Comparing Dual Water Equilibration Methods for Hydrogen Stable Isotope Analysis of Plant Carbohydrates and Proteinaceous Animal Samples

Abstract

Rationale

The hydrogen stable isotope composition (δ ²H) of organic samples carries information about processes including hydrology, climate, ecology, and plant metabolism. Numerous methods exist to control for hydrogen in organic samples that can isotopically exchange with ambient water or vapor.

Methods

We compared two established dual water steam equilibration approaches to control for exchangeable hydrogen in isotopic analysis: hot vacuum equilibration using the Uniprep autosampler and hot atmospheric pressure equilibration under varying equilibration temperatures and times. We evaluated how the extent of hydrogen isotope exchange and calculated exchange-corrected hydrogen stable isotope values varied among methods and sample types.

Results

More exchange could be achieved with atmospheric pressure methods compared to the Uniprep, and we confirmed that this was not a drying artifact. When direct comparisons were possible, atmospheric pressure methods yielded δ ²H values that agreed better with values from chemical derivatization, while Uniprep values agreed better with values provided for purchased reference materials based on room temperature equilibrations. Best long-term precision was achieved with fitted rather than prescribed isotope fractionation factors for equilibration.

Conclusions

Different equilibration methods can give reproducible yet distinct results. This is due to varying degrees to which different methods access non–carbon-bound hydrogen that is difficult to liberate from interior hydrogen bonds in complex biological structures such as cellulose or keratins. The optimal method depends on the application.