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Amino acid racemization dating of fossil bones, I. inter-laboratory comparison of racemization measurements
Earth and Planetary Science Letters, 43 (1979) 265 --268 0 Elsevier Scientific Publishing Company, Amsterdam - Printed
265 in The Netherlands
121
AMINO ACID RACEMIZATION
DATING OF FOSSIL BONES, I. INTER-LABORATORY OF RACEMIZATION MEASUREMENTS
JEFFREY Amino Acid Dating Laboratory,
COMPARISON
L. BADA, E. HOOPES, D. DARLING
Scripps Institution of Oceanography, CA 92093 (U.S.A.)
GRAHAM DUNGWORTH Laboratory of Exobiology,
University of California, San Diego, La Jolla,
l, HENK J. KESSELS
University of Nijmegen, Nijmegen (The Netherlands)
and KEITH A. KVENVOLDEN,
DAVID J. BLUNT
U.S. Geological Survey, Pacific-Arctic Branch of Marine Geology, 34.5 Middlefield Road, Menlo Park, CA 94025 (U.S.A.)
Received Revised version
November 20, 1978 received January 30, 1979
Enantiomeric measurements for aspartic acid, glutamic acid, and alanine in twenty-one different fossil bone samples have been carried out by three different laboratories using different analytical methods. These inter-laboratory comparisons demonstrate that D/L aspartic acid measurements are highly reproducible, whereas the enantiomeric measurements for the other amino acids show a wide variation between the three laboratories. At present. asoartic acid measurements are the most suitable for racemization dating of bone _ because of their superior analytical precision.
1. Introduction
During the last few years, a new technique for dating fossil bones has been developed, based on the racemization reaction of amino acids. (See Masters and Bada [I] for recent review of racemization bone dating.) The technique can be used to date fossil bones which have ages in the range of a few thousand years to several hundred thousand years. The actual dating range is dependent upon the temperature of the environment in which the bone was found. Although comparisons between racemization ages and r Present address: British Petroleum, Exploration Research and Development Division, Sunbury on Thames, Middlesex, England.
those produced from other evidence, including radiocarbon, historical records, and geologic evidence, have been found to be in close agreement at some 25 different sites throughout the world [ 11, arguments about the validity of the racemization dating methods have been published [2 1. This paper is the first in a series that will deal with some of the questions that have been raised concerning the validity of the racemization dating technique as applied to bone. One important phase of any new kind of geochemical measurement is the demonstration that the measurements that are carried out in one laboratory are reproducible in other laboratories which might use different analytical methods. In some earlier publications [3,4], it was shown that for a few samples, the racemization measurements carried out
266
at the Scripps Institution of Oceanography racemization laboratory were in close agreement with those carried out in another independent laboratory. In this paper, we present the results of the racemization measurements for aspartic acid, glutamic acid, and alanine carried out on various fossil bones from throughout the world by three different laboratories.
[5,6]. The aspartic acid was separated [7] from the total amino acid extracts obtained by the procedures described above by chromatography on Dowex-1. After isolation, the aspartic acid was reacted with Lleucine-N-carboxy-anhydride and the resulting dipeptides were separated on the amino acid analyzer [ 1 ,S]. The ratios of the other amino acids were determined by gas chromatography of the N-trifluoroacetyl-L-prolyl-peptide methyl esters [8].
2. Materials and methods Fossil bone samples from sites throughout the world were selected from the collection of samples present at the Scripps laboratory. Two types of samples were analyzed: (1) portions of the extracts from bone samples processed and analyzed at Scripps were sent to the Nijmegen and the U.S. Geological Survey (USGS) laboratories for analyses; and (2) bone pieces similar to the ones analyzed at Scripps were sent to the other laboratories for independent processing and analyses. The bone samples were processed according to the procedure described elsewhere [ 1 ,S]. Generally, approximately l-5 grams of dense, primary bone were first cleaned mechanically of any adhering soil or dirt. Next, the sample was subjected to a series of ultrasonic cleaning steps; the sample was repeatedly sonicated in water and approximately 1-2M HCl until the bone fragment was free of any extraneous surface materials such as carbonates, soils, etc. Following the cleaning procedure, the sample was dissolved in doubly distilled 6M HCl and hydrolyzed for either 4,6, or 24 hours; the different hydrolysis times were used in order to reduce the amount of acid-catalyzed racemization in some of the samples. Next, the sample was evaporated to dryness, reconstituted in a small volume of water, and desalted on Dowex-50 (H+) cation-exchange resin. After the desalting, the samples were analyzed using the procedures described below. 2.1. Enantiomeric analyses Scripps Laboratory. The enantiomeric analyses carried out at the Scripps laboratory utilized both the automatic amino acid analyzer and gas chromatography. The D/L aspartic acid measurements were carried out using the diastereomic dipeptide method
NQmegen Iaboratoly. Enantiomeric ratios in the total amino acid extracts were measured by gas chromatography of volatile derivations on capillary columns coated with an optically active stationary phase [9]. D and L NTFA methyl ester derivatives were prepared and separated by gas chromatography on 60 m stainless steel capillary columns coated with NTFA L-phenylalanyl-L-leucyl cyclohexyl ester previously conditioned at 125°C for two days [lo]. USGS laboratory. For this work, enantiomeric ratios of amino acid were determined by gas chromatog raphy of their N-pentafluoropropionyl-(+)-2-butyl ester derivatives. Preparation of the derivatives followed procedures outlined by Kvenvolden et al. [ 1 l] with the following exceptions: (1) esterification with 0.2 ml of (t)-2-butanol .4N HCl was carried out in an open system rather than in a closed vial; (2) pentafluoropropionic anhydride (0.2 ml) was used for acylation rather than trifluoroacetic anhydride. Diastereomeric derivatives of amino acids were resolved on two different stainless steel capillary columns: 200’ X 0.02” coated with UCON 75 H 90,000; and 200’ X 0.03” coated with Carbowax 20M. Quantitation of the amino acid enantiomers was obtained by measuring peak heights on gas chromatograms.
3. Results The results of the analyses by the three laboratories are summarized in Table 1. These interlaboratory comparisons demonstrate that measurements for aspartic acid are highly reproducible, regardless of the analytical method used to determine the aspartic acid enantiomeric ratio. For the extracts, the D/L aspartic acid measurements carried out in the
267
TABLE 1 Inter-laboratory
comparison of racemization analyses of fossil bones (all samples were hydrolyzed 24 hours except as indicated)
Sample locality and identification number
D/L glutamic acid
D/L aspartic acid
D/L alanine
Scripps
Nijmegen
USGS
Scripps
Nijmegen
USGS
Scripps
Nijmegen
USGS
0.475 0.416
0.477 0.433
0.48 0.44
0.18 0.21
0.131 0.165
0.16 0.16
0.22 0.15
0.118 0.114
0.13 0.14
0.470
0.393
_
_
0.204
_
_
0.260
_
0.092
0.099
_
_
0.031
_
-
0.0096
-
0.55
0.527
0.54
0.19
0.160
0.20
-
0.115
0.13
0.474 0.244
0.457 0.264
0.48 _
0.16 0.10
0.098 0.113
0.16 -
0.14 0.083
0.064 0.077
0.09 -
0.453 0.500 0.080
0.436 0.459 0.091
0.44 0.06
0.31 0.21 -
0.227 0.161 -
0.14 _ 0.03
0.18 _ -
0.223 0.234 0.011
0.24 0.02
0.73 0.244
0.621 0.233
0.67 _
-0.3 -
0.227 0.085
0.33 _
-0.5 _
0.474 0.039
0.43 _
0.504 0.437
0.557 0.41
0.48 -
-0.2 _
0.230 0.33
0.24 _
-0.5 -
0.419 0.27
0.45 _
0.503 0.556 0.467 0.417 0.244
0.45 0.58 0.364 0.395 0.221
_ _
0.23 0.21 0.30 0.10
0.17 0.25 0.091 0.157 0.068
_ -
0.41 0.21 0.33 0.083
0.39 0.34 0.046 0.151 0.039
_ _
0.28
0.372
-
_
0.10
-
-
0.11
-
0.189
0.208
0.12
-
0.060
0.04
-
0.030
0.03
0.56
-
0.52
0.33
_
0.33
0.47
-
0.28
Extracts
1. Tarkhan, Egypt, F1691 * 2. Tura, Egypt, Middle Kingdom 3. Palegawra, Iran, No. 12121 4. Ktilna Cave, Czechoslovakia, No. 11 ** 5. Warm Mineral Springs, Florida, human femur, 85019 6. Buhen Horse, Sudan 7. Gua Cha, Malaysia, BM13 Bones
1. Tarkhan, Egypt, F1556 * 2. Double Adobe, Arizona 3. Marin, California, No. 152 (UCLA 1891A) ** 4. Asych, U.S.S.R., III 1 5. Palegawra, Iran, No. 11224 6. Vertesszollos, Hungary 7. Riverside, California, Paleoindian skeleton 8. Scripps Horse (a) Leg (b) Scapula 9. Gua Cha, Malaysia, BM2 10. Gua Cha, Malaysia, BM7 11. Gua Cha, Malaysia, BM13 12. Santa Rosa Island, California, Mammoth 13. La Jolla, California W-12 SDi-4669 SDM16709 14. Yuha Burial, California * Sample hydrolyzed 6 hours. ** Sample hydrolyzed 4 hours.
three laboratories for any particular sample agree with each other to within about *5% of the average. The agreement is slightly poorer for the bone samples, but in general the measurements made in the three laboratories fall within about F-10% of the average D/L aspartic acid value for any one sample. The results for both glutamic acid and alanine show a much poorer agreement than those for
aspartic acid. In some instances, the enantiomeric ratios for glutamic acid and alanine disagree by nearly a factor of 2 between the three laboratories. The reason for this disagreement is uncertain but it could reflect an inherent variability in the measurements which results from the three different gas chromatographic methods that were used. The enantiomeric ratios for several other amino acids were also deter-
268
mined for the various samples, but in general the extent of racemization was too small to make any definitive conclusions about the agreement between the three laboratories. The results indicate that for the racemization dating of fossil bones, D/L aspartic acid measurements are at present the most suitable because of their superior analytical precision. Before we can calculate racemization ages based on either glutamic acid and alanine, further work is required to refine the precision and reproducibility of the enantiomeric measurements. It is important to note that there is no consistent difference in the D/L aspartic acid values determined by the various laboratories when extracts and actual bone samples are analyzed. This indicates that the processing procedure can be reproducibly carried out in other laboratories and that no significant variability is produced in the racemization measurements from the sample-preparation steps.
4. Conclusions The inter-laboratory comparisons we have given here convincingly demonstrate that aspartic acid enantiomeric measurements can be accurately and reproducibly obtained when different laboratories carry out the sample processing and racetnization analyses. On the basis of these inter-laboratory comparisons, aspartic acid is the most suitable amino acid to use for the racemization dating of fossil bones. Further refinement is required in the enantiomeric analyses of other amino acids before these measurements can be used for racemization dating of fossil bone. Investigations similar to those presented here should be carried out by using other fossil materials so that the reliability of racemization dates can be ascertained on fossil materials other than bone.
Acknowledgements The Scripps investigation was partially supported by NSF grants EAR 73-00320 and EAR 77-14490.
We thank the numerous individuals who generously supplied the samples used in these investigations. J.L.B. is an Alfred P. Sloan Fellow 197551979.
References 1 P.M. Masters and J.L. Bada, Amino acid racemization dating of bone and shell, in: Archaeological Chemistry, II, C.F. Carter, ed., Adv. Chem. Ser. 171 (1978) 117138. 2 H.M. Williams and G.G. Smith, A critical evaluation of the application of amino acid racemization to geochronology and geothermometry, Origins Life 8 (1977) 9 l144. 3 J.L. Bada and P.M. Helfman, Amino acid racemization dating of fossil bones, World Arch. 7 (1975) 160-173. 4 P.M. Helfman and J.L. Bada, Aspartic acid racemization in tooth enamel from living humans, Proc. Natl. Acad. Sci. (USA) 72 (1975) 2891.-2894. reaction of 5 J.L. Bada and R. Protsch, Racemization aspartic acid and its use in dating fossil bones, Proc. Natl. Acad. Sci. (USA) 70 (1973) 1331-1334. 6 J.M. Manning and S. Moore, Determination of D- and Lamino acids by ion exchange chromatography as L-D and L-L dipeptides, J. Biol. Chem. 243 (1968) 5591-5597. 7 C.H.W. Hits, S. Moore and W.H. Stein, The chromatography of amino acids on ion exchange resisn. Use of volatile acids for elution, J. Am. Chem. Sot. 76 (1954) 606336065. 8 E.H. Hoopes, E.T. Peltzer and J.L. Bada, Determination of amino acid enantiomeric ratios by gas liquid chromatog raphy of the N-trifluoroacetyl-L-prolyl-peptide methyl esters, J. Chromatogr. Sci. 16 (1978) 556-560. E. Bayer and J. 9 W.A. Koenig, W. Parr, H.A. Lichtenstein, Oro, Gas chromatographic separation of amino acids and their enantiomers. Non-polar stationary phases and a new optically active phase, J. Chromatogr. Sci. 8 (1970) 1833 186. A.W. Schwartz and L. Van de Leemput, 10 G. Dungworth, Composition and racemization of amino acids in mammoth collagen determined by gas and liquid chromatography, Comp. Biochem. Physiol. 53B (1976) 473-480. E. Peterson and G.E. Pollock, Geo11 K.A. Kvenvolden, chemistry of amino acid enantiomers: gas chromatography of their diastereomeric derivatives, in: Advances in Organic Geochemistry 1971 (Pergamon Press, New York, N.Y., 1972) 387-401.
265 in The Netherlands
121
AMINO ACID RACEMIZATION
DATING OF FOSSIL BONES, I. INTER-LABORATORY OF RACEMIZATION MEASUREMENTS
JEFFREY Amino Acid Dating Laboratory,
COMPARISON
L. BADA, E. HOOPES, D. DARLING
Scripps Institution of Oceanography, CA 92093 (U.S.A.)
GRAHAM DUNGWORTH Laboratory of Exobiology,
University of California, San Diego, La Jolla,
l, HENK J. KESSELS
University of Nijmegen, Nijmegen (The Netherlands)
and KEITH A. KVENVOLDEN,
DAVID J. BLUNT
U.S. Geological Survey, Pacific-Arctic Branch of Marine Geology, 34.5 Middlefield Road, Menlo Park, CA 94025 (U.S.A.)
Received Revised version
November 20, 1978 received January 30, 1979
Enantiomeric measurements for aspartic acid, glutamic acid, and alanine in twenty-one different fossil bone samples have been carried out by three different laboratories using different analytical methods. These inter-laboratory comparisons demonstrate that D/L aspartic acid measurements are highly reproducible, whereas the enantiomeric measurements for the other amino acids show a wide variation between the three laboratories. At present. asoartic acid measurements are the most suitable for racemization dating of bone _ because of their superior analytical precision.
1. Introduction
During the last few years, a new technique for dating fossil bones has been developed, based on the racemization reaction of amino acids. (See Masters and Bada [I] for recent review of racemization bone dating.) The technique can be used to date fossil bones which have ages in the range of a few thousand years to several hundred thousand years. The actual dating range is dependent upon the temperature of the environment in which the bone was found. Although comparisons between racemization ages and r Present address: British Petroleum, Exploration Research and Development Division, Sunbury on Thames, Middlesex, England.
those produced from other evidence, including radiocarbon, historical records, and geologic evidence, have been found to be in close agreement at some 25 different sites throughout the world [ 11, arguments about the validity of the racemization dating methods have been published [2 1. This paper is the first in a series that will deal with some of the questions that have been raised concerning the validity of the racemization dating technique as applied to bone. One important phase of any new kind of geochemical measurement is the demonstration that the measurements that are carried out in one laboratory are reproducible in other laboratories which might use different analytical methods. In some earlier publications [3,4], it was shown that for a few samples, the racemization measurements carried out
266
at the Scripps Institution of Oceanography racemization laboratory were in close agreement with those carried out in another independent laboratory. In this paper, we present the results of the racemization measurements for aspartic acid, glutamic acid, and alanine carried out on various fossil bones from throughout the world by three different laboratories.
[5,6]. The aspartic acid was separated [7] from the total amino acid extracts obtained by the procedures described above by chromatography on Dowex-1. After isolation, the aspartic acid was reacted with Lleucine-N-carboxy-anhydride and the resulting dipeptides were separated on the amino acid analyzer [ 1 ,S]. The ratios of the other amino acids were determined by gas chromatography of the N-trifluoroacetyl-L-prolyl-peptide methyl esters [8].
2. Materials and methods Fossil bone samples from sites throughout the world were selected from the collection of samples present at the Scripps laboratory. Two types of samples were analyzed: (1) portions of the extracts from bone samples processed and analyzed at Scripps were sent to the Nijmegen and the U.S. Geological Survey (USGS) laboratories for analyses; and (2) bone pieces similar to the ones analyzed at Scripps were sent to the other laboratories for independent processing and analyses. The bone samples were processed according to the procedure described elsewhere [ 1 ,S]. Generally, approximately l-5 grams of dense, primary bone were first cleaned mechanically of any adhering soil or dirt. Next, the sample was subjected to a series of ultrasonic cleaning steps; the sample was repeatedly sonicated in water and approximately 1-2M HCl until the bone fragment was free of any extraneous surface materials such as carbonates, soils, etc. Following the cleaning procedure, the sample was dissolved in doubly distilled 6M HCl and hydrolyzed for either 4,6, or 24 hours; the different hydrolysis times were used in order to reduce the amount of acid-catalyzed racemization in some of the samples. Next, the sample was evaporated to dryness, reconstituted in a small volume of water, and desalted on Dowex-50 (H+) cation-exchange resin. After the desalting, the samples were analyzed using the procedures described below. 2.1. Enantiomeric analyses Scripps Laboratory. The enantiomeric analyses carried out at the Scripps laboratory utilized both the automatic amino acid analyzer and gas chromatography. The D/L aspartic acid measurements were carried out using the diastereomic dipeptide method
NQmegen Iaboratoly. Enantiomeric ratios in the total amino acid extracts were measured by gas chromatography of volatile derivations on capillary columns coated with an optically active stationary phase [9]. D and L NTFA methyl ester derivatives were prepared and separated by gas chromatography on 60 m stainless steel capillary columns coated with NTFA L-phenylalanyl-L-leucyl cyclohexyl ester previously conditioned at 125°C for two days [lo]. USGS laboratory. For this work, enantiomeric ratios of amino acid were determined by gas chromatog raphy of their N-pentafluoropropionyl-(+)-2-butyl ester derivatives. Preparation of the derivatives followed procedures outlined by Kvenvolden et al. [ 1 l] with the following exceptions: (1) esterification with 0.2 ml of (t)-2-butanol .4N HCl was carried out in an open system rather than in a closed vial; (2) pentafluoropropionic anhydride (0.2 ml) was used for acylation rather than trifluoroacetic anhydride. Diastereomeric derivatives of amino acids were resolved on two different stainless steel capillary columns: 200’ X 0.02” coated with UCON 75 H 90,000; and 200’ X 0.03” coated with Carbowax 20M. Quantitation of the amino acid enantiomers was obtained by measuring peak heights on gas chromatograms.
3. Results The results of the analyses by the three laboratories are summarized in Table 1. These interlaboratory comparisons demonstrate that measurements for aspartic acid are highly reproducible, regardless of the analytical method used to determine the aspartic acid enantiomeric ratio. For the extracts, the D/L aspartic acid measurements carried out in the
267
TABLE 1 Inter-laboratory
comparison of racemization analyses of fossil bones (all samples were hydrolyzed 24 hours except as indicated)
Sample locality and identification number
D/L glutamic acid
D/L aspartic acid
D/L alanine
Scripps
Nijmegen
USGS
Scripps
Nijmegen
USGS
Scripps
Nijmegen
USGS
0.475 0.416
0.477 0.433
0.48 0.44
0.18 0.21
0.131 0.165
0.16 0.16
0.22 0.15
0.118 0.114
0.13 0.14
0.470
0.393
_
_
0.204
_
_
0.260
_
0.092
0.099
_
_
0.031
_
-
0.0096
-
0.55
0.527
0.54
0.19
0.160
0.20
-
0.115
0.13
0.474 0.244
0.457 0.264
0.48 _
0.16 0.10
0.098 0.113
0.16 -
0.14 0.083
0.064 0.077
0.09 -
0.453 0.500 0.080
0.436 0.459 0.091
0.44 0.06
0.31 0.21 -
0.227 0.161 -
0.14 _ 0.03
0.18 _ -
0.223 0.234 0.011
0.24 0.02
0.73 0.244
0.621 0.233
0.67 _
-0.3 -
0.227 0.085
0.33 _
-0.5 _
0.474 0.039
0.43 _
0.504 0.437
0.557 0.41
0.48 -
-0.2 _
0.230 0.33
0.24 _
-0.5 -
0.419 0.27
0.45 _
0.503 0.556 0.467 0.417 0.244
0.45 0.58 0.364 0.395 0.221
_ _
0.23 0.21 0.30 0.10
0.17 0.25 0.091 0.157 0.068
_ -
0.41 0.21 0.33 0.083
0.39 0.34 0.046 0.151 0.039
_ _
0.28
0.372
-
_
0.10
-
-
0.11
-
0.189
0.208
0.12
-
0.060
0.04
-
0.030
0.03
0.56
-
0.52
0.33
_
0.33
0.47
-
0.28
Extracts
1. Tarkhan, Egypt, F1691 * 2. Tura, Egypt, Middle Kingdom 3. Palegawra, Iran, No. 12121 4. Ktilna Cave, Czechoslovakia, No. 11 ** 5. Warm Mineral Springs, Florida, human femur, 85019 6. Buhen Horse, Sudan 7. Gua Cha, Malaysia, BM13 Bones
1. Tarkhan, Egypt, F1556 * 2. Double Adobe, Arizona 3. Marin, California, No. 152 (UCLA 1891A) ** 4. Asych, U.S.S.R., III 1 5. Palegawra, Iran, No. 11224 6. Vertesszollos, Hungary 7. Riverside, California, Paleoindian skeleton 8. Scripps Horse (a) Leg (b) Scapula 9. Gua Cha, Malaysia, BM2 10. Gua Cha, Malaysia, BM7 11. Gua Cha, Malaysia, BM13 12. Santa Rosa Island, California, Mammoth 13. La Jolla, California W-12 SDi-4669 SDM16709 14. Yuha Burial, California * Sample hydrolyzed 6 hours. ** Sample hydrolyzed 4 hours.
three laboratories for any particular sample agree with each other to within about *5% of the average. The agreement is slightly poorer for the bone samples, but in general the measurements made in the three laboratories fall within about F-10% of the average D/L aspartic acid value for any one sample. The results for both glutamic acid and alanine show a much poorer agreement than those for
aspartic acid. In some instances, the enantiomeric ratios for glutamic acid and alanine disagree by nearly a factor of 2 between the three laboratories. The reason for this disagreement is uncertain but it could reflect an inherent variability in the measurements which results from the three different gas chromatographic methods that were used. The enantiomeric ratios for several other amino acids were also deter-
268
mined for the various samples, but in general the extent of racemization was too small to make any definitive conclusions about the agreement between the three laboratories. The results indicate that for the racemization dating of fossil bones, D/L aspartic acid measurements are at present the most suitable because of their superior analytical precision. Before we can calculate racemization ages based on either glutamic acid and alanine, further work is required to refine the precision and reproducibility of the enantiomeric measurements. It is important to note that there is no consistent difference in the D/L aspartic acid values determined by the various laboratories when extracts and actual bone samples are analyzed. This indicates that the processing procedure can be reproducibly carried out in other laboratories and that no significant variability is produced in the racemization measurements from the sample-preparation steps.
4. Conclusions The inter-laboratory comparisons we have given here convincingly demonstrate that aspartic acid enantiomeric measurements can be accurately and reproducibly obtained when different laboratories carry out the sample processing and racetnization analyses. On the basis of these inter-laboratory comparisons, aspartic acid is the most suitable amino acid to use for the racemization dating of fossil bones. Further refinement is required in the enantiomeric analyses of other amino acids before these measurements can be used for racemization dating of fossil bone. Investigations similar to those presented here should be carried out by using other fossil materials so that the reliability of racemization dates can be ascertained on fossil materials other than bone.
Acknowledgements The Scripps investigation was partially supported by NSF grants EAR 73-00320 and EAR 77-14490.
We thank the numerous individuals who generously supplied the samples used in these investigations. J.L.B. is an Alfred P. Sloan Fellow 197551979.
References 1 P.M. Masters and J.L. Bada, Amino acid racemization dating of bone and shell, in: Archaeological Chemistry, II, C.F. Carter, ed., Adv. Chem. Ser. 171 (1978) 117138. 2 H.M. Williams and G.G. Smith, A critical evaluation of the application of amino acid racemization to geochronology and geothermometry, Origins Life 8 (1977) 9 l144. 3 J.L. Bada and P.M. Helfman, Amino acid racemization dating of fossil bones, World Arch. 7 (1975) 160-173. 4 P.M. Helfman and J.L. Bada, Aspartic acid racemization in tooth enamel from living humans, Proc. Natl. Acad. Sci. (USA) 72 (1975) 2891.-2894. reaction of 5 J.L. Bada and R. Protsch, Racemization aspartic acid and its use in dating fossil bones, Proc. Natl. Acad. Sci. (USA) 70 (1973) 1331-1334. 6 J.M. Manning and S. Moore, Determination of D- and Lamino acids by ion exchange chromatography as L-D and L-L dipeptides, J. Biol. Chem. 243 (1968) 5591-5597. 7 C.H.W. Hits, S. Moore and W.H. Stein, The chromatography of amino acids on ion exchange resisn. Use of volatile acids for elution, J. Am. Chem. Sot. 76 (1954) 606336065. 8 E.H. Hoopes, E.T. Peltzer and J.L. Bada, Determination of amino acid enantiomeric ratios by gas liquid chromatog raphy of the N-trifluoroacetyl-L-prolyl-peptide methyl esters, J. Chromatogr. Sci. 16 (1978) 556-560. E. Bayer and J. 9 W.A. Koenig, W. Parr, H.A. Lichtenstein, Oro, Gas chromatographic separation of amino acids and their enantiomers. Non-polar stationary phases and a new optically active phase, J. Chromatogr. Sci. 8 (1970) 1833 186. A.W. Schwartz and L. Van de Leemput, 10 G. Dungworth, Composition and racemization of amino acids in mammoth collagen determined by gas and liquid chromatography, Comp. Biochem. Physiol. 53B (1976) 473-480. E. Peterson and G.E. Pollock, Geo11 K.A. Kvenvolden, chemistry of amino acid enantiomers: gas chromatography of their diastereomeric derivatives, in: Advances in Organic Geochemistry 1971 (Pergamon Press, New York, N.Y., 1972) 387-401.