A new approach coupling ion-exchange chromatography purification and off-line conversion of individual amino acids to N2O for robust δ15N analysis

Compound-specific nitrogen (N) isotope analysis (δ¹⁵N) of individual amino acids (AA) is a powerful tool for tracing N source, N utilization patterns, and trophic fractionation in biogeochemical cycles and foodwebs. The δ¹⁵N of phenylalanine (Phe) retains the baseline δ¹⁵N values, decoupled from the effects of trophic transfer, while glutamic acid (Glu) demonstrates significant ¹⁵N enrichment with each trophic transfer. The δ¹⁵N of Glu and Phe in one single sample can thus yield information of both the N source and trophic position, which provides a better interpretive power than bulk δ¹⁵N. However, most published δ¹⁵N_AA results were determined using Gas Chromatography -Combustion-Isotopic Ratio Mass Spectrometry (GC-C-IRMS), which can only analyze 12-14 proteinaceous AAs due to the need of chemical derivatization.

We developed a method using Ion Exchange Chromatography -Pulsed Amperometric Detector to separate and collect underivatized AAs. Collected AAs are directly oxidized to nitrite by hypochlorite, which skips the evaporation to dryness step required by the GC approach. Hypochlorite specifically oxidize the amino-N only, greatly reducing the potential interferences from other N species. Resulted nitrite can be converted to N₂O via azide and δ¹⁵ N₂O is measured using Purge Trap-IRMS. Our method can analyze 17 AAs with high precision (<±0.5‰). The accuracy of our method was tested by analyzing 3 individual USGS AA standards (Glu, Valine, and Glycine) with a wide range of δ¹⁵N at natural abundance levels. Their δ¹⁵N values measured by our method are linearly correlated with the values provided by USGS with slopes of 1, proving that δ¹⁵N can be retained with high reliability after multi-preparation steps. We further tested our method with samples in various matrices, such as fish, sediment, and cyanobacteria. This method allows a precise and sensitive δ¹⁵N measurement of most amino acids, especially the two important "source (Phe)" and "trophic (Glu)" AAs. This allows us to reconstruct the variations of trophic structures and nutrient sources from sinking particles and sediments caused by El Niño and other physical forcing. AAs purified by our method can also be converted to CO₂ for δ¹³C and radiocarbon analysis, which can greatly simplify the approaches to obtain multiple isotope patterns within amino acids.