Improved chromatographic analysis of 15N:14N 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 (RTshift) 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 RTshift 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.