Procedure for Converting NO3 to N2O for oxygen and nitrogen isotopic analysis using Azide
Sample Collection Protocol nitrate water samples:
1. Collect samples in 60-100 ml acid cleaned screw cap bottles. More sample (i.e., 100 ml bottle) is better when NO3 concentrations are low.
2. Filter samples in the field with a 0.2 µM filter.
3. Cover bottle mouth in parafilm and screw cap over parafilm.
4. Freeze samples for storage.
Nitrogen species in sample are reduced to NO2-. If starting with NH4+ oxidize to NO3- first. For NO3- reduction, cadmium is added to samples and the samples are shaken overnight. The following day the yield of NO2- is measured colormetrically. Sample NO2- is transferred into vials and diluted with seawater to achieve a concentration of 4 µM. Vials are sealed and crimped with septa. A solution of sodium azide and acetic acid is injected into each vial to convert NO2- to N2O. Vials are placed in a bath for 1 hour. The reaction is stopped after one hour with sodium hydroxide. N2O is purged by He from the sample and injected into the isotope ratio mass spectrometer.
These procedures involve strong acid and bases as well as sodium azide which is a toxic substance if inhaled, ingested or absorbed through the skin. It is highly reactive and can decompose vigorously and explosively without warning. It should be kept away from sources of heat. Furthermore, this procedure involves cadmium metal which is highly toxic (see warning below). Lab coat, nitrile gloves, and goggles MUST be worn at all times.
A. Reduce NO3- to NO2-
USGS 34= 1.33 x10-4 M
USGS 35= 8.12 x10-5 M
N3 = 1.04 x10-4 M
1) 1M Imidizole (pH adjusted to 8 with 10% HCl)
2) Powdered Cd. Mix up cadmium. Place about 50g of powdered Cd into a 500ml beaker and add 10% HCl while swirling the Cd with a scoop. Once the solution is clear and the Cd is chunky pour off the HCl and rinse with DI water 4 times. Each rinse should remain clear. If it does not, rinse with HCl again. Once the pH of the DI is over 6 it is sufficiently rinsed. Leave DI water covering the Cd.
Note: All standards and reagents should be stored in 4°C.
1) 20ml screw top glass vials (cleaned and combusted) N = samples+ 5 standards + 2 blank.
1) Place 20 ml of sample (if [NO3- +NO2-] > 20µM dilute sample to 20µM with nutrient free seawater). Dilute standards to make 20 ml of 20µM (duplicate each standard)
2) Using pipette add 0.2 ml of Imidizole buffer to each vial.
3) Check the pH of a few samples after addition of the imidizole. It should be around 8. pH is usually between 8.2 and 8.5.
4) Wearing gloves weigh out 1g of Cd and place in each vial. Cd addition can be between 0.95g and 1.1g, error on the side of excess Cd. (1g of Cd is sufficient for samples up to 100uM NO3- )
5) Cap the vials with screw tops and place vials horizontally on a shaker table over night. Make sure they are secure. Samples should shake for 17 hours. Over reduction can occur so after 17 hours it is best to move to the next step in a timely way.
B. Centrifuge samples for 10 minutes at ~2000 rpm.
C. Measure the Yield of NO2-
1) NO2- concentration standards (diluted to 20µM for use)
-current stock bottle is 8.8 x10-5 M. For 10ml of colorimetry standard:
2.27 ml of the standard
7.73 ml of NaCl Solution.
1) Plastic test tubes, caps, plastic cell for spectrophotometer
1) Nitrite color reagent: Salfanilamide (1-Naphyhyl) ethylenediamine dihydrochloride (NED).
Note: All standards and reagents should be stored in 4°C.
1) Place 5 ml of sample/standard/blank in acid washed test tubes- be sure not to disturb the Cd at the bottom of the vial.
2) Add 0.1 ml of reagent to sample/standard/blank
3) Shake and let sit for 15 minutes.
5) Using a spectrophotometer, measure absorbance of each sample/standard/blank. First tune the instrument for 543 nm. Fill the cell with DI water and auto zero the instrument. Next measure the absorbance of the samples/standards/blank. After measuring each sample record absorbance in lab notebook and dispose of sample in a beaker- when finished empty the beaker into the hazardous waste carboy.
6) Enter data into excel, to calculate concentration. See the prepared excel spreadsheet.
D. Reduction of NO2- to N2O
1) Acetic Acid/Azide solution: 1:1 2M NaN3 and 20% acetic acid.
2) 6M NaOH (NO3- samples)
3) NaCl solution (35g NaCl in 1L)
Note: Reagent bottles should have piercable caps.
Note 2: All standards and reagents should be stored in 4°C.
1) Acid washed (dont have to be combusted) glass vials with flat tops. N = samples + standards+ 2 x(3 Nitrite standards) + blank
2) crimp tops and septa
3) 2 1ml syringes, with needle tips
4) 10 ml Pipette tips
1) Bubble N2 gas through the azide solution to be sure there is no N2O in the vial. Place azide solution in a vial with a septa and crimp top seal. Insert two needles in the septa. One needle should be attached to the N2 gas line, the other is a vent. In the hood turn on the gas flow so that the solution is gently bubbling. Allow bubbling to continue until ready for use (1 hr is sufficient).
2) Using NO2- concentration yield from Cd reduction, dilute samples and standards into glass flat top vials to achieve 15 ml of 4µM solution. Dilution should be done with a NaCl solution. All pipetting can be done with a 10ml pipette. If no dilution is required add a few drops of NaCl solution. If the pipette looks clean it doesnt have to be changed between samples.
* time saving tip: Fill all vials with 15ml of sea water and then subtract out and replace the needed sample volume. This avoids resetting the pipette for each vial.
3) Cap vials with septa and crimp closed.
4) In the hood, wearing gloves, lab coat, and safety goggles, use syringe to inject 0.8 ml of the azide solution, shake each vial briefly after injection.
5) Place vials in bath for 1 hour at 30°C.
6) In hood, wearing gloves, lab coat, and safety goggles, stop azide reaction by injecting 0.3 ml 6M NaOH.
7) Samples are ready for the isotope ratio mass spectrometer.
Appearance: silver white granules.
Danger! Flammable solid. May be fatal if inhaled. Harmful if swallowed. Causes eye, skin, and respiratory tract irritation. Contains cadmium. Cancer hazard. Avoid creating dust. Can cause lung and kidney disease. Inhalation of fumes may cause metal-fume fever. Air sensitive. May cause reproductive and fetal effects.
Target Organs: Blood, kidneys, liver, lungs, skeletal structures, prostate.
Potential Health Effects
Eye: Causes eye irritation.
Skin: Causes skin irritation.
Ingestion: Harmful if swallowed. May cause gastrointestinal irritation with nausea, vomiting and diarrhea. Ingestion may produce fluid loss, acute renal failure, and cardiopulmonary depression.
Inhalation: May be fatal if inhaled. Inhalation of fumes may cause metal fume fever, which is characterized by flu-like symptoms with metallic taste, fever, chills, cough, weakness, chest pain, muscle pain and increased white blood cell count. Damage may be delayed. May cause nausea, vomiting, abdominal pain, diarrhea, chest tightness, weakness, and delayed pulmonary edema. In humans inhalation causes proteinuria, an excess of protein in the urine.
Chronic: May cause respiratory tract cancer. Repeated inhalation may cause chronic bronchitis. Chronic inhalation may cause nasal septum ulceration and perforation. Cadmium and compounds may cause lung, liver and kidney damage and lung and prostate cancer in humans. May cause loss of smell, emphysema, anemia, bone demineralization, and lung fibrosis. The primary target organ for chronic cadmium disease is clearly the kidney.
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