OVERVIEW OF THE AVAILABLE PHYSICAL METHODS USED TO PROVIDE "ABSOLUTE" DATING
are several established techniques for determining a result for the "absolute"
age of an object. They can be summarised as follows :-
(expressed hereinafter as 14C),
is produced naturally in the upper atmosphere through the following reaction
+ n => 14C
+ p (where
a neutron and p
is a proton). The 14C
formed is rapidly oxidised to
and enters the earth's plant and animal lifeways through photosynthesis
and the food chain as a proportion of the total carbon within the atmospheric
carbon dioxide entering plants through the process of
All living organisms contain the current ratio of the unstable or radioactive
to its other naturally ocurring stable isotopes of 12C
These isotopes are present in the following amounts
= 98.89%, 13C
= 1.11% and 14C
= 0.00000000010%. Thus, one 14C
atom exists in nature for every 1,000,000,000,000 12C
atoms in living material.
rapidity of the dispersal of 14C
into the atmosphere has been demonstrated by measurements of radioactive
carbon produced from thermonuclear bomb testing. 14C
also enters the Earth's oceans in an atmospheric
and as dissolved carbonate (the entire 14C
inventory is termed the "carbon exchange reservoir"). Plants and animals
which utilise carbon in biological food-chains take up
during their lifetimes. They exist in
with the 14C
concentration of the atmosphere, that is, the numbers of 14C
atoms and non-radioactive carbon atoms stays approximately the same over
time. As soon as a plant or animal dies, they cease the
function of carbon uptake; there is no replenishment of radioactive carbon,
only the decay of what was absorbed during its lifetime.
radiocarbon method is based on the rate of decay of the radioactive or
unstable Carbon isotope-14 (14C),
which is formed in the upper atmosphere through the effect of cosmic ray
neutrons upon Nitrogen-14 (expressed hereinafter as 14N).
When an organism dies, it stops taking up the 14C
from the environment and that which it has already absorbed begins to decay
at a constant value, known as its "half-life".
Anderson and Arnold (1949) were the first to measure
the rate of this decay. They found that after 5568 years, half the 14C
in the original sample will have decayed and after another 5568 years,
half of that remaining material will have decayed, and so on. The half-life
(t 1/2) is the name given to this value which Libby measured at 5568 ±30
years. This became known as the "Libby" or "conventional" half-life. The
application of the Radiocarbon Method for dating has been proven to have
an acceptable level of accuracy only as far back in time as 7 half-lives,
after which there is a very small amount of radioactive carbon present
in a sample. So, at about 40 000 years, the limit of the technique is reached.
Beyond this time, other radiometric techniques must be used for dating.
By measuring the 14C
concentration or residual radioactivity of a sample whose age is not known,
it is possible to obtain the count-rate or number of decay events per gram
of Carbon. By comparing this with modern levels of activity (1890 wood
corrected for decay to 1950 AD) and using the measured half-life it becomes
possible to calculate a date for the death of the sample.
decays it emits a weak beta particle (ß), or electron, which possesses
an average energy of 160keV.
decay can be shown thus : 14C
=> 14N + ß
decays back to 14N.
There is a quantitative relationship between the decay of 14C
and the production of a beta particle. The decay is constant but spontaneous.
That is, the probability of decay for an atom
in a discrete sample is constant, thereby requiring the application of
statistical methods for the analysis of counting data.
follows from this that any material which is composed of carbon may be
dated. Herein lies the true advantage of the radiocarbon method, it is
able to be uniformly applied throughout the world.
method has been successfully used on materials containing organic (carbon-based)
structures, such as bone and charcoal. Greater details on the method and
limitations of Radiocarbon Dating can be found in
(1972) and is also briefly outlined below
Mass Spectrometry Dating.
is a measuring technique which directly counts the number of carbon atoms
in relatively small amounts (5 to 10 milligrams) of a sample to produce
a date based on the Radiocarbon Method. Samples from cave paintings and
fossil bones can be submitted without noticeable damage to the original
technique covers an age range from 1 000 to 800 000 years ago and has been
used on dating stalagmite and other cave sinter deposits, coral and teeth.
This method relies on the constant accumulation of
the by-products of radioactive decay of certain elements within the structure
of the object. The technique in this case counts the number of electrons
trapped over time in the mineral crystals of the object and
a constant radiation dose from the surrounding environment. This method
has been successfully used on the dating of cave stalagmite / sinter deposits,
wherein the initial crystallization of calcium carbonate
is used as the "zero point" and their subsequent growth can be determined
over time and dated.
technique covers an age range from the historic to 250 000 years ago and
has been used on dating pottery, burnt rock and sediments. This method
also relies on the constant accumulation of electrons, the
of radioactive decay of certain elements within the structure of the object.
Here, the technique relies for its "zero point" on an event such as burning
or exposure to sunlight.
Spin Resonance Dating.
technique covers an age range from 1 000's to 100 000's of years ago and
has been used on dating tooth enamel and cave sinter deposits such as stalagmite.
Once again, this method also relies on the constant
of electrons, the by-products of radioactive decay of certain elements
within the structure of the object. This method has been successfully used
on the dating of cave stalagmite / sinter deposits and even
enamel, wherein the initial crystallization of the crystals in the object
is used as the "zero point" and their subsequent growth can be determined
over time and dated.
technique covers an age range from 35 000 to 1 000 000's years ago and
has been used on dating volcanic rocks. K-Ar has a long half-life of 1
300 million years and has been best used to date the decay of gases
in volcanic deposits older than 750 000 years. This technique has been
crucial in determining the evolution of early Homo in the volcanic
regions of Africa and Asia, where archaeological deposits are often
between layers of volcanic ash.
obtaining the "absolute" age of an object using any of the above methods,
there are varying margins of error inherent with the technique used, together
with practical limitations on when they can be suitable for use.
radioactive methods involve a certain amount of statistical uncertainty
when calculating a date. Although Radiocarbon Dating is one of the most
commonly used techniques, it has its own additional inherent problems.
reference to para 13.2.1. above, we know that the atmospheric
ratio of carbon isotopes has not always been the same as they are now.
So, the direct results from the use of this technique have always been
to calibration graphs which correct the data accordingly (and to the best
of our current knowledge) before being issued by the testing laboratory.
is always recommended, wherever possible, to obtain more than one result
using different methods. Even where this is not possible and a result can
only be obtained using a single method, several samples should be
Cambridge Radiocarbon Conference in 1962 agreed that : (i) all dates should
be calculated using the Libby Half-life value of 5568 years; (ii)
Ages are then quoted as "years BP" (Before the Present) with a modern
point of AD 1950.
detailed technical overview on the composition of bone and what is required
for bone to be dated, is given at the bottom of this page.
13.3.1. A view of specimen PM 1297, a 7cm long
proximal fragment of Human Radius sitting on its
City Museum Accession Card, recorded on 20 May 2003.
to the Museum by Robert Burnard on 06 February 1899, this hominin fossil
fragment is from Worth's
dark-brown "staining" is a thin covering of calcareous sinter or stalagmite.
scale is in centimetres.)
RADIOCARBON DATING OF THE FOSSIL BONES OF WORTH'S CATTEDOWN BONE CAVE
FROM THE DISCOVERIES OF 1886-87 :
The Problem with Radiocarbon Dating Worth's Hominin Collection :
the bombing of Plymouth during the Second World War, the Museum of the
old Plymouth Institution received a direct hit in 1941. The building
became a burning inferno and it was only through the heroic attempts
one of its Members, who entered the smoking remains of the ruined building
afterwards, that the bulk of the Cattedown Hominin Skulls survive today
in the care of the Plymouth City Museum. The rest of this fantastic
collection is lost to science forever. So, for the most part, the entire
bulk of Worth's extensive donation of the Cattedown Cave
Hominin fossils to the Plymouth Athenaeum will never be seen again.
In retrospect, if
had donated them to the Plymouth City Museum, they would have survived
the ravages of the bombing.
to their exposure to the heat of the burning Athenaeum building,
the surviving Cattedown Hominin fossils would not be suitable for radiocarbon
dating, even if they were within the scope of the acceptable limitations
the Radiocarbon method, ie. < 38 000 years BP in age.
The 1994 Attempt at 14C
Dating Worth's Hominin Collection.
has been confirmed that at least one of the Cattedown fossil bones from
Burnard's donation to the Plymouth City Museum was of human
origin. We have yet to determine if there are any more. Remember that
Burnard's Collection and subsequent donation of Cattedown fossil
material did not follow the "ownership route" of Worth's
material. Burnard's fossils went from the cave excavation
into his own private
where it remained for about 12 years, before being donated directly to
the Plymouth City Museum in 1899. The provenance is quite different.
one of the attempts at dating the Hominin material in 1994, a proximal
fragment of Human Radius from the Burnard Collection (Accession
No. PM 1297) and originally derived from Worth's Cattedown Bone Cave,
removed from the premises of the Plymouth City Museum by M. Bishop
and given to K. Ray on 02 March 1994. It was sent to Andrew Chamberlain
at Sheffield University. However, the sample bone was found to
"unsuitable" for dating.
a personal electronic communication dated 31 January 2005, we have received
the following helpful information from Mr Andrew Chamberlain, who sheds
some additional light upon this attempt at dating one of the
Hominins. We are grateful for his following contribution :-
to correct the record on the Human radius for which I attempted to gain
a radiocarbon date (PM 1297). The problem encountered by the Oxford University
Radiocarbon Accelerator Unit in 1994 was
the recent provenance of this fossil, Specimen
PM 1297 had not
suffered the fate of the surviving fragments of Worth's Cattedown
Hominin Collection, which had been greatly affected by the heat of the
blitzed Athenaeum Museum, because this specimen was one of the few
hominin fragments donated directly to the Plymouth City Museum
by Burnard in 1899, thus circumventing the intermediate
and ill-fated custodianship by the Plymouth Institution's Athenaeum
Worth's main Hominin Collection. Unfortunately,
for the reasons given in para 13.2.1. above, the methodology
used in this attempt at dating was totally unsuitable for the probable
age of the fossil specimen under test and was doomed to failure.
the specimen did not contain enough collagen to allow dating to proceed.
While it is possible that the lack of collagen is due to great antiquity,
it is more likely that it is a result of the circulation of
groundwater that has destroyed the collagen, rendering the bones chalky
and brittle. (I expect that you may have noticed this on other unburnt
bones in the Burnard Collection). Thus the antiquity
the Human skeletal remains is unresolved. It is possible that in the intervening
years the Oxford lab has become more proficient in extracting collagen
from bone, so a repeat dating exercise may be
on this material."
The Problem with Radiocarbon Dating Worth's Faunal Collection :
Exciting New Opportunities of Dating Fossil Material from Worth's Cattedown
Bone Cave :
RADIOCARBON DATING THE FOSSIL BONES OF THE CATTEDOWN REINDEER RIFT CAVE
FROM THE DISCOVERIES OF 1973 :
DATING No. 1....[BM-729.
Cattedown Cave, Devonshire] :
complete tibia from among the new (Rangifer tarandus) Reindeer
finds was sent to Richard Burleigh at the Radiocarbon Laboratory at the
British Museum (Bloomsbury) who in due course announced a date
125 ± 390 years B.P. (δ13C
following extract of data is taken from the publication "Radiocarbon",
(3). pp. 339 and 341., from information provided by Richard
Burleigh and Andrew Hewson of the Research Laboratory, the British
shows the Reindeer to have been of Late Pleistocene age. It lived about
3 000 years after the greatest advance of the ice sheets of the Last Glaciation,
which in Europe had reached their maximum about
date was obtained by liquid scintillation counting of benzene using a Model
3315 Hewlett Packard 2100A computer system for on-line processing of counting
data. Sample materials were pre-treated
dilute acid and alkali as appropriate; only the Collagen fraction of bone
was used for dating.
Date is expressed in radiocarbon years relative to AD 1950 based on the
Libby half-life for 14C
of 5570 years and is corrected for isotope fractionation ( δ13C
values are relative to PDB). No corrections
been made for natural
variations. The modern reference standard is NBS oxalic acid. The error
quoted with the date is based on counting statistics alone and is equivalent
to ± 1 standard deviation (± 1σ).
CATTEDOWN CAVE, Devonshire :
from tibia of reindeer [Rangifer tarandus L.] from Cattedown
Cave, Plymouth, Devonshire, England. Tibia (ref. CBR 15.6.74/3) was from
an associated group of bones representing the hind part of a single skeleton
from late Pleistocene fill. Collected 1974 by B. Lewarne; submitted by
A.J. Sutcliffe, Dept. Palaeontology, British Museum (Natural History) as
part of programme for dating late-glacial and
mammals in British Isles.
corresponds with time of glacial advance and low sea level inferred from
field evidence (Sutcliffe & Lewarne, 1977); human skeletal remains
of possible Pleistocene age were found in 1886 in cave
ca 60 metres away in same Devonian Limestone formation (Worth, 1887; 1888)."
000 years ago. By this time the ice was already on the retreat but the
climate would still have been very severe and the occurrence of the Arctic
Reindeer [Rangifer tarandus] in Devon at this time fits well with
evidence. With vast volumes of the sea water remaining locked up on the
land as ice, the sea would still have been 30 metres (100 feet) or more
below its present level and much of the North Sea would have been dry land.
The Cattewater would also have been dry at this time, except for the waters
of the River Plym and its confluence with the River Tamar, the mouth of
the combined flow of which would have extended further
in the area of the Eddystone Rock.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - -
DATING No. 2....[OxA-17160.
Cattedown Reindeer Rift, UK.] :
April 2005., the Society was approached by Dr. R. Jacobi, a Member of the
AHOB Project, with a request for the Society to submit a sample of the
Cattedown Reindeer skeleton for a more accurate dating procedure,
would involve the removal of only 500 mg. bone material by drilling into
the heart of a bone. As a consequence, on Friday 22 April 2005., the Society
submitted the fossil Reindeer's Right Calcaneum for testing.
was to be sent to the Oxford University Accelerator Unit of their Research
Laboratory for Archaeology and the History of Art. On 11 July 2007., the
Society received a reply.
Oxford Accelerator Unit announced a date of 14 550 ± 55 years B.P.
data-set calibration information is reproduced in the Image to the left.
full data is published in "Archaeometry" journal, about which
we shall give more information when we have it.
the accompanying communication, Dr. Jacobi offered the following information
new radiocarbon determination fits not too badly with the original age
obtained by the British Museum Research Laboratory. Both dates indicate
that your Reindeer lived during the time of extreme cold
the end of the DIMLINGTON STADIAL (26,000 - 13,000 radiocarbon years ago).
Interestingly, we have just obtained a date of OxA-14826 = 14,395.±
60 years B.P.
for an Astragulus of a reindeer
Kent's Cavern, which is almost identical to the Cattedown result and overlaps
with it at 20. The Kent's Cavern date came as something of a surprise as
we had expected the bone to be of younger
1. Detailed Technical Background Information about the
Components of Bone Material and Their Role in the Radiocarbon Dating Technique
is a connective tissue largely composed of an organic protein; collagen
and the inorganic mineral hydroxyapatite, which combine to provide a mechanical
and supportive role in the body. Depending on the orientation of collagen
fibres, two types of bone can be
lamellar bone (cortical bone) and non lamellar (trabecular or cancellous
bone), which is found in vertebrae, at the ends of long bones, the mammalian
foetus, at fracture joints, and in many lower vertebrates. Three types
of cavities exist within the bone
Haversian canals, marrow cavities and the lacuna, which contain the bone
cells (osteocytes) from which canalicuae (small tunnels) extend. A number
of different bone extracts have been used to obtain Carbon-14 determinations.
Initially analyses were carried
on whole bone, later the organic (collagen) or inorganic component of bone
(hydroxyapatite) were separated and dated. A lack of success saw the dating
of different fractions obtained during bone pretreatment, including the
acid soluble (humics) as well as the
(acid insoluble component). More recently Carbon-14 determinations have
been carried out on mixtures of amino acids; specific amino acids, for
example hydroxyproline and proline; a series of individual amino acids;
larger or smaller (peptide) parts of
and non-collagenous proteins such as osteocalcin.
Some Extracted Fractions of Bone :
30% of bone is composed of organic compounds, of which 90 to 95% is collagen,
the rest being non-collagenous proteins. Collagen is a fibrous protein
which provides the bone with strength and flexibility, and is an important
component of many other tissues,
skin and tendon. Individual collagen molecules contain three polypeptides
of about 1000 amino acids per chain with a high glycine and hydroxyproline
content. Bundles of these collagen molecules are arranged in fibrils with
a molecular weight close to 97.1
These fibrils are twisted into a right handed coil (fibre) with a total
weight of between 95 000 and 102 000 Daltons.
the collagen fibre to fully mature a number of chemical bonds must form.
These include hydrogen bonds involving hydroxyproline, which stabilise
the helix, and cross linkages involving hydroxylysine and lysine, which
stabilise the fibrillar structure. These processes
throughout the growth and maturity of an individual, consequently the density
and stability of the bone tends to increase while the solubility decreases.
Once these bonds form only a small fraction of collagen can be extracted
by neutral salt solutions and organic acids or acid-citrate buffers. The
insoluble collagen which remains from such dissolutions, can however, be
solubilised by heating above 58'C. At this temperature the triple helix
denatures, but will partly reform into a gel when cooled.
in its unaltered state is also very resistant to proteolytic enzymes, however
a group of enzymes exist which degrade native collagen fibrils under physiological
conditions of temperature and pH; these are the collagenases. An enzyme
secreted by the gas
bacteria (Clostridium perfringens and Cl. histolyticum) and
melaninogenicus, a bacterium common in the gingival crevice of the
tooth, will also cleave the triple helix. The peptide's produced in such
cleavage are then open to proteolytic attack
the more conventional enzymes.
AMINO ACIDS :
molecules are composed of linear, unbranching sequences of approx 20 naturally
occurring amino acids. The structure of the molecule is stabilised by hydrogen
bonds; the most common being between the amino group (-NH2) of one residue
and the carboxyl
(-COOH) of a second residue, resulting in both acidic and basic properties.
Uncharged side-chains also interact with one another, but by excluding
water from their mutual interfaces (i.e. hydrophobic reaction). All amino
acids, except glycine, exhibit optical
existing in the natural state as laevo-rotary compounds, a property apparently
restricted to amino acids of a biological origin, a property which is exploited
in amino acid racemisation dating.
general the composition of mammalian collagens shows little variability.
Of special significance to recent AMS works is the amino acid hydroxyproline.
Hydroxyproline is found rarely in other proteins but comprises about 10%
of all amino acids in collagen. However,
a practical point of view the use of hydroxyproline for Carbon-14 analysis
is limited as it does not occur in large quantities in fossil bones, has
been detected in natural waters, is excreted in the urine, and can be found
in some plants.
percent of bone is made up of the inorganic mineral hydroxyapatite, which
includes calcium phosphate, calcium carbonate, calcium fluoride, calcium
hydroxide and citrate. This inorganic component [Ca3(P)4)2]3.Ca(OH)2
is predominantly crystalline, though
be present in amorphous forms. The crystals are platelets or rods, about
8 to 15Å thick, 20 to 40Å wide and 200 to 400Å long. The substitution
mechanisms that occur in the hydroxyapatite of bone include intercrystalline
exchange and a recrystallisation due to
and reformation of crystals, with the addition of new ions into the crystal
structure replacing Ca²+ or being adsorbed on the crystal surfaces.
tooth is constructed of three layers; the pulp cavity, containing blood
vessels and nerves; this is covered with the dentine; where the tooth is
exposed the dentine is covered by enamel; and the submerged roots are covered
with cementum. The cementum closely
cortical bone in composition, except that dentine is hard and dense, being
almost 75% mineral but with a higher collagen content than bone (30% compared
to 15% in bone). The enamel is denser and harder and is almost 98% mineral
crystals being much larger than those of dentine, cementum or bone and
consequently more resistant. Fully formed enamel contains a small amount
of low-molecular weight peptides that are almost devoid of proline and
Bone Weathering :
attention has been given to the environmental conditions of bone preservation.
However, the quantities and composition of surviving organic materials
in a specimen are dependent on their burial environment. Environmental
factors which have been suggested
influencing the rate at which collagen degrades include the composition,
pH and hydrology of the matrix; oxygenation; temperature; and changes brought
about by soil flora and fauna.
a generalised view of bone degradation the protein component undergoes
relatively slow hydrolysis to peptides, which then break down into amino
acids. At the same time there is spontaneous rearrangement of the inorganic
crystalline matrix which weakens the
bond and leaves the bone susceptible to dissolution by the action of internal
and external agents. Alterations during diagenesis are believed
to include random cross-linking, humification of parts of the molecule,
attachment of exogenous humic
and hydrolysis with preferential loss of some amino acids.
major groups of contaminants exist; humic and non-humic substances. Humic
substances are dark coloured acids moderately high-molecular-weight polymers
of indefinite structure. They represent an extremely heterogeneous mixture
of molecules, including amino
which, in any given soil or sediment may range in molecular weight from
2,000 to over 3 000 000 and have a range of properties depending on size
and attached functional group. While humic substances are generally insoluble
in acid and soluble in alkali, one
the fulvic acids, are soluble in both mediums making them extremely difficult
to separate from collagen. Non-humic substances include all classes of
organic compounds. The major contaminants are polyphenols, polysaccharides,
lignins as well as degraded
and other broken down bone components.
Radiocarbon Dating :
first radiocarbon measurements on bone were on naturally burned bone.
Soon after Libby (1952) stressed concern over the low organic
carbon content, porous structure and possible effects of putrefaction and
chemical alteration on the bone. Only two samples of
bone had been measured at this time, and both gave young dates. Consequently,
while there had been little work in this area, bone did not appear in Libby's
1952 listing of suitable sample materials, though burned bone was ranked
alongside charcoal at the top.
the obvious importance of bone to the chronology of many sites saw a continued
interest in bone as a dating medium. The major problem was traced to the
use of whole bone to generate Carbon Dioxide for Carbon-14 measurements,
both carbonates and organics could enter the date. Initial efforts to remove
the indigenous organics from the bone included techniques such as the artificial
pyrolysis of bone by May (1955) whose process was designed
to minimise loss of residual organics, acid
and dialysis and the gelatinization of "collagen". Despite these attempts
problematic dates still persisted. In a review of the literature up to
1960, Olson noted that bone dates were most often rejected.
Proof that humates were the predominant contaminant in
bone was finally given by De Vries. A variety of techniques
were developed to remove this matter: Initially the pre-treatment procedure
used on charcoal was adopted whereby decalcified bone is extracted with
0.1 to 0.5 M NaOH; conversion of the sample
gelatin by Longin (1971); and later Protsch
(1975) combined the HCl, NaOH and gelatinization steps. This is the general
"collagen" extraction procedure used today in carbon dating and dietary
the mid-70's a number of reviews and evaluations of bone dating were being
undertaken. One group at the Uppsala laboratory proposed the use of different
fractions (acid soluble and acid insoluble) for the majority of bones on
the basis that it would be improbable
contaminants to cause the same error in different fractions. Unfortunately
the yields from the different fractions were often insufficient for conventional
descriptions of experiments demonstrating the feasibility of accelerator
or cyclotron-radiocarbon-based isotopic measurements appeared in 1977.
The advent of AMS enabled dating of small amounts of material, of material
with very low organic carbon content,
multiple Carbon-14 determinations of different organic fractions. While
this was a clear advantage in the dating of bone, the use of smaller samples
required a clearer separation of the organic and inorganic portions. Therefore,
more emphasis had to be placed on
purity of the sample.
exchange chromatography had initially been introduced for the dating of
problem samples. The large sample sizes and excellent preservation of collagen
in the bones at the La Brea tar pits, California, made these Carbon-14
measurements possible, but proved
be too expensive and impractical for the large samples required with conventional
dating methods. The advent of AMS changed this. Initial chromatographic
techniques involved the hydrolysis of the extracted "collagen", but the
incomplete removal of humic acids
gelatinization, alkali and acid treatments often resulted in cross linkages
with residual impurities when hydrolysed. Attempts to remove humates prior
to hydrolysis using XAD resins and decolourising charcoal still
failed to remove exogenous amino acids associated
soil contaminants. More recently to aid in the understanding of the series
of reactions that can take place during diagenesis and pretreatment, van
Klinken (1994) has used sample yields during enzymatic cleavage
to screen the degree of cross linking.
counteract possible contamination products, techniques based on the molecular
weight and size of the collagen molecule have been used. Brown, Nelson,
Vogel and Southon (1988) modified the Longin method
of "collagen" extraction by adding an ultrafiltration
(gel electrophoresis) designed to exclude low molecular weight species.
Another approach developed to purify collagen for stable isotope analysis
involves the use of collagenase, which preferentially isolates tripeptides
of known length from the surviving collagen
attempts have concentrated on identifying relatively uncontaminated parts
of bone. The isolation of "aggregates" which were identified as having
potentially a better protected environment for collagen survival was undertaken
by DeNiro and Weiner, but do not
reliable results from bone with a low collagen content. Recently several
researchers have noted the use of non-collagenous components for dating
seriously degraded bone. Long, Wilson, Ernst, Gore and Hare
have suggested that phospho-proteins may be
from degradation as they bond to the apatite structure. Gillespie noted
the existence of osteocalcin, osteonectin and other phosphoproteins, proteoglycans,
and glycoproteins as well as blood proteins, which may display differential
survival characteristics to
first suite of Carbon-14 measurements of a non-collagenous protein were
undertaken on osteocalcin by Ajie, Kaplan, Slota and Taylor
Osteocalcin makes up 1% of total bone protein and appears to bind tightly
to hydroxyapatite, suggesting a good
of being protected from contamination. Further, it has not been detected
in many species of bacteria, plants or invertebrates. However, osteocalcin
values on two skeletons from the Haverty site (Los Angeles) gave disproportionably
old values, which if correct
signify the oldest human remains in the western hemisphere. It may be that
for osteocalcin to be a suitable medium, isolation of essential amino acids
may need to be performed.
Carbon-14 studies using the inorganic or carbonate fraction of bone were
in most cases clearly false, usually too young. Haynes investigated the
reliability of using the bone apatite fraction and concluded that erroneous
apatite dates can result from carbon
in the apatite structure during recrystallization, and/or surface exchange
reactions. Studies into separation of the in situ primary apatite
fraction from diagenetic carbonates were initiated in the 1960's and 1970's.
and Banewics (1980) reported more
results, and the demonstration that careful etching with acetic acid can
enable the residual carbonate to maintain a biogenetic d13C signal suggests
possibilities. But no-one has so far demonstrated that the indigenous carbonate
can be extracted reliably
separated from diagenetic carbonate.
results have generally been obtained from teeth, though Carbon Dioxide
exchange with the atmosphere may be more efficient in teeth than initially
thought. Recent studies on Carbon Dioxide from teeth do, however, indicate
that secondary carbonates may be
from stable isotope values, suggesting that reliable Carbon-14 determinations
may possibly be obtained on tooth enamel.
The AHOB Project Approach to Carbon Dating :
AHOB (= ANCIENT HUMAN OCCUPATION OF BRITAIN) Project, funded by the Leverhulme
Trust, is using a new improved methodology developed by project workers.
Analytical chemistry of Bone - the search for Collagen :
it became obvious that the state of preservation of collagen is vital for
Carbon-14 accuracy, researchers began to examine biochemical indices that
might be useful in characterising collagen. Those "finger-prints" which
have been adopted to assess the degradation
bones include measurement of the nitrogen content of bone, stable carbon
and nitrogen isotopes and the nitrogen/carbon ratio, a collagen like amino
acid pattern, the presence and relative concentration of hydroxyproline,
infra red spectra and tests for metal ions
from humic contaminants.
COLLAGEN CONTENT :
can be estimated by percentage nitrogen in the whole sample, or by measuring
the nitrogen content in the decalcified extract. Fresh, dry, defatted,
compact bone from large mammals contains on average between 4 and 5% organic
nitrogen by weight,
variations do occur depending on maturity and size of mammal. However,
such measurements neither indicate if the nitrogen is wholly present as
collagen, nor the extent of non-nitrogenous organic material.
CARBON AND NITROGEN ISOTOPES :
basic assumption in the stable or radiometric isotope analysis of bone
is that collagen is thought to retain Carbon-13 / Carbon-12 and Nitrogen-15
/ Nitrogen-14 values postmortem even though collagen is known to degrade
with time after death. However, as each
acid has a unique isotopic value, diagenesis of collagen will theoretically
alter the isotope value of the resulting organic fraction, while
humates also have an effect on the isotopic composition of bone depending
upon their concentration, Carbon-13, Carbon-14
Nitogen-15 compositions. In some cases, the traditional pre-treatments
(i.e. HCl, EDTA, NaOH and gelatinization) may further change the observed
isotopic values, though ion exchange chromatography does not seem
to cause any major variations.
/ NITROGEN RATIO :
values can be taken either on the whole bone or from an extract. Carbon/nitrogen
values of 2.9-3.6 from gelatinous extracts of bone are though to be indicative
of collagen with diagenetically unaltered carbon and nitrogen values, while
>>4) indicate extensive diagenesis, or a high proportion of exogenous carbon
possibly from sample preparation, non-collagenous proteins or contaminants.
studies have investigated the possibility of using amino acid composition
and/or racemisation values as a means of characterising indigenous organics
in bone samples. Some workers suggest that the absence of the collagen
amino acid signature indicates the
of contamination. Others have suggested that in some cases where the organic
content is extremely low (below 0.4 to 0.1% N), the amino acid pattern
may reflect the indigenous non-collagenous protein residue rather than
contamination. A number of factors
also alter the collagenous amino acid "finger-print": The different pretreatments
effect the total amino-acid composition of the bone, while differential
loss of amino acids and peptides may occur during diagenesis due to differences
in solubility, effect of
and susceptibility to oxidation or deamination, to name a few. Attempts
to identify a non-collagenous composition has seen the use of the Gly/Asp
ratio. Glycine is abundant in collagen, whereas aspartate is abundant both
in bone non-collagenous proteins
in most (including bacterial) protein, and therefore discrepancies in the
relative amounts of each are a sensitive test for contaminants.
IR spectroscopy has been used to estimate the purity of the protein under
analysis, as well as to assess the degree of recrystallization of hydroxyapatite.
However, with archaeological materials complex spectra may be obtained
due to diagenesis and
so at present this technique cannot identify impurities less than the >5-10%
BEAM ANALYSIS :
of light elements (F, N, P and Na) and trace metals using X-ray spectroscopy
has been done by Redvers-Newton and Coote (1994)
in order to identify the presence of metal complexes which form in the
presence of humic materials. Again the complex spectra
be obtained and due to diagenesis and exogenous organic matter.
these analytical techniques for collagen assessment have met with
only limited success, depending on the preservation state of the bone itself.
In an attempt to achieve a better chemical characterisation of the fraction
selected for dating Stafford, Brendel and
(1988) used a number of these criteria to classify bone preservation. Unfortunately,
there is currently no consensus as to bio-geochemical methods which can
be routinely used in bones exhibiting very low or trace amounts of collagen
(i.e. lost >95% of their
As a consequence Hedges and van Klinken suggest
an age limit of 18ka as older dates are more sensitive to modern contamination.