This working solution is applicable to all embodiments; the molar ratio of effective chlorine to ammonium ions is controlled to be 1.5:1 by adjusting the injection volume.

[0028] Buffer solution (optional): To stabilize the pH of the reaction system, a 0.05M phosphate buffer solution can be prepared, and the pH can be adjusted to 8.0~8.5 with sodium hydroxide. The solution should be fully degassed before use.

[0029] Example 1

[0030] theory 15The N2 concentration is 10 μmol / L. Completely fill a 50 mL sealed reaction flask with degassed water (or phosphate buffer), ensuring there is no headspace or air bubbles. Inject 10 μL of 100 mmol / L N2 solution through the diaphragm using a microsyringe. 15 Nitrogen-labeled ammonium chloride mother liquor was precisely injected into 3 μL of 0.5 mol / L sodium hypochlorite working solution using a microsyringe. The reaction temperature was controlled at 25℃. After 30 min of reaction, MIMS continuously sampled and monitored the m / z 30, m / z 53, m / z 85, and m / z 88 signals. The steady-state signal intensity was recorded after the signals stabilized (approximately 5 minutes). The measured residual chloramine concentrations were 0.10 μmol / L, 0.02 μmol / L, and 0 μmol / L, respectively. Calculations were then performed... 15 The actual concentration of N2 is 9.94 μmol / L.

[0031] Example 2

[0032] theory 15 N2 concentration 50 μmol / L. Completely fill a 50 mL sealed reaction flask with degassed water (or phosphate buffer), ensuring no headspace and no air bubbles. Inject 50 μL of 100 mmol / L N2 solution through the diaphragm using a microsyringe. 15 N-labeled ammonium chloride mother liquor was precisely injected into 15 μL of 0.5 mol / L sodium hypochlorite working solution using a microsyringe. The reaction temperature was controlled at 25℃. After 30 min of reaction, MIMS continuously sampled and monitored the m / z 30, m / z 53, m / z 85, and m / z 88 signals. The steady-state signal intensity was recorded after the signals stabilized (approximately 5 minutes). The residual chloramine concentrations were measured to be 0.20 μmol / L, 0.05 μmol / L, and 0 μmol / L, respectively. Calculations were then performed... 15 The actual concentration of N2 is 49.88 μmol / L.

[0033] Example 3

[0034] theory 15 The N2 concentration is 100 μmol / L. Completely fill a 50 mL sealed reaction flask with degassed water (or phosphate buffer), ensuring there is no headspace or air bubbles. Inject 100 μL of 100 mmol / L N2 solution through the diaphragm using a microsyringe. 15N-labeled ammonium chloride mother liquor was precisely injected into 30 μL of 0.5 mol / L sodium hypochlorite working solution using a microsyringe. The reaction temperature was controlled at 25℃. After 30 min of reaction, MIMS continuously sampled and monitored the m / z 30, m / z 53, m / z 85, and m / z 88 signals. The steady-state signal intensity was recorded after the signals stabilized (approximately 5 minutes). The measured residual chloramine concentrations were 0.30 μmol / L, 0.08 μmol / L, and 0 μmol / L, respectively. Calculations were then performed... 15 The actual concentration of N2 is 99.81 μmol / L.

[0035] Example 4

[0036] theory 15 The nitrogen concentration is 200 μmol / L. Completely fill a 50 mL sealed reaction flask with degassed water (or phosphate buffer), ensuring there is no headspace or air bubbles. Inject 200 μL of 100 mmol / L nitrogen solution through the diaphragm using a microsyringe. 15 N-labeled ammonium chloride mother liquor was precisely injected into 60 μL of 0.5 mol / L sodium hypochlorite working solution using a microsyringe. The reaction temperature was controlled at 25℃. After 30 min of reaction, MIMS continuously sampled and monitored the signals at m / z 30, m / z 53, m / z 85, and m / z 88. The steady-state signal intensity was recorded after the signal stabilized (approximately 5 minutes). The measured residual chloramine concentrations were 0.40 μmol / L, 0.10 μmol / L, and 0.01 μmol / L, respectively. Calculations were performed... 15 The actual concentration of N2 is 199.75 μmol / L.

[0037] Example 5

[0038] theory 15 The N2 concentration is 500 μmol / L. Completely fill a 50 mL sealed reaction flask with degassed water (or phosphate buffer), ensuring there is no headspace or air bubbles. Inject 500 μL of 100 mmol / L N2 solution through the diaphragm using a microsyringe. 15 N-labeled ammonium chloride mother liquor was precisely injected into 150 μL of 0.5 mol / L sodium hypochlorite working solution using a microsyringe. The reaction temperature was controlled at 25℃. After 30 min of reaction, MIMS continuously sampled and monitored the m / z 30, m / z 53, m / z 85, and m / z 88 signals. The steady-state signal intensity was recorded after the signals stabilized (approximately 5 minutes). The measured residual chloramine concentrations were 0.50 μmol / L, 0.12 μmol / L, and 0.02 μmol / L, respectively. Calculations were performed... 15 The actual concentration of N2 is 499.68 μmol / L.

[0039] Example 6

[0040] theory 15 The N2 concentration was 1000 μmol / L. A 50 mL sealed reaction flask was completely filled with degassed water (or phosphate buffer), ensuring no headspace and no air bubbles. Using a microsyringe, 1000 μL of 100 mmol / L 15N-labeled ammonium chloride stock solution was injected through the diaphragm. Then, 300 μL of 0.5 mol / L sodium hypochlorite working solution was precisely injected using the same microsyringe. The reaction temperature was controlled at 25℃. After 30 min of reaction, continuous MIMS sampling was performed, simultaneously monitoring the m / z 30, m / z 53, m / z 85, and m / z 88 signals. Once the signals stabilized (approximately 5 minutes), the steady-state signal intensity was recorded. The measured residual chloramine concentrations were 0.60 μmol / L, 0.15 μmol / L, and 0.03 μmol / L, respectively. The calculated values ​​were... 15 The actual concentration of N2 is 999.61 μmol / L.

[0041] Example 7

[0042] Describe the prepared 15 The N2 standard (such as 500 μmol / L in Example 5) was stored in a sealed container at 4°C in the dark. The m / z 30 signal was measured on days 1, 3, 7, and 30, and the signal drift was found to be <3%, which proves that the in-situ generated standard has good short-term to medium-term stability.

Claims

1. A method for dissolving double-labeled nitrogen gas in water ( 15 The method for preparing and quantifying N2 standard samples is characterized by, Includes the following steps: (1) 15 N abundance ≥ 98% 15 N-labeled ammonium chloride ( 15 Dissolve NH4Cl in degassed high-purity water to prepare... 15 NH4Cl mother liquor; (2) Take the steps from (1) 15 NH4Cl mother liquor was added in different volumes to sealed reaction flasks, completely filling them with liquid and eliminating headspace. Sodium hypochlorite (NaClO) solution was then added, controlling the Cl:N molar ratio at 1.5:1, pH 8.0-8.5, and temperature at 20-30°C. The reaction produced... 15 N2 gas; (3) The products generated by the reaction 15 N2 gas dissolves in situ in the reaction solution, and the dissolved state in the liquid phase is directly determined by membrane mass spectrometry (MIMS). 15 N2, record the intensity of characteristic ion signals at m / z 30; (4) At the same time, the concentrations of residual monochloramine (NH2Cl, m / z 53), dichloramine (NHCl2, m / z 85) and trichloramine (NCl3, m / z 88) in the reaction solution were determined by MIMS. (5) According to the principle of nitrogen conservation, calculate using the following formula. 15 The actual concentration of N2: in, For the initial 15 NH4Cl concentration, , , The concentrations of monochloramine, dichloramine, and trichloramine, as measured by MIMS, are shown in μmol / L.

2. The method according to claim 1, characterized in that: The background dissolved N2 concentration of the degassed high-purity water in step (2) is less than 0.01 μmol / L, and it is obtained by vacuum boiling or high-purity helium bubbling degassing treatment.

3. The method according to claim 1, characterized in that: In step (2), the Cl:N molar ratio is 1.5:1, the pH is 8.0~8.5, the temperature is 25°C, and the reaction time is 30-60 minutes.

4. The method according to claim 1, characterized in that: In step (3), the membrane injection mass spectrometry uses a silicone rubber membrane, the detection mode is selected ion monitoring (SIM), the injection flow rate is 0.5-2.0 mL / min, the membrane temperature is 25-40℃, the mass spectrometry ionization method is electron impact ionization (EI), and the electron energy is 70 eV. Simultaneously, m / z 28 ( 14 N 14 N background), m / z 30 ( 15 N 15 N), m / z 53 (NH2Cl), m / z 85 (NHCl2) and m / z 88 (NCl3).

5. The method according to claim 1, characterized in that: In step (4), when the signals of m / z 53, m / z 85, and m / z 88 are all below the detection limit (0.01 μmol / L as Cl2), the reaction is considered complete. 15 NH4 + Completely transformed 15 N2.

6. The method according to claim 1, characterized in that: By adjusting step (2) 15 The volume of NH4Cl mother liquor added was used to prepare a series of concentrations. 15 N2 standard samples, with a concentration range of 10-1000 μmol / L.

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