• C4 n -alkane mix is suitable as a working standard for Py-CSIA δ 13 C. • A slight 13 C-enrichment trend was observed at 500°C, but not significant. • At 600°C, Py-CSIA showed the best balance of fidelity and reproducibility. • Low-temperature depletion likely reflects incomplete volatilization/transfer. Pyrolysis compound-specific isotope analysis (Py-CSIA) is a relatively novel hyphenated technique for which no standard materials have yet been developed. For conventional CSIA (GC-C/IRMS), isotopically characterized standards are routinely used to construct calibration curves and to validate and correct analytical data. In this work, we test the suitability of the C4 CSIA standard (Indiana University, Stable Isotope Laboratory), a mixture of five odd-chain n -alkanes (C 17 –C 25 in decreasing concentration dissolved in hexane), as a reference material for δ 13 C measurements by Py-CSIA and evaluate the effect of pyrolysis temperature on carbon isotope composition. Aliquots of the C4 standard were deposited onto glass fibre discs in deactivated steel capsules and pyrolysed at five temperatures (250, 300, 400, 500 and 600°C) in a micro-furnace pyrolizer coupled online to a GC-C/IRMS system; an additional direct injection (DI) GC-C/IRMS analysis of C4 was used as the reference. Each condition was analysed in ten replicate runs. Overall, δ ¹³C values obtained by Py-CSIA showed excellent agreement with DI measurements, with no significant isotope fractionation observed across the tested temperature range, and coefficients of determination (R 2 ) greater than 0.96 for all temperatures. No statistically significant temperature effect was observed for C 19 , C 21 , C 23 , or C 25 , whereas C 17 was the only compound showing a significant response. A slight visual 13 C-enrichment trend was observed at 500°C, although this was not statistically significant, while at 600°C the δ ¹³C values converged towards the DI reference values and reproducibility improved. The more negative δ 13 C values observed at 250°C are more consistently explained by incomplete volatilization and/or incomplete transfer of the heavier n -alkanes than by clear pyrolytic fractionation, although minor thermal transformation effects cannot be fully excluded. These results indicate that the C4 saturated linear n -alkane mixture is suitable as a working standard for Py-CSIA under the analytical conditions tested, and that elevated pyrolysis temperatures, particularly 600°C, provide the most robust balance between isotopic fidelity and reproducibility for this compound class. Further work is needed to determine whether this behaviour also applies to other compound classes and more complex organic matrices.
San-Emeterio et al. (Sun,) studied this question.