5:15 PM - 7:15 PM
[MIS22-P11] A simple and rapid method for measuring the stable nitrogen isotopic composition of ammonia in aqueous samples
Keywords:Nitrogen isotope ratios, Ammonia, Derivertization
Nitrogen cycles in environments with changing several redox forms through, for example, the fixation of ammonia by primary producers (NH3 to NOrg), the decomposition of organic matter by living organisms (NOrg to NH3), and the denitrification from nitrate by denitrifying bacteria (NO3- to N2). The stable isotopic composition (d15N) of these nitrogen chemicals has been employed as a conventional tool for identifying their sources and for evaluating the flux of nitrogen-involving processes in marine and terrestrial nitrogen cycles. However, the isotope analysis of NH3/NH4+ is still technically challenging, even though NH3/NH4+ is the central form of the nitrogen cycle. On the other hand, NH3 is an important nitrogen source for primary producers and a toxic chemical for many animals in ecosystems, which requires us to reduce the technical challenging for evaluating the sources and input/output balance of NH3/NH4+ and the other forms in ecosystems and environments.
During the last several decades, although a large variety of methods have been developed, precipitation and bacterial methods have been frequently used for measuring the isotopic composition of NH3/NH4+. In the former method, NH3/NH4+ is purified by distillation or diffusion technique into an acidified trap and is introduced as a solid chemical (e.g., an ammonium tetraphenylborate and ammonium sulfate) into an elemental analyzer - isotope ratio mass spectrometer (EA-IRMS). In the later method, NH3/NH4+ is converted to nitrate (NO3-) with hypobromite (BrO-) or persulfate (S2O82-) and subsequently to nitrous oxide (N2O) with the specific denitrifying bacterium Pseudomonas aureofaciens that lacks N2O-reductase activity, and is introduced as N2O into a purge-and-trap gas chromatograph - isotope ratio mass spectrometer (PT-GC-IRMS). However, these traditional methods require high skills and a long time for chemical/biological treatments prior to the isotope analysis. In the present study, we therefore have developed a simple and rapid method for measuring the d15N values of NH3/NH4+ in aqueous samples. Conventional chemical derivatization methods rely on acylation involving dehydration reactions. It is difficult to make acylation of NH3 in aqueous solution. However, by utilizing acylation involving not a dehydration reaction but a dehydration reaction, the derivatization of NH3 can be easily achieved even in aqueous solution. Specifically, isopropyl chloroformate was used as the acylating agent and pyridine as the catalyst to acylate NH3 in aqueous solution. This converted NH3 to isopropoxycarbonyl-NH2 derivatives in a one-step reaction at room temperature in a few tens of minutes. The isopropoxycarbonyl-NH2 derivative was extracted with dichloromethane/n-hexane (3/2) and introduced into a gas chromatograph-isotope ratio mass spectrometer (GC-IRMS) for isotope analysis.
We currently have the following four results:
(1) The derivatization is achieved by one step reaction at room temperature for 40 minutes.
(2) The d15N values of isopropoxycarbonyl-NH2 derivative can be determined with a standard deviation of 0.3‰-0.9‰.
(3) The stability of derivative is relatively short (within 16 hours) at room temperature.
(4) The yields of derivatives is not stable (30%-100%) in the procedure of this study.
Thus, this method developed does not require high skills and a long time for chemical/biological treatments prior to the isotope analysis and will be potentially applicable for the isotope analysis of NH3/NH4+ in various biological and environmental samples. In the next step, we will improve the derivatization condition such as pH of samples or temperature of reactions and will apply the improved method for natural aqueous samples such as seawater, freshwater, and biological samples.
During the last several decades, although a large variety of methods have been developed, precipitation and bacterial methods have been frequently used for measuring the isotopic composition of NH3/NH4+. In the former method, NH3/NH4+ is purified by distillation or diffusion technique into an acidified trap and is introduced as a solid chemical (e.g., an ammonium tetraphenylborate and ammonium sulfate) into an elemental analyzer - isotope ratio mass spectrometer (EA-IRMS). In the later method, NH3/NH4+ is converted to nitrate (NO3-) with hypobromite (BrO-) or persulfate (S2O82-) and subsequently to nitrous oxide (N2O) with the specific denitrifying bacterium Pseudomonas aureofaciens that lacks N2O-reductase activity, and is introduced as N2O into a purge-and-trap gas chromatograph - isotope ratio mass spectrometer (PT-GC-IRMS). However, these traditional methods require high skills and a long time for chemical/biological treatments prior to the isotope analysis. In the present study, we therefore have developed a simple and rapid method for measuring the d15N values of NH3/NH4+ in aqueous samples. Conventional chemical derivatization methods rely on acylation involving dehydration reactions. It is difficult to make acylation of NH3 in aqueous solution. However, by utilizing acylation involving not a dehydration reaction but a dehydration reaction, the derivatization of NH3 can be easily achieved even in aqueous solution. Specifically, isopropyl chloroformate was used as the acylating agent and pyridine as the catalyst to acylate NH3 in aqueous solution. This converted NH3 to isopropoxycarbonyl-NH2 derivatives in a one-step reaction at room temperature in a few tens of minutes. The isopropoxycarbonyl-NH2 derivative was extracted with dichloromethane/n-hexane (3/2) and introduced into a gas chromatograph-isotope ratio mass spectrometer (GC-IRMS) for isotope analysis.
We currently have the following four results:
(1) The derivatization is achieved by one step reaction at room temperature for 40 minutes.
(2) The d15N values of isopropoxycarbonyl-NH2 derivative can be determined with a standard deviation of 0.3‰-0.9‰.
(3) The stability of derivative is relatively short (within 16 hours) at room temperature.
(4) The yields of derivatives is not stable (30%-100%) in the procedure of this study.
Thus, this method developed does not require high skills and a long time for chemical/biological treatments prior to the isotope analysis and will be potentially applicable for the isotope analysis of NH3/NH4+ in various biological and environmental samples. In the next step, we will improve the derivatization condition such as pH of samples or temperature of reactions and will apply the improved method for natural aqueous samples such as seawater, freshwater, and biological samples.