Item talk:Q66778

Determination of the δ¹⁵N and δ¹⁸O of nitrate in water; RSIL lab code 2900

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.