Nitrate Isotope Analysis

The denitrifier method uses Pseudomonas aureofaciens bacteria to denitrify nitrate (NO₃) to nitrous oxide (N₂O). The general workflow, assuming all supplies are on hand, is to inoculate bottles on day 1, harvest those bottles and inject your samples on day 2, start the mass spectrometer on day 3, bake out the mass spectrometer, and lastly, if no other runs are scheduled, set the system to idle on day 4. Some flexibility exists within this framework. This particular method focuses on day 3+.

The isotope ratio mass spectrometer (Brave Irene) Finnigan DeltaPlus can be configured to run N₂O as N₂O (m/z 44, 45, and 46) or as O₂ and N₂ (m/z 32, 33, 34, and 28, 29) via hot gold tube pyrolysis of N₂O. This flow diagram may be helpful.

Appropriate precautions should be taken to protect yourself against extreme temperatures as you will encounter a range from very cold liquid nitrogen temperatures to very hot baking out parts. Wear eye protection and leather gloves when handling liquid nitrogen. You will also be using sodium hydroxide and, when doing so, should wear eye protection and nitrile gloves.

On the computer, in the program IsoDat Acquisition, change the Gasbench valco valve button (located in the Gas bench window) to the appropriate setting by clicking on it once. For N₂O analysis, make sure valve is in "Load" position. For O₂ / N₂ analysis (gold tube method), make sure valve is in "Inject" position. Make sure to run through the daily log backgrounds AFTER switching the valco valve.

The lab uses a daily log for each instrument or preparation line to allow users a first glance at the readiness of the instrument. By comparing the current state of the instrument to historical states, you are more informed about the instrument and whether or not it is functioning properly and ready to run your samples.

Each daily log is web based and browser accessible. No link is provided here by design. Open the browser on the controlling computer and you should see at least two tabs already open. One tab is this SOP and the other is the daily log. If the browser has more than two tabs open, it may have additional SOPs. Use the bookmark toolbar as needed if tabs have been closed.

Work through each cell of the daily log. If you are uncertain where to find certain information, hover over the column header tip, denoted by a circled 'i' (ⓘ).

Make certain to press the 'save to log' button when you are finished entering data.

You are welcome to make notes if you have observed something with or done something to the instrument and would like to document that information. Use the "insert note" link at the top of the daily log to make a note. You may enter notes at any time.

Open "N2O_Samples.seq" or "O2N2_Samples.seq" sequence from the left side of the screen within the "File Browser" window under the "Sequences" tab.

• Make sure every row in the peak center column is checked.
• Make sure the "AS Sample" column increments by 1 for each row from 1 to the # of vials you have.
• Make sure the "AS Method" column has ">Internal No 9" selected.
• Identifier 1 – Sample / Standard name.
• Identifier 2 – Nitrate concentration of the sample/standard in μM.
• Comment – Sample / Standard injection volume in mL.
• Make sure the Method has "N2O_samples.met" or "O2N2_samples.met" selected.
• Highlight the rows containing the vials you wish to run.

Last chance checklist:

• the water trap should be at -60 °C
• the GC should be set to 0 °C (room temperature)
• the IRMS heaters should be off
• the VOC trap should be at room temperature.

Start the run:

• Highlight rows if not running the entire sequence
• click Start
• enter your sample set name in the field next to "Folder Name" and "Export File Name"
• click OK

You are NOT done yet:

• Wait for the first sample to start.
• After the needle has been inserted into the first vial, watch the ball flow meter until it stabilizes.
• Adjust the Precon Carrier pressure until the ball flow meter reads and stabilizes at 113 (this corresponds to 60 mL / min).
• Adjust the pressure SLOWLY. If you turn it down, you must give the system time for that excess helium pressure to dissipate.
• If you can not reach 60 mL / min, something is wrong and you should stop the run AFTER this first vial.
• Assuming you were able to reach 60 mL / min, stick around the lab until you see peaks for the first vial that look reasonable.
• Come back later in the day to top off the liquid nitrogen in the dewar.

Shortly after your run has finished, either complete a bake-out followed by putting the system into idle mode, or if you do not have time for the bake-out, just put the system into idle mode. The system is using helium at a very high rate until you show up to put it in idle mode.

After your run is finished, use matlab to reduce the data.

1. Here is the path to you data file C:\Thermo\Isodat NT\Global\User\Gas Bench\Results\[YOUR FOLDER STARTING WITH DATE]\Excel\[YOUR .CSV FILE].
2. You can use the "Results" shortcut on the desktop to get you most of the way.
3. You only need the .csv file. You do not need the entire folder of Irene specific data files (.dxf).
4. Copy this file to the server S:\Data\projects\[YOUR PROJECT FOLDER]\raw.
5. Go to the "North Going Zax", "South Going Zax", or "West Going Zax" computers and open Matlab.
6. Type irene and hit enter.
7. Follow prompts.
8. Output summary file is located S:\Data\projects\[YOUR PROJECT FOLDER]\reduced.

A "bake-out" is the process of heating up portions of a system in order to remove contaminants or otherwise undesirables. Most of the time water is the undesirable. Here we need to heat up water trap to remove trapped water, the VOC trap to remove the trapped volatile organic compounds, the GC to remove adhered CO₂, and the mass spectrometer for good measure. This bake-out should be completed after every run or before the next run. Note, if the instrument was not baked out following the previous run, AND, it is presently in idle mode, you will need to wake it up by turning the precon helium to running pressure.

1. Water trap -- The water trap is held at -60 °C during analysis and acts to trap any water vapor that is carried away from the sample vial. It also acts to prevent liquid media from traveling into the PreCon in the event of excessive foaming while purging. This trap needs to be thawed and baked out between each run. You can leave the immersion cooler on if you are going to run again the same day.
   • Screw the plastic white cap onto the vent on the top of the PreCon. (Note: ensure the He carrier dial on the front of the PreCon is turned up to "running".)
   • Then, remove the water trap out of the ethanol dewar, taking care not to tangle the tubing with anything, and place the trap into the heat-tape-foil-wrapped pipe. You will start it heating with the mass spec software in just a minute. Helium is flushing out both holes of the needle, so any water should evaporate and exit out the needle. If you are recovering from no peaks due to a clogged trap, allow the trap to become very hot, use gloves, pick up the trap while hot and CAREFULLY invert it such that any liquid inside will flow out the exit arm and then out the needle (look at the needle to see if it's dripping).

2. 

   

   Helium clean-up trap -- This is the loop that sits in the bottom of the blue PreCon liquid nitrogen dewar and acts to scrub the helium of any condensables. Lift it free of the dewar and allow it to dangle and warm up to room temperature. Leave it out of the dewar for the entire bake-out procedure. Replace it back into the dewar after the bake-out procedure.
3. VOC Trap -- The VOC (Volatile Organic Compound) trap retains any organics that are carried away from the sample vial. If these compounds make it to the gold tube, the organics will react and harvest all oxygen from your sample. Turn the Thermolyne controller located between the PreCon and the autosampler to 1.5. The VOC trap is supposed to bake at 180 °C. Use the yellow thermocouple meter and plug the VOC thermocouple in to see the temperature.

4. 

   GC columns -- The GasBench GC box has two GC columns in it. One for N₂O and one for O² / N². Both always have helium flowing through them and can be baked out at 200 °C. At the east side of the GasBench, press the "P" button on the "Jumo iTron 16". WATCH OUT FOR THE TINY CAPILLARY TUBING! Press the up arrow until the set point temperature reads 200 °C, press the "P" button again to apply this selected temperature. Repeat this sequence when you are finished baking out the GC's except the set point will be 0 °C (no heat at all, the GC box can not be cooled, this is lab temperature).
5. IRMS -- Some of the heaters inside the mass spec will also be on during the bake routine in the next step.
6. Start the bake sequence -- Using the mass spec computer, find the sequence called "BakeInstrument.seq" and run both lines by hitting start and then ok. This will heat the water trap for 10 minutes (and get very hot 300 °C) and also heat the mass spec. Then those heaters turn off and the instrument continues to provide a trace for another 50 minutes. Once this trace is complete (and the hour long bake routine is done), turn the GC to 0 °C as described above and turn the VOC trap off. You may need to set the water trap on top of the gas bench to allow it to cool before placing it back into the ethanol dewar.

Irene Gasbench Helium Cylinder Pressure is LOW

Helium must either be flowing through the Gasbench at all times OR the needle valve in the mass spectrometer case needs to be closed. If the Irene Gasbench Helium cylinder pressure is low, you have two options:

1. Switch to the backup helium cylinder - Generally choose this option. It is the easiest and least invasive. The Irene Gasbench has two helium cylinders connected to it. Both are located on the south wall. The main cylinder is in front and the backup is in the back. Once you have successfully switched to the backup helium cylinder, you can safety change the main cylinder.
2. If both the primary and backup cylinders need to be changed or if you know you are going to interrupt helium flow to the Gasbench, it is important that you protect the mass spectrometer from atmospheric air. Follow these steps to increase happiness.



• Close needle valve - on North side of Irene, unlatch door with built in fan. Small needle valve is located within (see photo to help identify). Close to finger tightness. How do you know it's closed? Check the vacuum gauge on IsoDat Acquisition, it should read something like Vac 2.0e-008 mBar.
• Swap the helium cylinder(s).
• Turn both cylinders on and check for leaks.
• allow helium to purge for at least an hour.
• Turn off the source.
• Open needle valve all the way.

No sample peaks during a run

• Make sure the liquid nitrogen dewar has liquid nitrogen in it otherwise the mass spectrometer cannot produce sample peaks and the sample cannot be analyzed.
• Did the needle insert completely into the sample vial? If you did not oil the septum (or did not wipe all NaOH and antifoam off the septum before oiling) the needle may have jammed half way in.
• Are you getting samples peaks for N2O in helium but not bacteria? Could be the batch.

Low / Small Peaks

• Look to see that the vials are bubbling when the needle is injected, if it is not, the needle is clogged.
• Check for leaks using the helium leak detector.
• Does the N₂O-in-He produce peaks.

High Backgrounds

• May indicate there is a leak. Use the helium leak detector and check for leaks.
• Pay particular attention to the valves as they have a history of leaking.