A test of comparative equilibration for determining non-exchangeable stable hydrogen isotope values in complex organic materials.

Comparative equilibration has been proposed as a methodological approach for determining the hydrogen isotopic composition (deltaD) of non-exchangeable hydrogen in complex organic materials, from feathers to blood and soils. This method depends on using homogenized standards that have been previously calibrated for their deltaD values of non-exchangeable H, that are compositionally similar to unknown samples, and that span an appropriate isotopic range. Currently no certified organic reference materials with exchangeable H exist, and so isotope laboratories have been required to develop provisional internal calibration standards, such as the keratin standards currently used in animal migration studies. Unfortunately, the isotope ratios of some samples fall outside the range of keratin standards currently used for comparative equilibration. Here we tested a set of five homogenized keratin powders as well as feathers from Painted Buntings and Dark-eyed Juncos to determine the effects of extrapolating comparative equilibration normalization equations outside the isotopic range of keratin standards. We found that (1) comparative equilibration gave precise results within the range of the calibration standards; (2) linear extrapolation of normalization equations produced accurate deltaD results to approximately 40 per thousand outside the range of the keratins standards used (-187 to -108); and (3) for both homogenized keratin powders and heterogeneous unknown samples there was no difference in variance between samples within and outside the range of keratin standards. This suggested that comparative equilibration is a robust and practical method for determining the deltaD of complex organic matrices, although caution is required for samples that fall far outside the calibration range.