Nitrate Isotope Analysis
The denitrifier method uses Pseudomonas aureofaciens bacteria to denitrify nitrate (NO3) to nitrous oxide (N2O). The general flow of work, 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, once the bake out is done, turn the Precon helium dial down 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 (mass 44, 45, and 46) or as O2 and N2 (mass 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 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 encounter corrosive reagents and should wear protective clothing and nitrile gloves as well as work in a fume hood.
- Bake-out Complete -- Make sure everything associated with the baking out procedure (see Baking Out the Instrument) is ready for samples. You should bake the instrument before (or after) every run.
- -60 °C chiller is on AND cold -- Plug in the chiller using the brown extension cord coming up out of the cabinet below. Use the digital thermometer to verify the temperature is below -40 °C before running. If the ethanol appears solid, it needs to be replaced with fresh ethanol. To replace the ethanol: remove the water trap, loosen the clamp on the dewar, dump the ethanol into a white dish pan until its room temperature, add fresh ethanol.
- Helium clean-up liquid nitrogen trap -- This is a loop that is within the large precon liquid nitrogen dewar. Be careful when removing it to allow it to thaw between runs.
- GasBench GC temperature -- The GasBench GC should be at lab temperature. The set point is 0 °C and the measured temperature should be ~ 22 °C.
- PreCon liquid nitrogen dewar -- Fill the liquid nitrogen dewar on the PreCon. This dewar often collects water at the rim. This ice should be removed before each run by putting the liquid nitrogen back into a capped handled dewar and then allowing the PreCon dewar to warm up (you can fill it with hot water, dump the water, wipe out the dewar to dry it).
- PreCon Reagents -- The reagents should be checked weekly and changed when necessary. Pack a 17.5 cm long 3/8" OD pyrex tube with half ascarite and half magnesium perchlorate separated by glass wool and glass wool at either end. Use the vortex to pack down the ascarite and magnesium perchlorate. Remove existing trap, install the new trap, tighten with wrenches, leak check. All waste goes into a waste container.
- Measuring N2O or O2 / N2 -- Make sure the Gas Bench valco valve is ready for you to measure the species of interest (N2O vs O2 / N2). If you are interested in the 17O of nitrate, you must measure your samples as O2 and N2 by passing the N2O through the gold tube. If, however, you are measuring nitrate quantities lower than 50 µmol, you probably want to use the N2O method. See the "Switching GasBench Valco Valve" section below for specific instructions on how to change the valve.
Switching GasBench Valco Valve
If you need to switch between the N2O and O2 / N2 GC modes or if the computer was rebooted, carefully follow these steps:
- Close needle valve - on North side of Irene, unhinge door with built in fan. Small needle valve is located within, attached above larger equipment. 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.
- On the gas bench, turn the helium dial all the way down to 0. Wait for it to get to 0. You must be patient. Don't keep dialing the knob. It takes time for the pressure to go down.
- On the computer, in the program IsoDat Acquisition, change 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.
- Turn helium on gas bench back to 1, wait for it to get to 1.
- Open needle valve all the way.
- Lyse bacteria -- All bacteria must be dead to prevent any further activity that might affect your samples. Lyse the bacteria with 0.2 mL 10M NaOH and shake well. Wipe off all NaOH from tops of septa. Make sure vials are right side up.
- Antifoam -- After the vial side-walls have had a chance to drain from previous step, add a few drops of well mixed antifoam-B into media solution and 5-10 drops to the inside-walls of the vial. Do not shake at all. This will allow fresh antifoam to reside on the side-walls of the vial. Anitfoam-B must be well mixed to be effective. Make sure you shake the syringe before using it. Wipe off all antifoam from tops of septa.
- Fill rack with vials -- The first and last position vials should be an N2OinHe and you may choose to include them throughout the run (e.g. every 10 vials). They help to monitor instrument drift as well as presence / absence of sample peaks. Flush an empty, freshly capped vial with N2OinHe for 30 seconds using the cylinder in the mass spec lab. If you must make an N2OinHe using pure N2O, inject at most 0.5 µL into a 20 mL vial.
- Oil -- Put 2 drops of vacuum pump oil on the top of each septum. The oil acts to lubricate the needle as it pierces the septum. The autosampler does not have the strength to push a dry needle through a dry septum.
- The daily log is Irene_DailyLog.xlsx.
- Turn all reference gases off in the Gas Bench window on the left side of Isodat Acquisition
- Record the date, your name, and the source settings
- Record the gas pressures from the compressed gas cylinders as well as from the gas bench and precon peripherals.
- Enter backgrounds for water (m/z 18), argon (m/z 40), nitrogen (m/z 28, 29, 30), oxygen (m/z 32, 33, 34), and N2O / CO2 (m/z 44, 45, 46) by selecting the appropriate gas from the little menu located in the lower left corner of Isodat Acquisition. If any of the values are unreasonably high relative to historical measurements, see troubleshooting below.
- If you are measuring your samples as N2O, turn on N2O reference gas and once it is stable, enter these values into the daily log under signals. If you are measuring O2 and N2, turn on O2 and enter the signals. Turn O2 off, switch to N2, turn N2 on, and enter those signals. All of these numbers should be in the thousands.
- Conduct a jump calibration -- Open Instrument Control, turn the N2 reference gas on, select Scan the Jump Calibration, highlight row 001, click the circular arrows at bottom of window, click the the prompts to proceed, wait for the calibration to finish. Enter today's date for jump calibration assuming you just did one.
- Enter the number of samples (excluding standards) and the species of bacteria used
- Enter notes related to the dataset itself
Samples sequence -- 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 one to the number 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
- Preparation - volume of 18 MΩ injected 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
- Start the run -- Highlight rows if not running the entire sequence; click start; enter your dataset name, making sure that the dominant folder starts with "yymmdd".
After your run is finished, use matlab to reduce the data.
- 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]
- You can use the "Results" shortcut on the desktop to get you most of the way.
- You only need the .csv file. You do not need the entire folder that largely contains irene specific data files.
- Copy this file to the server S:\Data\projects\[YOUR PROJECT FOLDER]\raw
- Go to either the "North Going Zax" or the "South Going Zax" computers and open matlab
- Type 'irene' and hit enter
- Follow prompts
- Output summary file is located S:\Data\projects\[YOUR PROJECT FOLDER]\reduced
Bake the water trap, the VOC trap, the GC, and the mass spec after each run.
- 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 traps 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 its dripping).
- 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.
- 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 HI.
- 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).
- IRMS -- Some of the heaters inside the mass spec will also be on during the bake routine in the next step.
- 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.
Running Samples -- NOTE, to run samples, 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 white cap on the PreCon vent should be removed, and the VOC trap should be at room temperature.
- Sample vial media with NaOH -- This media, while sterile (because of the NaOH) is very basic and must be neutralized. Use the crimp cap removal tool to pull off the caps. Discard the caps into the trash and pour the media into a 1 L glass medial bottle which needs to be labeled "High pH Waste". When this bottle is full, neutralize the liquid with Hydrochloric acid (HCl) waste (this is a 10-20% HCl solution that has previously been used to acidify soil or rock samples). Use pH paper to verify it is neutral. If you overshoot and make it acidic, use the carbonate that is in the hood to bring it back to neutral. Pour the neutral pH liquid down the drain and flush with water. Enter the appropriate information into the drain log.
- All glassware -- Wash all glassware as per the Cleaning Laboratory Glassware method.
- Centrifuge tubes -- Rinse caps and centrifuge tubes with alcohol. Rinse caps and vials 3x with DI water. Invert tubes in rack. Rinse rack with DI water. Place caps upside down on rack. Place rack with tubes and caps in clean-dish hood to dry overnight.
- Syringe Needles -- All syringe needles go in the red plastic sharps container.
- 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 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 N2OinHe 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.
- Sigman DM, Casciotti KL, Andreani M, Barford C, Galanter M, Böhlke JK. (2001) A Bacterial Method for the Nitrogen Isotopic Analysis of Nitrate in Seawater and Freshwater. Analytical Chemistry 73: 4145-4153. doi: 10.1021/ac010088e.
- Casciotti KL, Sigman DM, Galanter Hastings M, Böhlke JK, Hilkert A. (2002) Measurement of the Oxygen Isotopic Composition of Nitrate in Seawater and Freshwater Using the Denitrifier Method. Analytical Chemistry 74: 4905-4912. doi: 10.1021/ac020113w.
- Kaiser J, Hastings MG, Houlton BZ, Röckmann T, Sigman DM. (2007) Triple Oxygen Isotope Analysis of Nitrate Using the Denitrifier Method and Thermal Decomposition of N2O. Analytical Chemistry 79: 599-607. doi: 10.1021/ac061022s.