Investigation of preparation techniques for δ²H analysis of keratin materials and a proposed analytical protocol

Abstract

Accurate hydrogen isotopic measurements of keratin materials have been a challenge due to exchangeable hydrogen in the sample matrix and the paucity of appropriate isotopic reference materials for calibration. We found that the most reproducible δ(2)H(VSMOW-SLAP) and mole fraction of exchangeable hydrogen, x(H)(ex), of keratin materials were measured with equilibration at ambient temperature using two desiccators and two different equilibration waters with two sets of the keratin materials for 6 days. Following equilibration, drying the keratin materials in a vacuum oven for 4 days at 60 °C was most critical. The δ(2)H analysis protocol also includes interspersing isotopic reference waters in silver tubes among samples in the carousel of a thermal conversion elemental analyzer (TC/EA) reduction unit. Using this analytical protocol, δ(2)H(VSMOW-SLAP) values of the non-exchangeable fractions of USGS42 and USGS43 human-hair isotopic reference materials were determined to be -78.5 ± 2.3 ‰ and -50.3 ± 2.8 ‰, respectively. The measured x(H)(ex) values of keratin materials analyzed with steam equilibration and N(2) drying were substantially higher than those previously published, and dry N(2) purging was unable to remove absorbed moisture completely, even with overnight purging. The δ(2)H values of keratin materials measured with steam equilibration were about 10 ‰ lower than values determined with equilibration in desiccators at ambient temperatures when on-line evacuation was used to dry samples. With steam equilibrations the x(H)(ex) of commercial keratin powder was as high as 28%. Using human-hair isotopic reference materials to calibrate other keratin materials, such as hoof or horn, can introduce bias in δ(2)H measurements because the amount of absorbed water and the x(H)(ex) values may differ from those of unknown samples. Correct δ(2)H(VSMOW-SLAP) values of the non-exchangeable fractions of unknown human-hair samples can be determined with atmospheric moisture equilibration by normalizing with USGS42 and USGS43 human-hair reference materials when all materials have the same powder size.

Influence of feed and water on the stable isotopic composition of dairy milk

“…This technique may cause exchange of protons 123 that would not be readily exchanged at room temperature. Room temperature equilibration has 124 more recently been used successfully (Qi & Coplen, 2011), and comparable results using either 125 method have been obtained in these authors' laboratory. Moreover, equilibration under 126 ambient conditions on milk may encourage the activity of naturally occurring lipase that might 127 have survived pasteurisation (72−78 °C) and may lead to hydrolysis of milk triglycerides into 128 free fatty acids.…”

Full Issue

“…Sample capsules were then quickly closed (,10 min exposure to lab air) and loaded into the helium atmosphere of the Costech zero-blank autosampler. Tests of the rate of back-reaction (or water adsorption) of dried samples exposed to lab air showed minimal measurable change after 30 min (2%), in agreement with Qi and Coplen (2011).…”

“…Using the principle of identical treatment for unknowns and standards (Werner and Brand 2001), the Environmental Isotope Laboratory calibrated keratin control standards at room temperature, equilibrating standards with 3 water vapors with very different d 2 H values, similar to the method described by Qi and Coplen (2011). Control standards were placed in sealed chambers with 3 waters (liquid) ranging from À139 to þ48% to equilibrate for !4 days.…”

Critique: Measuring Hydrogen Stable Isotope Abundance of Proteins to Infer Origins of Wildlife, Food and People

“…While the first two problems are easily overcome with drying and careful sample handling, the latter problem has attracted a wide variety of disparate approaches that range from dealing with this challenge [47,[52][53][54][55][56] to not addressing the problem [57,58]. One might argue that for studies purely concerned with looking at differences in H isotopic composition between and among samples, knowledge of the molar exchange fraction and 'true' d 2 H-values of nonexchangeable hydrogen might not be an issue, so long as all samples have been treated in an identical fashion, and having been stored, exposed to the same humidity under identical conditions (temperature, relative humidity and stable isotopic composition of humidity), thus …”

“…To Subsequent to equilibration and prior to H isotope ana lysis, samples must be dried down either in an evacuated desiccator containing noncaking phosphorous pentoxide as a desiccant or in a container connected to a continuously operating vacuum line (vacuum drying oven or freeze dryer arrangement) for at least 7 days to remove residual moisture traces from the samples [47,54,55]. To avoid back-exchange of equilibrated samples with ambient humidity, samples have to be loaded quickly (e.g., in <30 min) on to a Zero-Blank autosampler that can be sealed (i.e., isolated from ambient) and, once sealed, can be purged with a dry helium so samples are kept in a dry, inert, atmosphere while waiting their turn to be analyzed [47,54,55,77]. Following this protocol permits strictest adherence to the PIT and will therefore avoid any ambiguity that would otherwise arise or could otherwise be construed.…”