Qualitative application based on IR spectroscopy for bone sample quality control in radiocarbon dating

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Abstract

Bone samples suffer from contamination and deterioration, depending on their conservation state and previous restoration and consolidation processes. The sample preparation laboratory of the CEDAD (Center for Dating and Diagnostics) of the University of Lecce is developing a quality control protocol for bone samples based on Fourier transform infrared (FTIR) spectroscopy to identify the presence of collagen in bone samples and to assess its quality. FTIR measurements were carried out on collagen extracted from many ancient samples dated at CEDAD. Efforts to shift the FTIR quality control test from the filtration step to a check-in treatment are proceeding to optimize the time for preparation and to reduce the overall turnaround time. A standard fast demineralization treatment was set up and applied to a variety of ancient samples of different origin and age.

Introduction

CEDAD is a research and service center for AMS radiocarbon dating where many osteological samples are measured every year [1]. Organic samples undergo AAA (acid alkali acid) treatment; bones are first mechanically cleaned and crushed to reduce them in powder, then prepared by following the Longin method [2], [3], [4]. Bone is a composite material made of an organic (20–30 wt%), mainly collagen, an inorganic fraction (60–70 wt%), and water (∼10%). The mineral phase consists of carbonated hydroxylapatite in which the basic apatite structure, Ca10(PO4)6(OH)2, is modified by the substitution of the phosphate with the carbonate group [5], [6]. Bone apatite is poorly cristalline, and its small crystals are reactive. Ancient bones undergo complex processes of diagenesis, recrystallization, hydrolisis, denaturation and degeneration of both the organic and mineral phase [7]. These processes are different and influenced by various parameters and conditions like humidity and temperature, pH and soil composition at the burial site [8]. For radiocarbon dating the information about the age of the sample is carried by collagen. Many previous studies concerned with the preservation of ancient bones focused on parameters such as C/N ratio, δ13C and δ15N, used also for paleodietary considerations [9]. Spectroscopic parameters, such as the infrared splitting factor (IRSF) and the carbonate/phosphate (C/P) ratio, estimated from the FTIR spectrum of bone powder or deproteinated bone have been also suggested as an index of bone diagenesis. The IRSF is calculated from the phosphate peak splitting at 565–605 cm−1 and it is an index of mineral recrystallization [10]. The higher the value, the larger and more ordered are the apatite crystals. IRSF ranges from 2.6 to 3.0 for fresh bones. The carbonate content C/P is the ratio of the absorbances of the CO3 and PO4 peaks in the FTIR spectrum [6]. In many cases the C/N, the δ13C and δ15N parameters and the spectroscopic parameters such as IRSF and C/P give a general indication about the presence and preservation of collagen in bones. Nevertheless, IRSF and C/P evaluate the condition of the mineral phase that is not always directly related to collagen preservation [10] and more reliable results are to be expected from direct collagen FTIR observations.

Section snippets

Experimental techniques

About 40% of samples submitted to CEDAD for AMS radiocarbon dating are osteological samples. Unfortunately some samples fail to produce CO2 for radiocarbon dating after chemical treatments. Therefore it is important to have a fast and reliable method to evaluate the preservation state and the collagen content of bone samples and to check the effectiveness of collagen extraction treatment. The aim of this work was to develop a “quality control” test of collagen extracted from bones, based on a

Bone samples IR spectra

A preliminary study on modern fresh collagen was carried out to evaluate the amount of material needed for FTIR measurements, minimizing the amount of ancient sample required. Several modern samples were prepared to create an internal, “clean” reference, without consolidant, diagenesis and contaminants, treated in the same way as ancient samples. Fig. 1 shows one of the spectra used to identify the collagen IR absorption bands, showing a good agreement with literature indications about collagen

The fast demineralization process

Once shown that FTIR quality control worked well on filtered collagen, we investigated if it was possible to shift the quality control test from the filtration step to a check-in treatment, in order to optimize the time for preparation and the amount of sample needed for AMS measurement. Complete demineralization and filtration can take a week. Following the approach of Yizhaq et al. [18], a standard fast demineralization treatment was set up. The aim was to extract the collagen FTIR

Conclusions

For AMS dating it is important to verify the presence of carbon contaminants in the bone samples. We developed a systematic quality control protocol based on FTIR spectroscopy of collagen extracted from samples of different ages and environments. A relationship between the presence of collagen in the FTIR spectrum and the total CO2 yield has been found. The quality control test works well with both good and bad samples, avoiding the waste of time and materials for problematic samples. It is our

Acknowledgements

This work was supported by the Italian Ministery for Research (MIUR) through the SIDART project and co-funded by European Union.

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