Methods for the Determination of Stable Isotope Ratios of Multiple Nitrogen Species in Rainwater Using Distillation and Evaporation

Nitrogen (N) isotope ratios (δ¹⁵N) of multiple N species including ammonium (NH₄⁺), nitrate (NO₃⁻), dissolved organic N (DON), and total dissolved N (TDN) can serve as indicators of the sources of wet N deposition. However, methods for sequential analysis of these multiple N species are not well developed. In this study, methods for the determination of δ¹⁵N of these multiple N species were proposed through a traditional distillation method for NH₄⁺ and NO₃⁻, direct evaporation for TDN, and an isotope mass balance equation for δ¹⁵N-DON. A series of experiments were conducted 1) to find evaporation conditions including pH (<3.5 vs. 5.4) and evaporation methods (oven drying at 60 and 100°C, infra-red chamber, and freeze-drying), 2) to investigate precision and accuracy of distillation for δ¹⁵N of NH₄⁺ and NO₃⁻, and 3) to determine δ¹⁵N-TDN by direct evaporation and δ¹⁵N-DON with mass balance equation. (NH₄)₂SO₄ (‒4.0 ± 0.03‰), KNO₃ (‒4.2 ± 0.03‰), and CO(NH₂)₂ (‒5.4 ± 0.04‰) were used as reference materials for NH₄⁺, NO₃⁻, and DON, respectively. In this study, for the analysis of δ¹⁵N of NH₄⁺ and NO₃⁻, the conventional distillation method was tested in an effort to save cost for laboratories equipped with the distillation system. Evaporation using oven at 60°C (but not 100°C), infra-red chamber, or freeze-drier after acidification to < pH 3.5 prevented ¹⁴NH₃ loss. Analysis of the δ¹⁵N-NO₃⁻ (‒4.4 ± 0.1‰ to ‒3.9 ± 0.5‰) was reliable for a wide range of N content (0.1–0.5 mg), but analytical errors for δ¹⁵N-NH₄⁺ were as high as 2.1‰ when N content was small (e.g., 0.1–0.3 mg N) due to background contamination and potential interference by co-existing DON. Direct evaporation of solution containing NH₄⁺, NO₃⁻, and DON to dryness produced reliable δ¹⁵N-TDN with accuracy <0.15‰ and precision <0.21‰. However, the analytical errors of δ¹⁵N-DON were highly dependent on the content of co-existing NH₄⁺ as well as DON content. Therefore, the proposed protocol can be applied for rainwater containing a high NH₄⁺ concentration (>2.0 mg N L⁻¹ assuming that 200 ml of sample is used for distillation).