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

Stable isotope analysis of leaf material has many applications including assessment of plant water-use efficiency and palaeoclimatology. 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.

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.

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.

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.