The effect of processing medium on the ²H/¹H of carbon-bound hydrogen in α-cellulose extracted from higher plants

Rationale: Although the ²H/¹H ratio of the carbon-bound hydrogens (C-Hs) in α-cellulose extracted from higher plants has long been used successfully for climate, environmental and metabolic studies, the assumption that bleaching with acidified NaClO₂ to remove lignin before pure α-cellulose can be obtained does not alter the ²H/¹H ratio of α-cellulose C-Hs has nonetheless not been tested. Methods: For reliable application of the ²H/¹H ratio of α-cellulose C-H, we processed plant materials representing different phytochemistries and photosynthetic carbon assimilation modes in isotopically contrasting bleaching media (with an isotopic difference of 273 mUr). All the isotope ratios were measured by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). Results: Our results show that H from the bleaching medium does appear in the final pure α-cellulose product, although the isotopic alteration to the C-H in α-cellulose due to the incorporation of processing H from the medium is small if isotopically “natural” water is used to prepare the processing medium. However, under prolonged bleaching such an isotope effect can be significant, implying that standardizing the bleaching process is necessary for reliable ²H/¹H measurement. Conclusions: The currently adopted method for removing lignin for α-cellulose extraction from higher plant materials with acidified NaClO₂ bleaching is considered acceptable in terms of preserving the isotopic fidelity if isotopically “natural” water is used to prepare the bleaching solution.