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.

Method Overview:

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.

Safety Warning:

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. Lab coat, nitrile gloves, and goggles MUST be worn at all times.

Day One

A. Reduce NO₃^- to NO₂^-

Nitrate Standards:
USGS 34= 1.33 x10^-4 M
USGS 35= 8.12 x10^-5 M
N3 = 1.04 x10^-4 M

Reagents:
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.

Supplies:
1) 20ml screw top glass vials (cleaned and combusted) N = samples+ 5 standards + 2 blank.

Method:
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.

Day Two

B. Centrifuge samples for 10 minutes at ~2000 rpm.

C. Measure the Yield of NO₂^-

Standards:
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.

Supplies:
1) Plastic test tubes, caps, plastic cell for spectrophotometer

Reagents:
1) Nitrite color reagent: Salfanilamide (1-Naphyhyl) ethylenediamine dihydrochloride (NED).

Note: All standards and reagents should be stored in 4°C.

Method:
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

Reagents:
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.

Supplies:
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

Method:
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.

NEWS & EVENTS

November 2009

UCSC's SIL retires its 15 year-old dual-inlet Optima following a very productive career that generated over 40,000 data points.

November 2009

Two Tsavo Lions In Famed Killings Get Partial Reprieve according to UCSC Graduate Student Justin Yeakel as inteviewed on NPR's All Things Considered. Listen here:

July-August 2009

SIL co-director Christina Ravelo (Ocean Sciences) sails as co-chief scientist on Integrated Ocean Drilling Project (IODP) Expedition 323 to investigate Bearing Sea Paleoceanography.

December 2008

SIL co-director Christina Ravelo (Ocean Sciences) gives the 2008 Emiliani Lecture at the American Geophysical Union Meeting in San Francisco on "Lessons from the Pliocene Warm Period and the Onset of Northern Hemisphere Glaciation".

September 2008:

UCSC SIL has been funded by the National Science Foundation for a new Dual-Inlet Isotope Ratio Mass Spectrometer with individual acid drip system for very small calcium carbonate samples.

July 2008:

SIL co-director Jim Zachos (Earth and Planetary Sciences) recieves prestigious Humbolt Research Award. See Humbolt Award for details.

May 19, 2006: UCSC Stable Isotope Laboratory Symposium

In recognition of the new continuous flow instruments added to the UCSC stable isotope facility a Symposium is being run to highlight the new analytical capabilities.