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Methods for the Production and Use of Nitrogen‐13 In Studies of Denitrification
Author(s) -
Tiedje J. M.,
Firestone R. B.,
Firestone M. K.,
Betlach M. R.,
Smith M. S.,
Caskey W. H.
Publication year - 1979
Publication title -
soil science society of america journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.836
H-Index - 168
eISSN - 1435-0661
pISSN - 0361-5995
DOI - 10.2136/sssaj1979.03615995004300040016x
Subject(s) - chemistry , analytical chemistry (journal) , ion chromatography , elution , chromatography , thermal conductivity detector , gas chromatography , nitrogen , ion exchange , yield (engineering) , radiochemistry , ion , materials science , organic chemistry , metallurgy
Methods were developed for use of the radioactive isotope of nitrogen, 13 N, for short‐term studies of denitrification. 13 N was generated by irradiation of water with 12 to 15 MeV proton beams from a sector‐focused cyclotron. Under typical operating conditions of 0.7 to 3 µA beam currents for 10 min, the 13 N ionic species produced were NO 3 ‐ , 75–90%; NO 2 ‐ , 5–10% and NH 4 + , 0.5–25%. Traces of [ 13 N] N 2 O and [ 13 N] N 2 were also produced. The measured yield varied from 2 to 16 mCi/10 min irradiation depending on beam current. Vacuum evaporation at high pH was used to obtain 13 NO 3 ‐ + 13 NO 2 ‐ at > 99.8% purity, and high performance liquid chromatography (HPLC) was used to obtain 13 NO 3 ‐ or 13 NO 2 ‐ at > 99% purity. The HPLC system used a Partisil SAX anion exchange column eluted with phosphate buffer at pH 3.0 and was coupled to a coincidence NaI(T1) detector for counting 13 N species in the effluent. Separation of NH 4 + , NO 2 ‐ , and NO 3 ‐ was achieved within 5 min. This system was used to monitor purity of 13 N substrates and for studies of dissimilatory nitrate reduction to ammonia. A gas chromatograph‐proportional counter detector system was developed to separate and measure [ 13 N] N 2 , [ 13 N] N 2 O and 13 NO. Separation was by Poropak Q and Molecular Sieve 5A columns and was achieved in 5 min. Denitrification rates and products of soils and bacterial cultures incubated in sealed flasks were monitored with this system. Continuous rates of [ 13 N] N 2 and [ 13 N] N 2 O production were monitored using a differential trapping, gas stripping system. Soil slurries amended with 13 NO 3 ‐ or 13 NO 2 ‐ were stripped of gases by continuously sparging with helium. N 2 O was collected in a liquid nitrogen trap. Nitrogen gas passed through this trap but was retained in a Molecular Sieve trap immersed in liquid nitrogen. 13 N gases collected in each trap were continuously counted by NaI (T1) detectors. Linear rates of gas production were typically observed from 15 min after addition of the 13 N substrate to termination of the experiment after 1 to 1.5 hours. 13 N has the advantage in denitrification studies of allowing direct measurement of N 2 , very sensitive short‐term rate measurements, and isotope exchange experiments at low substrate concentrations.