Compound specific isotope analysis of individual amino acids (CSI-AA) is a powerful tool for tracing nitrogen (N) source and transformation in biogeochemical cycles. Specifically, the d15N value of phenylalanine (d15NPhe) represents an increasingly used proxy for baseline d15N values, with particular promise for paleoceanographic applications. Further, multiple studies have shown that d15N values of Phe and glutamic acid (Glu) can be coupled to provide precise estimates of trophic position (TP), while simultaneously decoupling baseline d15N values from the effects of trophic transfer. However, current derivatization/gas chromatography (GC) methods require expensive and relatively uncommon instrumentation, and have relatively low precision, posing significant issues for many potential applications. Here we present a new approach to CSI-AA based on a high-pressure liquid chromatography (HPLC) purification of underivatized amino acids, followed by offline elemental analysis-isotope ratio mass spectrometry (EA-IRMS). The utility of this approach is demonstrated in two different applications using natural materials: 1) the construction of a high precision paleoceanographic record from the d15NPhe in proteinaceous coral tissue and 2) the TP estimation of five marine organisms using the coupled d15N values of Glu and Phe. Both demonstrations were compared to values measured by an established GC based method. In all cases both methods produced equivalent values, however, values generated by the new HPLC/EA-IRMS approach had higher average precision in all cases. These results demonstrate that this approach represents a viable alternative to traditional GC based methods for d15NAA measurement and may therefore allow significant expansion of CSI-AA applications, requiring only commonly available instrumentation to produce high precision d15NAA values.

Marine dissolved organic matter (DOM) is the largest pool of actively cycling organic carbon and nitrogen in the ocean. Despite the importance of DOM in biogeochemical cycles, understanding of its composition and cycling is limited, in part due to difficulty in isolating representative material. Here we present a new approach to DOM isolation via a sequential combination of ultrafiltration and solid phase extraction, which allowed the selective isolation of two fractions with different compositions and reactivities. By limiting the influence of reactivity mixtures, we were able to directly investigate the composition and cycling of both labile and refractory DOM. Measurements of bulk isotopic (Δ14C, δ13C, δ15N) and compositional (C/N ratio, 13C and 15N NMR) parameters confirmed the uniqueness of the two fractions. The ultrafiltered high molecular weight (HMW) fraction was younger and dominated by carbohydrate-like and protein-like molecules. In contrast, the low molecular weight (LMW) fraction was older and dominated by alicyclic carbon and heterocyclic nitrogen containing molecules.

Spectroscopic studies have shown that the nitrogen-containing fraction of DOM (DON) is dominated by labile proteinaceous material. Despite this, other evidence suggests that a large fraction of DON is resistant to degradation. However, the paradigm of DON composition is based on measurements of HMW DON alone. Measurements of isotopic (δ15N, AA-Δ14C) and compositional (15N NMR, AA-D/L ratios) parameters of LMW DON provided a new perspective on the composition and cycling of DON in the ocean. Amino acids in LMW DON had both old radiocarbon ages and high D/L ratios, suggesting a bacterial mechanism capable of preserving proteinaceous material on millennial timescales. NMR analysis and nitrogen isotopic ratios demonstrated that LMW DON is dominated by heterocyclic nitrogen-containing molecules likely with intrinsically refractory molecular structures. These observations present a new paradigm for marine DON composition and suggest a mechanism for its long-term persistence.