Chemical techniques to extract organic fractions from fossil bones for accurate 14C datingIntroductionExperimental methodResults and discussionHCl concentrations on decalcification of fossilsNaOH extraction on different treatment timesComparison to the XAD-2 treatment methodConclusionAcknowledgementsReferencesChemical techniques to extract organic fractions fromfossil bones for accurate14C datingMasayo Minamia,*, Hiroo Mutob, Toshio NakamurabaDepartment of Earth and Environmental Sciences, Graduate School of Environmental Studies, Nagoya University,Chikusa, Nagoya 464-8602, JapanbCenter for Chronological Research, Nagoya University, Chikusa, Nagoya 464-8602, JapanAbstractWe examined different concentrations of HCl, such as 0.4, 0.6, 0.8, 1.0 and 1.2 M, for decalcification of fossil bonesand different times of 0.1 M NaOH treatment on collagens to determine the best conditions for purifying collagenthrough extraction of humic contaminants, and compared the alkali treatment method with the XAD-2 treatmentmethod for several types of fossils. The yield of acid-insoluble bone fractions did not change over the range from 0.4 to1.0 M HCl and decreased suddenly with 1.2 M HCl on decalcification, and the14C ages of the extracted gelatins fromthe five decalcified fractions were unchanged, suggesting that <1.0 M, and probably about 0.4 M, is recommended tothe best concentration of HCl to decalcify fossil bones efficiently. The alkali treatment was done with 0.1 M NaOH atroom temperature. The NaOH-treated collagens, with a considerable loss of organic bone protein especially for longtreatment time, gave almost the same14C ages as those of the XAD-purified hydrolysates. The NaOH-treatment timeshould be less than several hours to avoid a loss of collagen. The fossil bones used are relatively well-preserved, but thealkali treatment could bring about a lot of loss of organic bone proteins for poorly-preserved bones. The XAD-2treatment method is effective for accurate radiocarbon dating of fossil bones, if the XAD-2 resin is completely pre-cleaned.Ó 2004 Elsevier B.V. All rights reserved.PACS: 91.65.Dt; 91.65.Hy; 91/70.BfKeywords: Fossil bone; Gelatin; XAD-2 resin;14C age1. IntroductionBone has large surface area and porous struc-ture, and a great medium for sorption and pre-cipitation of dissolved colloidal particles of humicmaterials derived from surroundings. The majorityof fossil bones, therefore, are often contaminatedwith substantial foreign organic materials, whichcause significant errors in radiocarbon (14C) andstable isotope analyses. Accurate isotope mea-surement on fossil bones requires that all foreignorganic materials be removed. The Longin’smethod of collagen extraction by 8% HCl andsubsequent purification by acidic hot water iscommonly used to remove adsorbed humic con-taminants from bone protein [1]. However,14Cages for gelatins extracted from less well-preserved*Corresponding author. Tel.: +81-52-789-3030; fax: +81-52-789-3033.E-mail address: [email protected] (M. Minami).0168-583X/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.nimb.2004.04.060Nuclear Instruments and Methods in Physics Research B 223–224 (2004) 302–307www.elsevier.com/locate/nimbfossil bones containing little collagen are oftenyounger than the estimated stratigraphic ages [2,3].It is likely that the use of gelatin extraction alonedoes not fully remove contaminants from bone.Arslanov and Svezhentsev [4] reported that bonecollagens could be efficiently extracted by thetreatment of decalcified bone with 0.1 M NaOH atroom temperature for 24 h, then purified using theLongin’s method. On the other hand, Staffordet al. [5,6] reported that the NaOH extraction ofcollagen is recommended only for well-preservedbones because removal of humic contaminants isless reproducible and gelatin is lost easily fromless-preserved bones, and that XAD-2 resin iseffective to remove humic contaminants and givesthe highest yields of protein from bones withvariable types of preservation. Minami and Na-kamura [7] compared the Longin’s gelatin-extrac-tion method with the XAD-2 chromatographymethod, and assumed that in the Longin’s methodthe gelatins extracted from fossils with a low col-lagen content and a gelatin yield of less than 1%gave unsuitable younger14C ages than the actualage, while the XAD-2 resin is effective in the re-moval of organic carbon contamination from suchfossils with low collagen. For well-preserved fossilbone, however, there was no large difference be-tween the two methods.The acid–alkali treatment of collagen mightcause a great loss of bone protein. The amount ofdissolved collagen increases greatly when HClconcentration exceeds 0.5 M and when NaOHconcentration exceeds 0.1 M [4]. To evaluate thebest conditions for extracting bone collagens withhigh yield and high purity, we investigated thesolubility of bone protein on decalcification withdifferent concentrations of HCl and the amount ofdissolved collagen by NaOH extraction with dif-ferent treatment times, and then compared theXAD-2 treatment method with the combinedmethod of NaOH treatment and gelatin extrac-tion.2. Experimental methodTo investigate the solubility of bone organicmatter as a function of the concentrations of HCl,an animal bone fragment, collected from a shell-mound excavated at the Awazu submarine arche-ological site in the southern basin of Lake Biwa,Shiga Prefecture, Japan, was used. The14C age ofthe fossil bone (AWA-12) was measured to be4540 ± 70 BP for the XAD-purified hydrolysate [7],and wood sample that was collected together withthe bone sample from the same layer was dated ca.4600 BP [8]. We also used a dolphin fossil exca-vated from the Mawaki archeological site, Ishik-awa Prefecture, Japan, to evaluate the amount ofdissolved collagen by NaOH extraction on differ-ent treatment time. The wood sample collectedfrom the same layer was dated to be 5600 ± 100BP. A molar fossil of Naumann’s elephantcollected from the bottom of Uwa-sea, EhimePrefecture, Japan, was also analyzed. It is likelythat collagen content decreases with age andmore humic contaminants are added to older fos-sils.The fossil bones were repeatedly ultrasonicatedin distilled water followed by 0.2 M NaOH treat-ment, and rinsed with distilled water, then thesamples were lyophilized and pulverized. TheAWA-12 was treated with different concentrationsof HCl solutions in a cellulose tube in a beaker ordirectly