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Comparison of different bone pretreatment methods for AMS 14C dating
Nuclear Instruments and Methods in Physics Research B 172 (2000) 424±427
www.elsevier.nl/locate/nimb
Comparison of dierent bone pretreatment methods for AMS 14 C dating q Sixun Yuan a
a,*
, Xiaohong Wu a, Shijun Gao a, Jinxia Wang b, Lianzhen Cai b, Kexin Liu c, Kun Li c, Hongji Ma c
Department of Archaeology, Peking University, Haidian, Beijing 100871, People's Republic of China Institute of Archaeology, Academy of Social Sciences, Beijing 100710, People's Republic of China c Institute of Heavy Ion Physics, Peking University, Beijing 100871, People's Republic of China
b
Abstract Bone sample preparation has been studied in detail to ®nd a suitable method for the processing of smaller sample amounts such as oracle bones. In a recent research project only 1 g of bone material will be available for radiocarbon dating of these valuable bones with the AMS method. The standard methods for the preparation of gelatin were compared with the amino acids treated by XAD-2 resin. Additionally, it was tested if the alkaline step in the procedure may be skipped or not. According to our results we may conclude that for well preserved bones the gelatin production yields reliable results. We could also show that the XAD-2 treatment is an eective method for the removal of humic acids. Ó 2000 Elsevier Science B.V. All rights reserved. PACS: 06.60.Ei Keywords: AMS;
14
C dating; Bone sample pretreament
1. Introduction Samples of bone, horn, teeth, etc. from various species are important 14 C dating materials. Because of their complicated compositions, the different kinds of contamination incorporated from the surrounding sediment and the changes of their
q Supported by the Xia-Shang-Zhou Chronology Project of China. * Corresponding author. Tel.: +86-10-6275-1667; fax: +8610-6275-1667. E-mail address: [email protected] (X. Wu).
components during burial, the reliability of radiocarbon dating of fossil bones with poor preservation still has some problems up to now. The key issue is to determine what kind of pretreatment yields reliable components for dating. Hedges and Klinken [1] presented an overview of approaches in the pretreatment of bone in 1992. At present in most AMS labs, according to the pretreatment methods selected for fossil bones, the materials used for 14 C dating are: A. Collagen extracted from bone, decalci®ed with acid and subsequently soaked with alkali. B. Gelatin extracted from collagen hydrolyzed with pH 2±3 HCl at 90°C. The resulting
0168-583X/00/$ - see front matter Ó 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 0 0 ) 0 0 3 4 8 - 7
S. Yuan et al. / Nucl. Instr. and Meth. in Phys. Res. B 172 (2000) 424±427
solution is ®ltered or centrifuged afterwards and evaporated to dryness. C. Puri®ed amino acids obtained by hydrolyzing the gelatin with 6N HCl at 110°C, and further cleanup with XAD-2 resin. D. Speci®c individual amino acids (e.g., glycine, proline, hydroxyproline) obtained by separating amino acids further. In the separation processes, the ®rst two methods are the simpler procedures. Whereas the latter two, especially the last one, are very time consuming procedures which also require more than 10 times the amount of sample material than the simpler methods. Some labs try a variety of further puri®cation approaches to remove contamination from humic acid residues possibly still present in the sample after the application of the standard pretreatment methods. These puri®cation methods include activated charcoal adsorption, ion exchange separation, ultra®ltration as well as XAD-2 resin treatment, etc. [2,3]. In the course of a big research project [4] a large quantity of bone samples such as horse, sheep, ox, human bone, etc. ± also very valuable samples of oracle bone with ancient Chinese inscriptions ± will be dated. Generally, the gelatin content of the samples is more than 20 mg/g of bone. For the rare artifacts (oracle bones) only about one gram of each sample can be obtained. Since these samples are of insucient weights for typical dating methods and high measurement precision must be achieved with these smaller sample amounts, it is necessary to select pretreatment methods that meet both the measurement precision and smaller sample quantity requirements. Process B (i.e., gelatinextraction method) which is being used in many labs, and process C, in which the hydrolysates of gelatin are treated with XAD-2 resin to get amino acids, are supposed to be suitable for this purpose. In order to check their applicability, we tested both methods and compared them with each other. 2. Experiments and results 2.1. Basic research In connection with 14 C dating of bone samples, a number of studies concerning bone components,
425
bone alteration in burial process, separation of components for dating, nature and detection of contamination, etc. [5±9] have been performed. We measured and compared the molecular weight distributions as well as the dierence in amino acid compositions between modern and ancient bone samples using HPLC. We analyzed the content of humics in gelatin with infrared spectrometry and ultraviolet spectrometry. By these means we attempted to determine the extraneous contaminant content in pretreated bone-dating components according to the changes of bone composition in the burial process and the humic contents. Unfortunately, it is dicult to obtain quantitative results with these methods, and to determine whether there are traces of contamination present in the material used for dating. 2.2. Comparison measurement When a dating result is assessed, it is usually in contrast with the dating results of the sample materials or sample components of higher reliability. As a rule, charcoal samples are regarded as comparatively reliable dating materials. Among the four dating components and processes, compared with the ®rst two, the latter two undergo more puri®cation steps, so probably contain less contaminant. XAD-2 treatment is regarded as an eective method for removing humics. So the amino acids treated with XAD-2 can be regarded as better bone-dating material. 2.2.1. Comparison of dating results between gelatin and XAD-2 treated amino acids In the four methods of collagen, gelatin, amino acids and individual amino acids, collagen sometimes may retain unremoved contaminant containing carbon, while in the preparation of individual amino acids not only much bone material is necessary but also very complicated separation processes are involved. Therefore in our study we only selected gelatin and amino acids treated with XAD-2 for comparison with charcoal samples from the same archaeological periods. A similar work has been reported by Minami and Nakamura [10,11]. We treated the samples by means of process B to obtain gelatin which was
426
S. Yuan et al. / Nucl. Instr. and Meth. in Phys. Res. B 172 (2000) 424±427
divided into two parts: one part was converted directly into graphite; the other was transformed into amino acids by process C which were treated with XAD-2 and then converted into graphite, which was measured with the EN tandem accelerator mass spectrometry at Peking University (PKUAMS). The d13 C of samples were analyzed with a stable isotope mass spectrometer. The results are shown in Table 1. From the data in Table 1 we can see that in each group the data agree within 1r error. In group 2, the results of SA98094 and SA98155 also agree with the 14 C determination performed by the ISOTRACE lab, Toronto. 2.2.2. Comparison of measurement results of dierent processes or components The alkaline step of the collagen preparation procedure may cause a greater loss of collagen. During the pretreatment of poorly preserved bone samples, even part of the peptides of degraded collagen may be dissolved during the decalci®cation. In order to ®nd a suitable cleaning method for this kind of sample that yields
reliable dating results, we treated the same sample material with dierent methods and compared the results: 1. We studied the eect of skipping the alkali treatment. We used process A without the alkali step to prepare collagen, and processes B and C were used to produce the amino acids which were treated by XAD-2. The results were compared with the results of the amino acids produced by the unchanged processes A, B, C, and/or the gelatin produced by A and B. The results are listed in Table 2. 2. We investigated whether organics dissolved during the decalci®cation step may be used for dating. After decalcifying samples with acid, the organics in solution were evaporated with rotatory evaporator and lyophilized. From the organics obtained amino acids were prepared by procedure C and puri®ed with XAD-2. The result of these amino acids were compared with that of gelatin produced by processes A and B (Table 3). The comparison of data from Tables 2 and 3 shows that in each group every date agree within a 1±2r error.
Table 1 Comparison dating results between gelatin and XAD-2 resin-treated amino acids Group
1
Sample no.
SA98093 SA98093Ab
2
SA98094 SA98094Ab SA98155
3
SA98096 SA98096Ab
a b
d 13 C (&)
14
2.97
)10.0
2781 50
Amino acids
2.88
)9.1
2805 50
Gelatin
3.05
)14.2
2573 50
Amino acids
2.87
)12.1
2574 51
)25.1
2640 50
3.08
)15.7
2555 50
2.89
)16.6
2531 53
Sample material
Components
Human bone Human bone Horse bone Horse bone Charcoal
Gelatin
Horse bone Horse bone
Gelatin Amino acids
C/N
Charcoal
The pretreatment and determination of the samples were done in Toronto. A: The sample pretreated by XAD-2 method.
C Age (yr BP)
This lab
ISOTRACE Lab, Toronto, Canadaa
2570 50 (TO-7999) 2630 40 (TO-7998)
S. Yuan et al. / Nucl. Instr. and Meth. in Phys. Res. B 172 (2000) 424±427
427
Table 2 XAD-2 treatment eects on removing contamination from bone components Group
Sample no.
Matter
Components
C/N
d13 C (&)
14
1
SA98094 SA98094Aa SA98094AAb SA98096 SA98096Aa SA98096AAb SA98167 SA98167AAb
Horse bone Horse bone Horse bone Horse bone Horse bone Horse bone Sheep bone Sheep bone
Gelatin Amino acids Amino acids Gelatin Amino acids Amino acids Gelatin Amino acids
3.05 2.87 2.91 3.08 2.89 2.87 3.06 2.92
)14.2 )12.3 )12.6 )15.7 )16.6 )13.6 )16.4 )15.5
2573 50 2574 51 2440 43 2555 50 2531 53 2556 50 2868 48 2824 43
2 3 a b
C age (yr BP)
A: The sample pretreated by XAD-2 method. AA: The sample pretreated by XAD-2 resin, while alkali treatment was skipped in preparing collagen.
Table 3 Comparison of measurement results of gelatin and organics (amino acids) in solution
a
Group
Sample no.
Matter
Components
C/N
d13 C (&)
14
1
SA99068 SA99068sa
Bone Bone
Gelatin Amino acids
2.84 2.76
)5.6 )5.6a
3385 38 3366 63
C age (yr BP)
Not measured, assume that it was the same result as SA99068.
3. Conclusions 1. At present it is still dicult to detect trace contaminants containing carbon in dating components of pretreated bone samples. 2. In the case of good-preserved bone samples, gelatin prepared with procedures A and B can give a reliable age. 3. XAD-2 treatment is indeed an eective method for removing humic contamination in bone samples. 4. In the pretreatment of the samples, it is possible to skip the alkaline step as shown in 2.2.2(1) step. But the procedure of cleaning up puri®ed amino acids with XAD-2 resin is more time consuming than alkaline treatment.
Acknowledgements We wish to thank Dr. R.P. Beukens, ISOTRACE Lab, Toronto, Canada, for comparison
measurement of the samples SA98094 and SA98155. References [1] R.E.M. Hedges, G.J. Van Klinken, Radiocarbon 34 (3) (1992) 279. [2] T.W. Staord Jr., K. Brendel, R.C. Duhamel, Geochim. Cosmochim. 52 (1988) 2257. [3] T.A. Brown, D.E. Nelson, J.S. Vogel, J.R. Southon, Radiocarbon 30 (2) (1988) 171. [4] Z.Y. Guo, K. Liu, X. Lu, H. Ma, K. Li, S. Yuan, X. Wu, Nucl. Instr. and Meth. B 172 (2000) 724. [5] R. Gillespie, Radiocarbon 31 (3) (1989) 239. [6] M.J. DeNiro, S. Weiner, Geochim. Cosmochim. 52 (1988) 2197. [7] R.R. Burky, D.L. Kirner, R.E. Taylor, P.E. Hare, J.R. Southon, Radiocarbon 40 (1) (1998) 11. [8] KH.A. Arslanov, Yu.S. Svezhentsev, Radiocarbon 35 (3) (1993) 384. [9] T.W. Staord Jr., P.E. Hare, L. Currie, A.J.T. Jull, D.J. Donahue, J. Archaeol. Sci. 18 (1991) 35. [10] M. Minami, T. Nakamura, Chinese Sci. Bull. 43 (Suppl.) (1998) 89. [11] M. Ninami, T. Nakamura, Nucl. Instr. and Meth. B 172 (2000) 462.
www.elsevier.nl/locate/nimb
Comparison of dierent bone pretreatment methods for AMS 14 C dating q Sixun Yuan a
a,*
, Xiaohong Wu a, Shijun Gao a, Jinxia Wang b, Lianzhen Cai b, Kexin Liu c, Kun Li c, Hongji Ma c
Department of Archaeology, Peking University, Haidian, Beijing 100871, People's Republic of China Institute of Archaeology, Academy of Social Sciences, Beijing 100710, People's Republic of China c Institute of Heavy Ion Physics, Peking University, Beijing 100871, People's Republic of China
b
Abstract Bone sample preparation has been studied in detail to ®nd a suitable method for the processing of smaller sample amounts such as oracle bones. In a recent research project only 1 g of bone material will be available for radiocarbon dating of these valuable bones with the AMS method. The standard methods for the preparation of gelatin were compared with the amino acids treated by XAD-2 resin. Additionally, it was tested if the alkaline step in the procedure may be skipped or not. According to our results we may conclude that for well preserved bones the gelatin production yields reliable results. We could also show that the XAD-2 treatment is an eective method for the removal of humic acids. Ó 2000 Elsevier Science B.V. All rights reserved. PACS: 06.60.Ei Keywords: AMS;
14
C dating; Bone sample pretreament
1. Introduction Samples of bone, horn, teeth, etc. from various species are important 14 C dating materials. Because of their complicated compositions, the different kinds of contamination incorporated from the surrounding sediment and the changes of their
q Supported by the Xia-Shang-Zhou Chronology Project of China. * Corresponding author. Tel.: +86-10-6275-1667; fax: +8610-6275-1667. E-mail address: [email protected] (X. Wu).
components during burial, the reliability of radiocarbon dating of fossil bones with poor preservation still has some problems up to now. The key issue is to determine what kind of pretreatment yields reliable components for dating. Hedges and Klinken [1] presented an overview of approaches in the pretreatment of bone in 1992. At present in most AMS labs, according to the pretreatment methods selected for fossil bones, the materials used for 14 C dating are: A. Collagen extracted from bone, decalci®ed with acid and subsequently soaked with alkali. B. Gelatin extracted from collagen hydrolyzed with pH 2±3 HCl at 90°C. The resulting
0168-583X/00/$ - see front matter Ó 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 0 0 ) 0 0 3 4 8 - 7
S. Yuan et al. / Nucl. Instr. and Meth. in Phys. Res. B 172 (2000) 424±427
solution is ®ltered or centrifuged afterwards and evaporated to dryness. C. Puri®ed amino acids obtained by hydrolyzing the gelatin with 6N HCl at 110°C, and further cleanup with XAD-2 resin. D. Speci®c individual amino acids (e.g., glycine, proline, hydroxyproline) obtained by separating amino acids further. In the separation processes, the ®rst two methods are the simpler procedures. Whereas the latter two, especially the last one, are very time consuming procedures which also require more than 10 times the amount of sample material than the simpler methods. Some labs try a variety of further puri®cation approaches to remove contamination from humic acid residues possibly still present in the sample after the application of the standard pretreatment methods. These puri®cation methods include activated charcoal adsorption, ion exchange separation, ultra®ltration as well as XAD-2 resin treatment, etc. [2,3]. In the course of a big research project [4] a large quantity of bone samples such as horse, sheep, ox, human bone, etc. ± also very valuable samples of oracle bone with ancient Chinese inscriptions ± will be dated. Generally, the gelatin content of the samples is more than 20 mg/g of bone. For the rare artifacts (oracle bones) only about one gram of each sample can be obtained. Since these samples are of insucient weights for typical dating methods and high measurement precision must be achieved with these smaller sample amounts, it is necessary to select pretreatment methods that meet both the measurement precision and smaller sample quantity requirements. Process B (i.e., gelatinextraction method) which is being used in many labs, and process C, in which the hydrolysates of gelatin are treated with XAD-2 resin to get amino acids, are supposed to be suitable for this purpose. In order to check their applicability, we tested both methods and compared them with each other. 2. Experiments and results 2.1. Basic research In connection with 14 C dating of bone samples, a number of studies concerning bone components,
425
bone alteration in burial process, separation of components for dating, nature and detection of contamination, etc. [5±9] have been performed. We measured and compared the molecular weight distributions as well as the dierence in amino acid compositions between modern and ancient bone samples using HPLC. We analyzed the content of humics in gelatin with infrared spectrometry and ultraviolet spectrometry. By these means we attempted to determine the extraneous contaminant content in pretreated bone-dating components according to the changes of bone composition in the burial process and the humic contents. Unfortunately, it is dicult to obtain quantitative results with these methods, and to determine whether there are traces of contamination present in the material used for dating. 2.2. Comparison measurement When a dating result is assessed, it is usually in contrast with the dating results of the sample materials or sample components of higher reliability. As a rule, charcoal samples are regarded as comparatively reliable dating materials. Among the four dating components and processes, compared with the ®rst two, the latter two undergo more puri®cation steps, so probably contain less contaminant. XAD-2 treatment is regarded as an eective method for removing humics. So the amino acids treated with XAD-2 can be regarded as better bone-dating material. 2.2.1. Comparison of dating results between gelatin and XAD-2 treated amino acids In the four methods of collagen, gelatin, amino acids and individual amino acids, collagen sometimes may retain unremoved contaminant containing carbon, while in the preparation of individual amino acids not only much bone material is necessary but also very complicated separation processes are involved. Therefore in our study we only selected gelatin and amino acids treated with XAD-2 for comparison with charcoal samples from the same archaeological periods. A similar work has been reported by Minami and Nakamura [10,11]. We treated the samples by means of process B to obtain gelatin which was
426
S. Yuan et al. / Nucl. Instr. and Meth. in Phys. Res. B 172 (2000) 424±427
divided into two parts: one part was converted directly into graphite; the other was transformed into amino acids by process C which were treated with XAD-2 and then converted into graphite, which was measured with the EN tandem accelerator mass spectrometry at Peking University (PKUAMS). The d13 C of samples were analyzed with a stable isotope mass spectrometer. The results are shown in Table 1. From the data in Table 1 we can see that in each group the data agree within 1r error. In group 2, the results of SA98094 and SA98155 also agree with the 14 C determination performed by the ISOTRACE lab, Toronto. 2.2.2. Comparison of measurement results of dierent processes or components The alkaline step of the collagen preparation procedure may cause a greater loss of collagen. During the pretreatment of poorly preserved bone samples, even part of the peptides of degraded collagen may be dissolved during the decalci®cation. In order to ®nd a suitable cleaning method for this kind of sample that yields
reliable dating results, we treated the same sample material with dierent methods and compared the results: 1. We studied the eect of skipping the alkali treatment. We used process A without the alkali step to prepare collagen, and processes B and C were used to produce the amino acids which were treated by XAD-2. The results were compared with the results of the amino acids produced by the unchanged processes A, B, C, and/or the gelatin produced by A and B. The results are listed in Table 2. 2. We investigated whether organics dissolved during the decalci®cation step may be used for dating. After decalcifying samples with acid, the organics in solution were evaporated with rotatory evaporator and lyophilized. From the organics obtained amino acids were prepared by procedure C and puri®ed with XAD-2. The result of these amino acids were compared with that of gelatin produced by processes A and B (Table 3). The comparison of data from Tables 2 and 3 shows that in each group every date agree within a 1±2r error.
Table 1 Comparison dating results between gelatin and XAD-2 resin-treated amino acids Group
1
Sample no.
SA98093 SA98093Ab
2
SA98094 SA98094Ab SA98155
3
SA98096 SA98096Ab
a b
d 13 C (&)
14
2.97
)10.0
2781 50
Amino acids
2.88
)9.1
2805 50
Gelatin
3.05
)14.2
2573 50
Amino acids
2.87
)12.1
2574 51
)25.1
2640 50
3.08
)15.7
2555 50
2.89
)16.6
2531 53
Sample material
Components
Human bone Human bone Horse bone Horse bone Charcoal
Gelatin
Horse bone Horse bone
Gelatin Amino acids
C/N
Charcoal
The pretreatment and determination of the samples were done in Toronto. A: The sample pretreated by XAD-2 method.
C Age (yr BP)
This lab
ISOTRACE Lab, Toronto, Canadaa
2570 50 (TO-7999) 2630 40 (TO-7998)
S. Yuan et al. / Nucl. Instr. and Meth. in Phys. Res. B 172 (2000) 424±427
427
Table 2 XAD-2 treatment eects on removing contamination from bone components Group
Sample no.
Matter
Components
C/N
d13 C (&)
14
1
SA98094 SA98094Aa SA98094AAb SA98096 SA98096Aa SA98096AAb SA98167 SA98167AAb
Horse bone Horse bone Horse bone Horse bone Horse bone Horse bone Sheep bone Sheep bone
Gelatin Amino acids Amino acids Gelatin Amino acids Amino acids Gelatin Amino acids
3.05 2.87 2.91 3.08 2.89 2.87 3.06 2.92
)14.2 )12.3 )12.6 )15.7 )16.6 )13.6 )16.4 )15.5
2573 50 2574 51 2440 43 2555 50 2531 53 2556 50 2868 48 2824 43
2 3 a b
C age (yr BP)
A: The sample pretreated by XAD-2 method. AA: The sample pretreated by XAD-2 resin, while alkali treatment was skipped in preparing collagen.
Table 3 Comparison of measurement results of gelatin and organics (amino acids) in solution
a
Group
Sample no.
Matter
Components
C/N
d13 C (&)
14
1
SA99068 SA99068sa
Bone Bone
Gelatin Amino acids
2.84 2.76
)5.6 )5.6a
3385 38 3366 63
C age (yr BP)
Not measured, assume that it was the same result as SA99068.
3. Conclusions 1. At present it is still dicult to detect trace contaminants containing carbon in dating components of pretreated bone samples. 2. In the case of good-preserved bone samples, gelatin prepared with procedures A and B can give a reliable age. 3. XAD-2 treatment is indeed an eective method for removing humic contamination in bone samples. 4. In the pretreatment of the samples, it is possible to skip the alkaline step as shown in 2.2.2(1) step. But the procedure of cleaning up puri®ed amino acids with XAD-2 resin is more time consuming than alkaline treatment.
Acknowledgements We wish to thank Dr. R.P. Beukens, ISOTRACE Lab, Toronto, Canada, for comparison
measurement of the samples SA98094 and SA98155. References [1] R.E.M. Hedges, G.J. Van Klinken, Radiocarbon 34 (3) (1992) 279. [2] T.W. Staord Jr., K. Brendel, R.C. Duhamel, Geochim. Cosmochim. 52 (1988) 2257. [3] T.A. Brown, D.E. Nelson, J.S. Vogel, J.R. Southon, Radiocarbon 30 (2) (1988) 171. [4] Z.Y. Guo, K. Liu, X. Lu, H. Ma, K. Li, S. Yuan, X. Wu, Nucl. Instr. and Meth. B 172 (2000) 724. [5] R. Gillespie, Radiocarbon 31 (3) (1989) 239. [6] M.J. DeNiro, S. Weiner, Geochim. Cosmochim. 52 (1988) 2197. [7] R.R. Burky, D.L. Kirner, R.E. Taylor, P.E. Hare, J.R. Southon, Radiocarbon 40 (1) (1998) 11. [8] KH.A. Arslanov, Yu.S. Svezhentsev, Radiocarbon 35 (3) (1993) 384. [9] T.W. Staord Jr., P.E. Hare, L. Currie, A.J.T. Jull, D.J. Donahue, J. Archaeol. Sci. 18 (1991) 35. [10] M. Minami, T. Nakamura, Chinese Sci. Bull. 43 (Suppl.) (1998) 89. [11] M. Ninami, T. Nakamura, Nucl. Instr. and Meth. B 172 (2000) 462.