IRMS Analysis of Organic Acids

The analysis of δ¹³C values and concentrations of individual organic acids (citrate and malate) was performed by coupling an Isolink HPLC device to a DeltaV isotope ratio mass spectrometer (all Thermo-Fisher; Lehmann et al., 2015 (link), 2016b). Before analysis, the aqueous bulk organic acid fractions have to be passed through a 0.45 µm syringe filter (Infochroma, Zug, Switzerland). Organic acids were separated on a 4.6×300 mm Allure Organic Acids column (Restek, Bellefonte, PA, USA) at 8 °C using 100 mM KH₂PO₄ (pH 3) as a mobile phase with a flow rate of 500 µl min^–1. All organic compounds were oxidized to CO₂ at 99 °C using Na₂S₂O₈ under acidic conditions. The CO₂ was subsequently separated from the mobile phase and measured for its isotope ratio and concentration in the IRMS. Every 10 samples, a set of malate and citrate laboratory standards of different concentrations (10–180 ng C μl^–1) was analyzed. Offset corrections to EA-IRMS δ¹³C values and determination of concentrations were performed according to Rinne et al. (2012) (link). The applied method was shown to be free of isotope fractionation by analysis of standard materials with respect to the measurement precision, which was typically better than 0.4‰ (SD).