Isotope dilution to enhance δD measurability in low-abundance n-alkane samples

Compound-specific stable isotope analysis (CSIA) of organic biomarkers enables reconstruction of past environmental and climatic conditions by resolving the isotope composition of individual molecular compounds. Among these applications, hydrogen isotope (δD) measurements of leaf wax n-alkanes are widely used to infer changes in hydroclimate, including variations in precipitation isotopic composition, moisture source, and evaporative conditions. Because long-chain n-alkanes are resistant to degradation and preserve a terrestrial signal in sedimentary archives, their δD values provide a robust proxy for past continental hydroclimate across a wide range of depositional settings.

Despite this utility, CSIA of organic biomarkers is frequently limited by the low abundance of target compounds, particularly in sedimentary archives where concentrations vary strongly across stratigraphy and compound class. This limitation is especially acute for δD measurements, which typically require injected analyte masses of several hundred nanograms to achieve acceptable precision on gas chromatography-isotope ratio mass spectrometry (GC-IRMS) systems. Because hydrogen isotope analysis relies on pyrolytic conversion to H₂, δD measurements generally operate at lower absolute signal intensities than compound-specific δ¹³C analyses, placing them closer to instrumental sensitivity limits where background correction, baseline placement, and nonlinear response exert a proportionally greater influence on measured isotope ratios. As a result, δD analysis of individual low-abundance compounds is often precluded, necessitating pooled samples or coarse sampling intervals that suppress short-duration climate signals and limit the achievable resolution of paleoclimate reconstructions.

Here, we assess the feasibility, limitations, and uncertainty structure of isotope dilution (ID) for δD measurements of leaf wax n-alkanes using internationally recognized, isotopically characterized n-alkane standard mixtures. Isotope dilution offers a potential strategy to stabilize isotope measurements through controlled mixing of a low-abundance analyte with an isotopically characterized spike, thereby increasing the total amount of analyte contributing to the measurement and reducing uncertainty associated with low signal intensities. Controlled mixing experiments isolate the effects of nominal mixing ratio, isotope contrast between spike and sample, and signal intensity on back-calculated isotope values. These tests provide a framework for quantifying uncertainty propagation in ID-CSIA and for defining practical constraints on its application. Our results establish conditions under which isotope dilution can yield accurate and precise δD measurements for low-abundance compounds and provide methodological guidance for extending CSIA into concentration regimes that are otherwise analytically inaccessible, enabling higher-resolution paleoclimate reconstructions and expanding the range of sedimentary archives amenable to biomarker isotope analysis.