Nitrate - δ15N, δ18O, δ17O
We use the bacterial denitrifier method along with an autosampler / PreCon / GasBench II assembly coupled to a Finnigan Delta Plus Advantage to analyze nitrate δ15N, δ18O, and Δ17O. Pseudomonas aureofaciens denitrifying bacteria are grown from freezer stocks on agar petri plates and used to inoculate 500 mL of tryptic soy broth growth media. After 6-8 days bacterial cells are harvested by centrifugation at 8000 rpm and 18 °C. Bacterial cell pellets are resuspended in nitrate free media and divided into 22 20 mL crimp-seal vials. Vials are crimped and purged with helium for 4-5 hours to purge the vial completely of oxygen and to allow the cells to process any remaining nitrate to nitrous oxide. A volume of sample and standard is injected to yield a total of 20 nmoles of nitrate for direct analysis of N2O or 200 nmoles of nitrate for N2O pyrolysis. Vials are inverted and placed on an orbital shaker overnight. After at least 8 hours, 0.2 mL of 10 M NaOH is injected into each vial to lyse the cells and several drops of Antifoam are added. Vials are then loaded into a GC PAL autosampler. Each vial is purged with helium, forcing the effluent through a cold water trap (-60 °C), a carbon dioxide, water chemical trap (Ascarite, Magnesium perchlorate), a volatile organic compound trap, and a liquid nitrogen trap, before a vent. We use nitrates USGS35, USGS34, USGS32, and IAEA-NO-3 as our international standards for this method. All sample / standard N2O remains in the liquid nitrogen trap and is moved into a secondary focusing liquid nitrogen trap. A dichotomy exists from here.
Intact N2O for δ15N and δ18O - If samples are to be analyzed for δ15N and δ18O only, the cryo focused N2O moves through a porapak Q gas chromatography column for separation with any remaining CO2 and then into a Thermo Finnigan Delta Plus Advantage for mass / charge 44, 45, and 46 measurement. Raw δ15N values are corrected to the Air-N2 scale by generating a linear curve from measured versus accepted USGS34 and IAEANO3 international reference materials with values of -1.8 ‰ and +4.76 permil, respectively. The IsoDat software assumes all oxygen measurements are mass dependent. Because USGS35 exhibits mass independent fractionation between O17 and O18, δ values must be recalculated with an appropriate alpha value. USGS35 δ15N values are then corrected to the USGS34 and IAEANO3 curve and used as a quality control reference. This method assumes all samples have a Δ17O equal to 0.
Pyrolyzed N2O for δ15N, δ17O and δ18O - If samples are to be analyzed for δ15N, δ17O and δ18O, the cryo focused N2O moves through a gold tube held at 800 °C which pyrolyzes the N2O into O2 and N2. These two species are then separated in a 5A molecular sieve gas chromatography column and then sent into the isotope ratio mass spectrometer for mass / charge 32, 33, 34 and then 28, 29 measurement. Raw δ15N values are corrected to the Air-N2 scale as in the intact N2O method above without any corrections concerning mass dependence because all of the oxygen isotopes are measured directly when N2O is pyrolyzed. δ17O and δ18O raw values are corrected to VSMOW by generating a linear curve from measured versus accepted USGS35 and USGS34 international reference materials with values 51.1 ‰ and -14.6 ‰ for δ17O and +56.81 ‰ and -27.78 ‰ for δ18O, respectively (using CIAAW δ18O).
We prefer 20 nmoles of nitrate / nitrite (minimum 5 nmoles) to complete the δ15N and δ18O analysis. We prefer 100 nmoles of nitrate / nitrite (minimum 20 nmoles) to complete the Δ17O analysis in no more than 13 mL of sample. The minimum nitrate / nitrite concentrations, then, are 25 μg / L and 95 μg / L, respectively (be prepared to sacrifice precision for nitrate concentrations lower than our preferred amounts). 13 mL is the maximum volume we can inject into a single analysis vial but you should send enough sample for 2-3 replicates in the event of desired reruns. It is most efficient to have the nitrate / nitrite concentration prior to isotope analysis. If you do not provide sample concentration, additional replicates will be required. Analysis costs are here. No additional sample preparation costs are associated with this method. You are, however, responsible for the cost of additional analyses due to absent or inaccurate nitrate concentration measurements. Please have your samples filtered to 0.1 µm and /or frozen in a plastic container capable of freezing. If freezing is not possible / practical, you may choose to acidify with nitrate free HCl to pH 2 (this requires addition of nitrate free NaOH back to pH 7 prior to analysis). Make certain your container lids are water tight and remain so during shipment.
Standard Operating Procedures
- Nitrate Preconcentration
- Nitrate Sample Preparation
- Nitrate Isotope Analysis
- IsoGeoChem 2010 Poll of SOPs
- 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.