Simplified batch equilibration for D/H determination of non‐exchangeable hydrogen in solid organic material

Hydrogen isotopic analysis of organic materials has been widely applied in studies of paleoclimate, animal migration, forensics, food and flavor authentication, and the origin and diagenesis of organic matter. Hydrogen bound to carbon (C‐H) generally retains isotopic information about the water present during organic matter synthesis and associated biosynthetic fractionations, but hydrogen bound to other elements (O, S, or N) can readily exchange with atmospheric water vapor and reflects recent exposure to water or vapor. These two pools must be separated to obtain meaningful information from isotope ratios of organic materials. Previously published analytical methods either replace exchangeable H chemically or control its isotopic composition, usually by equilibration with water or waters of known isotopic composition. In addition, the fraction of H that is exchangeable can vary among samples and is itself of scientific interest. Here we report an improved and automated double‐equilibration approach. Samples are loaded in a 50‐position autosampler carousel in an air‐tight aluminum equilibration chamber. Water vapor of known isotopic composition is pumped through the chamber at 115°C for at least 6 h. After flushing with dry N₂ and being cooled, the carousel is rapidly transferred from the equilibration chamber to a He‐purged autosampler attached to a pyrolysis elemental analyzer connected to an isotope ratio mass spectrometer. By equilibrating two aliquots of each sample with two isotopically distinct waters, it is possible to calculate both (1) the D/H ratio of non‐exchangeable H, and (2) the fraction of H that is exchangeable. Relative to previous double‐equilibration techniques, this approach offers significant reductions in sample size and labor by allowing simultaneous equilibration of several tens of samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluating uncertainty in the calculation of non‐exchangeable hydrogen fractions within organic materials

We calculated the fraction of exchangeable hydrogen atoms in proteinaceous materials commonly analyzed for stable isotopic composition related to the region‐of‐origin of an animal. These included several types of α‐ and β‐keratin, and muscle tissue. We find that the fraction of H atoms in keratin available for exchange at a biologically relevant temperature (25°C) averaged 9% across a range of ground organic materials, but was as high as ～17% in cut hair; muscle tissue has ～12% exchangeable H atoms. Under most analysis conditions, the difference in exchangeable fractions due to physical sample processing has a minimal effect on the calculated δ²H values of the non‐exchangeable H atoms within a keratin‐containing tissue (<2‰). However, extreme mismatches between sample and reference material types could affect δ²H values. Copyright © 2009 John Wiley & Sons, Ltd.     

A test method for determining water in organic solvents

A test procedure is developed for determining water in acetone, dioxane, isobutanol, ethanol, petroleum ether, and ethyl acetate in the ranges 0.5–12; 0.5–10; 0.5–8; 0.5–6, and 0.5–4 vol %, respectively. Determination is based on the measurement of the acidity of a solution obtained upon adding citric acid or sodium hydrogen sulfate to organic solvents using an indicator test strip containing Congo red. The relative standard deviation of the results of analysis is 0.05–0.1; the duration of analysis is about 30 min.     

A strategy for selection of reference materials in stable oxygen isotope analyses of solid materials

The propagation of uncertainties associated with the stable oxygen isotope reference materials through a multi-point normalisation procedure was evaluated in this study using Monte Carlo (MC) simulation. We quantified the normalisation error for a particular selection of reference materials and their number of replicates, when the choice of standards is restricted to either nitrates, sulphates or organic reference materials alone, and in comparison with when this restriction was relaxed. A lower uncertainty in stable oxygen isotope analyses of solid materials performed using High-Temperature Pyrolysis (HTP) can be readily achieved through an optimal selection of reference materials. Among the currently available certified reference materials the best performing pairs minimising the normalisation errors are USGS35 and USGS34 for nitrates; IAEA-SO-6 and IAEA-SO-5 for sulphates; and IAEA-601 and IAEA-602 for organic materials. The normalisation error can be reduced further--by approximately half--if each of these two analysed reference materials is replicated four times. The overall optimal selection among all nine considered reference materials is the IAEA-602 and IAEA-SO-6 pair. If each of these two reference materials is replicated four times the maximum predicted normalisation error will equal 0.22‰, the minimum normalisation error 0.12‰, and the mean normalisation error 0.15‰ over the natural range of δ(18)O variability. We argue that the proposed approach provides useful insights into reference material selection and in assessing the propagation of analytical error through normalisation procedures in stable oxygen isotope studies.     

Differences in the stable isotope signatures of seabird egg membrane and albumen – implications for non‐invasive studies

In many bird species, egg membranes can be obtained non‐invasively after the chicks have hatched, and stable isotope analysis of egg membranes can be used to study the diet and foraging distribution of these birds during egg formation. It has been suggested that the enrichment factors of albumen and egg membranes differ for ¹³C, but are similar for ¹⁵N. In this study, we compared carbon and nitrogen stable isotopes of the membranes and albumen of individual eggs of three wild seabird species, the Southern Rockhopper penguin Eudyptes chrysocome, the Imperial shag Phalacrocorax atriceps albiventer, and the Thin‐billed prion Pachyptila belcheri. We also included chicken eggs for comparison. Egg membranes were generally enriched in ¹³C, compared with albumen. The difference varied between species, with 2.1‰ in Rockhopper penguins, 1.6‰ in Imperial shags, but only 0.5‰ in Thin‐billed prions and 0.4‰ in chicken eggs. Egg membranes were slightly enriched in ¹⁵N in Imperial shags (0.9‰) and chickens (0.5‰), compared with albumen, while there was no difference for Thin‐billed prions and Rockhopper penguins. The isotopic values of carbon and nitrogen were correlated between albumen and egg membranes of individual eggs, suggesting that egg membranes can be used reliably to investigate trophic differences between individuals, seasons or colonies. Species‐specific mathematical corrections could be used to compare results across studies that use different egg components. Copyright © 2009 John Wiley & Sons, Ltd.     

Revised δ34S reference values for IAEA sulfur isotope reference materials S‐2 and S‐3

Revised δ³⁴S reference values with associated expanded uncertainties (95% confidence interval (C.I.)) are presented for the sulfur isotope reference materials IAEA‐S‐2 (22.62 ± 0.16‰) and IAEA‐S‐3 (−32.49 ± 0.16‰). These revised values are determined using two relative‐difference measurement techniques, gas source isotope ratio mass spectrometry (GIRMS) and double‐spike multi‐collector thermal ionization mass spectrometry (MC‐TIMS). Gas analyses have traditionally been considered the most robust for relative isotopic difference measurements of sulfur. The double‐spike MC‐TIMS technique provides an independent method for value‐assignment validation and produces revised values that are both unbiased and more precise than previous value assignments. Unbiased δ³⁴S values are required to anchor the positive and negative end members of the sulfur delta (δ) scale because they are the basis for reporting both δ³⁴S values and the derived mass‐independent Δ³³S and Δ³⁶S values. Published in 2009 by John Wiley & Sons, Ltd.     

A novel approach for the homogenization of cellulose to use micro‐amounts for stable isotope analyses

Climate reconstructions using stable isotopes from tree‐rings are steadily increasing. The investigations concentrate mostly on cellulose due to its high stability. In recent years the available amount of cellulose has steadily decreased, mainly because micro‐structures of plant material have had to be analyzed. Today, the amounts of cellulose being studied are frequently in the milligram and often in the microgram range. Consequently, homogeneity problems with regard to the stable isotopes of carbon and oxygen from cellulose have occurred and these have called for new methods in the preparation of cellulose for reliable isotope analyses. Three different methods were tested for preparing isotopically homogenous cellulose, namely mechanical grinding, freezing by liquid nitrogen with subsequent milling and ultrasonic breaking of cellulose fibres. The best precision of isotope data was achieved by freeze‐milling and ultrasonic breaking. However, equipment for freeze‐milling is expensive and the procedure is labour‐intensive. Mechanical grinding resulted in a rather high loss of material and it is also labour‐intensive. The use of ultrasound for breaking cellulose fibres proved to be the best method in terms of rapidity of sample throughput, avoidance of sample loss, precision of isotope results, ease of handling, and cost. Copyright © 2009 John Wiley & Sons, Ltd.