Assessment of bias in carbon isotope composition of organic leaf matter due to pre‐analysis milling methods

Rationale Stable isotope analysis of leaf material has many applications including assessment of plant water-use efficiency and paleoclimatology. To facilitate interpretations of small shifts in the carbon isotope composition (δ13C) of leaves, accurate and repeatable results are required. Pre-sample homogenisation is essential to ensure a representative sample is analysed, but can also introduce error. Methods We investigate how different grinding methods (freezer-milling and ball-milling) affect the carbon content and δ13C of tree leaves from a wetland in Queensland, Australia, commenting on how increased temperature, sample contamination, sample loss, or poor homogenisation may impact results. Results No alteration of leaf δ13C is observed due to different milling methods, although there may be a significant increase in %C of samples processed using ball-milling. Conclusions We suggest %C variability is possibly due to contamination from abraded plastic vials or insufficient homogenisation during ball-milling, with no significant impact on δ13C. Overall, we suggest that intermittent ball-milling may be the best solution to reduce costs, preparation time and use of liquid nitrogen, aiming to achieve complete homogenisation using the shortest possible duration of milling.