Techniques and Methods 10–C17
The purpose of the Reston Stable Isotope Laboratory (RSIL) lab code 2900 is to determine the δ15N and δ18O of nitrate (NO3–) in water. The δ15N and δ18O of the dissolved NO3– are analyzed by converting the NO3– to nitrous oxide (N2O), which serves as the analyte for mass spectrometry. A culture of denitrifying bacteria is used in the enzymatic conversion of the NO3– to N2O, which follows the pathway shown in equation 1:
NO3– → NO2– → NO → 1/2 N2O (1)
Because the bacteria Pseudomonas aureofaciens lack N2O reductive activity, the reaction stops at N2O, unlike the typical denitrification reaction that goes to N2. After several hours, the conversion is complete, and the N2O is extracted from the vial, separated from volatile organic vapor and water vapor by an automated –65 °C isopropanol-slush trap, a Nafion drier, a CO2 and water removal unit (Costech #021020 carbon dioxide absorbent with Mg(ClO4)2), and trapped in a small-volume trap immersed in liquid nitrogen with a modified Finnigan MAT (now Thermo Scientific) GasBench 2 introduction system. After the N2O is released, it is further purified by gas chromatography before introduction to the isotope-ratio mass spectrometer (IRMS). The IRMS is a Thermo Scientific Delta V Plus continuous flow IRMS (CF-IRMS). It has a universal triple collector, consisting of two wide cups with a narrow cup in the middle; it is capable of simultaneously measuring mass/charge (m/z) of the N2O molecule 44, 45, and 46. The ion beams from these m/z values are as follows: m/z = 44 = N2O = 14N14N16O; m/z = 45 = N2O = 14N15N16O or 14N14N17O; m/z = 46 = N2O = 14N14N18O. The 17O contributions to the m/z 44 and m/z 45 ion beams are accounted for before δ15N values are reported.
Revised September 17, 2012 (version 1.1)
First posted 2007
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Coplen, T.B., Qi, Haiping, Révész, Kinga, Casciotti, Karen, and Hannon, J.E., 2012, Determination of the δ15N and δ18O of nitrate in water; RSIL lab code 2900, chap. 17 of Stable isotope-ratio methods, sec. C of Révész, Kinga, and Coplen, T.B. eds., Methods of the Reston Stable Isotope Laboratory (slightly revised from version 1.0 released in 2007): U.S. Geological Survey Techniques and Methods, book 10, 35 p., available only at http://pubs.usgs.gov/tm/2006/tm10c17/. (Supersedes version 1.0 released in 2007.)
Summary of Procedure
Reporting Units and Operational Range
Reference Materials and Documentation
Labware, Instrumentation, and Reagents
Sample Collection, Preparation, Analysis, Retention Times, and Disposal
Data Acquisition, Processing, Evaluation, Quality Control, and Quality Assurance
Health, Safety, and Waste-Disposal Information
Appendix A. Step-by-Step Procedure to Log-In Samples to LIMS-LSI
Appendix B. Step-by-Step Procedure to Print a Template and a Samples-to-Be-Analyzed List .
Appendix C. Step-by-Step Preparation Procedure for Nitrogen Isotopic Analysis of Nitrate by Bacterial Denitrification
Appendix D. Step-by-Step Procedure to Add Sample Information to Sequence Table
Appendix E. Step-by-Step Procedure to Transfer Data to LIMS-LSI, to Transfer Data to Back-Up Computer, and to Reevaluate Old Data
Appendix F. Step-by-Step Procedure to Determine and Apply Correction Factors and Evaluate Data
Appendix G. Daily Check List
Appendix H. Step-by-Step Procedure to Report Data