Standardization for ¹³ C-¹³ C clumped isotope analysis by the fluorination method

The ¹³ C-¹³ C isotopologues of C₂ molecules have recently been measured using a fluorination method. The C₂ compound is first fluorinated into hexafluoroethane (C₂ F₆ ), and its ¹³ C-isotopologues are subsequently measured using a conventional isotope ratio mass spectrometer. Here, we present an approach for standardizing the fluorination method on an absolute reference scale by using isotopically enriched C₂ F₆ .

We prepared physical mixtures of ¹³ C-¹³ C-labeled ethanol and natural ethanol. The enriched ethanol samples were measured using the recently developed fluorination method. Based on the difference between the calculated and measured ∆¹³ C¹³ C values, we quantified the extent to which isotopologues were scrambled during dehydration, fluorination, and ionization in the ion source.

The measured ∆¹³ C¹³ C value was approximately 20% lower than that expected from the amount of ¹³ C-¹³ C ethanol. The potential scrambling in the ion source was estimated to be 0.5-2%, which is lower than the observed isotopic reordering. Therefore, isotopic reordering may have occurred during either dehydration or fluorination.

For typical analysis of natural samples, scrambling in the ion source can only change the ∆¹³ C¹³ C value by less than 0.04‰, which is lower than the current analytical precision (±0.07‰). Therefore, the observed isotopic reordering may have occurred during the fluorination of ethene through the scrambling of isotopologues of ethene but not in the ion source of the mass spectrometer or during the dehydration of ethanol, given the small amount of C₁ and C₃₊ molecules. Thus, we obtained the empirical transfer function ∆¹³ C¹³ C_CSC  = λ × ∆¹³ C¹³ C with a λ value of 1.25 ± 0.01 for ethanol/ethene and 1.00 for ethane. Using the empirical transfer function, the developed fluorination method can provide actual differences in ∆ values.