Enabling scientific discovery through stable isotope analysis
Analysis of Nitrate δ15N, δ18O, Δ17O
Summary: 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.

Reference Materials: We use nitrates USGS35, USGS34, and IAEA-NO-3 as our international standards for this method.

Sample submission: We prefer 20 nmoles of nitrate / nitrite (minimum 5 nmoles) to complete the δ15N and δ18O analysis. We prefer 200 nmoles of nitrate / nitrite (minimum 10 nmoles) to complete the Δ17O analysis. It is most efficient to have the nitrate / nitrite concentration prior to isotope analysis. Please have your samples filtered and frozen in a plastic container capable of freezing. Please include in your shipment container the following form (pdf or doc).

Exhaustive description of analysis: Pseudomonas aureofaciens denitrifying bacteria are grown from freezer stocks on agar petri plates and used to inoculate 500 mL of trypic 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 a reciprical 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, Magneiusm perchlorate), a volatile organic compound trap, and a liquid nitrogen trap, before a vent. All sample / standard N2O remains in the liquid nitrogen trap and is moved into a secondary focusing liquid nitrogen trap. A dochotomy exists from here.

Intact N2O for δ15N and δ18O - If samples are to be analyzed for δ15N and δ18O only, the cryofocused N2O moves through a porapaq Q gas chromatography column for separation with any remaining CO2 and then into a Thermo Finnigan Delta Plus for mass / charge 44, 45, and 46 measurement.

Pyrolyzed N2O for δ15N, δ17O and δ18O - If samples are to be analyzed for δ15N, δ17O and δ18O, the cryofocused 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.

Calculations: Intact N2O for δ15N and δ18O - 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 - 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 istopes 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).

Suggested reading:

Sigman DM, Casciotti KL, Andreani M, Barford C, Galanter M, Böhlke JK. A Bacterial Method for the Nitrogen Isotopic Analysis of Nitrate in Seawater and Freshwater. Analytical Chemistry 73: 4145-4153. (2001).
Casciotti KL, Sigman DM, Galanter Hastings M, Böhlke JK, Hilkert A. Measurement of the Oxygen Isotopic Composition of Nitrate in Seawater and Freshwater Using the Denitrifier Method. Analytical Chemistry 74: 4905-4912. (2002).
Kaiser J, Hastings MG, Houlton BZ, Röckmann T, Sigman DM. Triple Oxygen Isotope Analysis of Nitrate Using the Denitrifier Method and Thermal Decomposition of N2O. Analytical Chemistry 10.1021/ac061022s. (2007).