Improved chromatographic analysis of N:N ratios in ammonium or nitrate for isotope addition experiments

Estimating nitrogen transformation rates in aquatic ecosystems by isotope dilution techniques is simplified by directly measuring nitrogen isotopic ratios for NH₄⁺ in the water using high performance cation exchange liquid chromatography (HPLC). Modifications of HPLC conditions and implementation of a median-area method for retention time determination improved and linearized a previously reported sigmoid relationship between the retention time shift (RT_shift) of the NH₄⁺ peak and the ratio of ¹⁵NH₄⁺]: Total NH₄⁺] in seawater fortified with ¹⁵NH₄⁺. Increasing the temperature of the HPLC column from 47 to 85 °C increased mobile phase buffer flow rate relative to column back pressure, decreased the retention time for NH₄⁺, and allowed the buffer pH to be optimized relative to the pK of NH₄⁺. The use of median-area rather than maximum-height to define the retention time of NH₄⁺ further improved the linearity (r > 0.995) of the relationship between the ratio ¹⁵NH₄⁺]: Total NH₄⁺] and RT_shift over the range of isotope ratios. Reduction of NO₃⁻ to NH₄⁺ by adding zinc dust to acidified (pH 2) seawater or lakewater samples, followed by pH neutralization, and subsequent analysis of NH₄⁺ isotope ratios by HPLC, extended application of the method to isotope dilution experiments with NO₃⁻. Advantages of this direct-injection method over mass-measurement approaches traditionally used for isotope dilution experiments include small sample size and minimal sample preparation.