Abstract
Stable isotope analyses are used on precious archeological and paleontological materials despite their destructive nature, because the information gained by these methods on, for example, feeding habits, migration and health of individuals cannot otherwise be obtained. We approached this issue by devising a new sequential extraction scheme aimed at producing multiple (O, C, N) isotope proxies from small amounts of sample. The new extraction scheme includes dissolution of the bone in dilute HNO3 followed by separate treatments of the collagenous and phosphate fractions. The collagen fraction is treated further adopting the methods presented in the literature for collagen extraction, modified to accommodate small sample sizes. The phosphate-containing fraction is purified from organic contaminants by H2 O2 and the phosphate is precipitated as Ag3 PO4 following methods presented in the literature. The use of HF as demineralization agent is also tested. A starting amount of ca 2 mg produced enough material for meaurement by isotope ratio mass spectrometry of the collagen C and N isotope compositions and bone phosphate O isotope composition. We show that the isotopic data obtained from the sequential extraction scheme are comparable with the isotopic composition measured following conventional methodologies that are usually based on 100-500 mg sample sizes. The new sequential extraction scheme combines the preparation for stable isotope analysis of bone mineral and organic phases, thus minimizing the amounts of sample needed and damage caused on a sample piece. The method may allow analysis of skeletal samples previously excluded from isotope analysis due to material limitations.
Highlights
Isotopic compositions of skeletal remains have been extensively used in archeological and paleontological studies to reveal information on, for example, nutritional sources[1,2,3] and dietary disruptions,[4,5,6,7] migration,[8,9] paleoecology[10,11,12] and paleoclimatology.[13]
We show that the isotopic data obtained from the sequential extraction scheme are comparable with the isotopic composition measured following conventional methodologies that are usually based on 100–500 mg sample sizes
Our results indicate that the organic and mineral matrices of single skeletal material samples may be separated to allow isotopic analyses by (1) demineralization with dilute HNO3 (0.2 M, for 17 h), (2a) collecting the liquid, PO43−-containing fraction, purified from organic contaminant by H2O2 (15%, evaporated to dryness) and precipitated as Ag3PO4, and (2b) continuing the demineralization of the collagenous fraction with dilute HCl (0.25 M, 1–5 days or longer), washing with NaOH (0.0625 M, 24 h), gelatinization in acidic conditions at elevated temperature and lyophilization
Summary
Isotopic compositions of skeletal remains have been extensively used in archeological and paleontological studies to reveal information on, for example, nutritional sources[1,2,3] and dietary disruptions,[4,5,6,7] migration,[8,9] paleoecology[10,11,12] and paleoclimatology.[13]. Modifications of the “bulk” separation method for the analysis of skeletal material (e.g.50,51), which uses up to 40 mg of starting material, have been developed for sample sizes as small as 0.2–0.3 mg.[32,34,59,60] The method of Wiedemann-Bidlack et al,[33] which we have adapted for use in our laboratory, incorporates the microprecipitation scheme presented in Wenzel et al,[59] a step for the oxidative removal of organic matter with NaOCl as outlined in Stephan,[51] and a strongly buffered ammoniacal silver ammine solution for the final Ag3PO4 precipitation step With these components it is possible to slowly precipitate millimeter-scale crystals of pure Ag3PO4 – ideal for the accurate and precise determination of the 18O/16O ratio by isotope ratio mass spectrometry (IRMS).[31,32,33]. The musk ox bone material was brown/reddish brown in color, probably due to iron-containing impurities, such as hematite and/or iron oxyhydroxides
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