treatment methods applied to sample preparations for

Treatment Methods Applied to

Sample Preparations for Carbon-14 Determinations

Dr Sophie CERSOY

Muséum national d’Histoire naturelle, Paris (France)

IAEA seminar - TC Project RAS1025 - Enhancing the Capabilities of Radiocarbon Dating in Archaeological Applications (ARASIA) – 3rd Sept 2020

Plan of the seminar


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Principle of 14C dating

Sample contamination

Sample treatment methods

Effect of burial environnment

Collagen characterization

Data processing

Pr. Willard F. Libby (right), the physical chemist

who conceived of 14C dating(University of Chicago Library)

Pr. Hessel de VRIES, "the unsung hero of radiocarbon dating"

(Université de Gröningen)


Carbon (C) = a chemical elements, with more than one type of atom, called isotopes.

98.99 % 1.11 % 0.000000000001 % Content on earth

UNSTABLESTABLESTABLE

Carbon-12 (12C)

Carbon-13 (13C)

Carbon-14 (14C)

protons

neutrons

electrons


1



14N + 1 neutron 14C + 1 proton

Carbon-14 production

Cosmic rays

In the upper atmosphere (stratosphere) :



Carbon incorporation into living organisms

Photosynthesis

Feeding

Troposphère

CO2

Breathing



After death….

End of CO2

exchanges

The amount of 14C decreases over time: it is a natural process

called radioactive decay

CO2



Radioactive decay of carbon-14

Date of death

14C 14N + 1 electron + 1 anti-neutrino

14C12C

Time elapsedsince death

« Cambridge half-life »of radiocarbon

Regular math law !



Radioactive decay of carbon-14

Regular math law !

Date of death

14C 14N + 1 electron + 1 anti-neutrino

14C12C

Time elapsedsince death

VALID IF the amount of 14C in the atmosphere (t=0)is constant over time !!

« Cambridge half-life »of radiocarbon



Need for calibration

IntCal 13 radiocarbon

calibration curve

The quantity of carbon-14 in the atmosphere is NOT constant over time, in particular because of the activity of the sun and fossil fuel combustion

From : https://c14.arch.ox.ac.uk/oxcal/OxCal.html.

Result of the measurementin year "Before

Present“(BP = before 1950)

Calibrated date = calendar age

(in calBC or calAD)

Without calibration



Which archaeological materials can be dated ?

First requirement : Carbon-containing materials

Bone, ivory, sinew, antlers, teeth Shell, coral, otholith Horn, feather, nails, beak, claws

Hair, textiles like silk, wool, fur, leather, parchment, paper

Insects Wood, charcoal

Seeds, plant remains,

…

and all objects made of these materials!

Fats, peat, wax, lead white,

carbon black…

Second requirement : Materials less than 50,000 years old



-50 000 -40 000-30 000 -20 000-10 000 0

Neolith

ic

AN

TIQ

UITY

Homo Sapiens in

Europe

Pleistocene Holocene

Time

14C dating

Lascaux Cave

First burials

Paleolithic

PREHISTORY

MID

DLE

AG

ES

, M

OD

ER

N

an

d C

ON

TEM

PO

RA

RY

ER

A

Dendrochronology

Uranium-Thorium dating


Second requirement : Materials less than 50,000 years old



14C dating


Representativeness You date what you have chosen to sample !

Archaeological interpretationYou date the death of the living organism and not the date of use of the object.

Association of the sample dated with the archaeological event of interest ?



2

Main issue : Dating carbon that was present in the living organism

valid if all exogenous sources of carbon are removed !

From : (Wood, 2015)

80 14C yroverestimation

Effect of contamination on

a radiocarbon date






a radiocarbon date

From : (Wood, 2015)

80 14C yroverestimation

14 500 14C yrunderestimation

5 14C yrunderestimation

For accurate dates, contamination needs to be reduced to 0.1%





Challenging

From : (Wood, 2015)

PleistoceneHolocene


a radiocarbon date



What are the sources of contamination?

Soil organic matters, bacterial contamination

Aromatics

Alkanes/alkenes

Cholestanes

Alcohols/ketones

Bone sample from the Neolithic site of Bercy, France Chemical analysis of the composition of humic acids

(Py-GCxGC/MS)

From : (Cersoy, 2018)

• Buried material• Humification process (from plants mainly)• Dark coloured humic substances (humic acid, fulvic acid and humin)




Exogeneous carbonate

• Secondary carbonaceous material (diagenetic and non-biogenic)• Deposited on or in the material to be dated, after its fossilization

Atmospheric CO2

DissolvedCO2

E.g. : Leaching of bones by groundwater

Precipitation of « modern » calcite in the pores of the bone

Isotope exchange between the bone's carbonate and the dissolved carbon

Dissolution/reprecipitation of apatite




Carbon-based exogeneous products (anthropogenic)

Preparation or decoration of the material for its use

Preservation/restoration additives

Organic dyes

Plant residues in mummy hair

From : (Fresnais, 2015)

Bone preservatives recommendedby archaeologists since 1904

From : (Johnson, 1994)




Lab/ modern contamination

Filters and ultrafilters

= Keratin (from hair, woolen clothing, eyelashes, eyebrows), fats (on fingers)

= with organic membranes

Archaeologists and lab staff

© R. Barrois

© N. Goepfert

3 Sample treatment methods


From sampling to measurement

Sampleselection

Mechanicalcleaning

Chemical treatments / purification

Carbon isolation (combustion and graphitization)

Counting the number 14C atoms

(AMS)

Sample Cleanedsample

Endogenouscarbon phase

CO2 then C (graphite target)

Uncalibrateddate



From sampling to measurement

Sampleselection

Mechanicalcleaning

Chemical treatments / purification

Carbon isolation (combustion and graphitization)

Counting the number 14C atoms

(AMS)

Sample Cleanedsample

Endogenouscarbon phase

CO2 then C (graphite target)

Uncalibrateddate



Conventional AAA treatment

• Widespread• Effective for most samples (e.g. charcoal) • Basis of more complex treatments

Acid step

(A)

Alkali step

(A)

Acid step

(A)

Removeexogenous

carbonates and some fulvic acids

Removehumic acids

Effect : Remove somefulvic acids

and dissolvedC02

Variations between

protocols :

Choice of acid, concentration and duration

Optional Choice of pH, temperature and duration



Conventional AAA treatment

Tips and practical precautions

• Wear lab coat, nitrile gloves, safety goggles, closed shoes and outfit covering the legs

• Use dedicated glasswares baked out at 450-500°C for a minimum of 3 hours

• Carefully rinse between each wash (at least 3 times and ideally with ultrapure water)

• Separated soluble and insoluble phases by centrifugation, decanting or using pre-

cleaned filters (e.g. Ezee filters from Elkay, Millipore nitrocellulose filters, glass filters)

• Monitor the pretreatment to adjust the protocol if necessary (duration)

• Treat the samples in batches with known-age standards and/or similar material for

quality controls and to improve data correctionKnown age VIRI E mammouth sample



Case of charcoal : MNHN protocol

Acid step

(A)

Alkali step

(A)

Acid step

(A)

Hydrochloric acid(HCl) 1 M

20°C1h at least

(Several times)

Sodium hydroxyde (NaOH) 0,0005M - 0,03M

20 °C10 to 30 minutes

Several rinseuntil pH 7

Several rinseuntil pH 7

Hydrochloric acid(HCl) 1 M

20°C30 minutes

1) Sorting :Isolate charcoal samples

from sediment, plant remains and rootlets

2) Weighing :Weigh at least 10-20 mg of sample

4) DryingAt 50-70°C , one night

3) Chemical treatment :



Bleaching : for wood, parchment and plant remains

Bleaching

Sodium chlorite (NaClO2) pH 3

2.5–5.0% w/v70 to 80 °C

Up to 30 min

• Remove lignins, waxes, and resins• Concentration, temperature and duration MUST be

adjusted depending on the fragility of each sample !

Acid step

(A)

Alkali step

(A)

Acid step

(A)

Hydrochloric acid(HCl) 0,5 to 1 M20°C to 80 °C20 minutes

Sodium hydroxyde (NaOH) 0,2 M20 °C to 80 °C

20 minutes

Hydrochloric acid(HCl) 0,5 to 1 M20°C to 80 °C

1 h

From : (Brock, 2010)



Case of keratin samples : hair, wool, horn, nails, claws

Idea: extract keratin from the inner part of the hair (cortex)

Keratinextraction

Dithiothreitol (DTT), 10% in sodium

dodecylsulphate (SDS), and

Tris–HCl buffer50 °C5 – 7 d

Acid step

(A)

Alkali step

(A)

Acid step

(A)


20 minutes – 1h

Sodium hydroxyde (NaOH) 0,005 – 0,1 M

20 °C to 60 °C20 minutes – 1h


1 h

• Wear hairnet (to avoid modern hair contamination !)• 50- 100 mg of sample for hair

From : (Richardin, 2011)

Keratinprecipitation

Dichloroacetic acid (DCO), 2 %20°C

15 minThen trichloroacetic acid (TCA)

1hThen acetone

15 min



Case of collagen samples

What’s in a bone ?

80 % Mineral phase

- Hydroxylapatite

- Carbonates

Collagen17 to 18% of the material

20 % Organic phase

- 88% collagen (type I) = a protein

- 8% glycoproteins

- 3% non-collagenic proteins(osteocalcin, osteonectin)

- 1% lipids and sialoproteins



Case of collagen samples : protocols for bone, teeth, ivory

Purification

• Dating the organic phase of bone is more reliable• About 20 protocols for this phase proposed over the last 50 years

Demineralization

(A)

Contaminant removal

(A)

Gelatinization

(A)

Removeexogenous

carbonates and some fulvic acids+ remove the

mineral part of bone

Removehumic acids

Remove somefulvic acids

and dissolvedC02

+ collagensolubilization

Isolate pure collagenpeptides



Case of collagen samples : protocols for bone, teeth, ivory

PurificationDemineralization

(A)

Contaminant removal

(A)

Gelatinization

(A)

Removeexogenous

carbonates and some fulvic acids+ remove the

mineral part of bone

Removehumic acids

Remove somefulvic acids

and dissolvedC02

+ collagensolubilization

Isolate pure collagenpeptides

Hydrochloric acid(HCl) 0,2 to 2 M

4°C, 20°C or 80 °C20 minutes – 2 days

EDTA20°C

Several days/weeks

or

Sodium hydroxide(NaOH) 0,1 M

20°C 0 minutes – 1 night

Hydrochloric acid(HCl) pH 1 to 558 °C to 100 °C

50 minutes – 17 h

Macro (10-100 μm), micro (0,1-10 μm)

and/or ultrafiltration (0,001-0,1 μm)




Case of collagen samples : difficult samples

Consolidants, preservatives, chemical contaminants

Highly contaminatedsamples

Microsamples (< 60 mg)e.g. small vertebrates, precious objects

Solventextraction

Acetone 40°C, 30-60 min

+ Methanol 40-50 °C,

30-60 min+

Chloroform 20°C, 30- 60 min

(ultrasonication)

« Soft » protocol

1. Demineralization : HCl 0.2 M, 4°C, 2- 4 d

2. Contaminant removal : NaOH 0,1M, 4°C, 2-4 d

3. Solubilization :pH 1, 1h, 90 °C

4. Purification : glass microfilters


Single aminoacid dating

Idea : isolatinghydroxyproline,

an amino acid foundmostly in collagen

preparative high performance liquid chromatography (HPLC)

From : (Brock, 2018) From : (McCullagh, 2010)

© J Rofes



Contamination

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• Cross-linking between collagen and organic acids from the soil (within hours)

• Difficult to study

• Uptake until circa 20 %

• « Complexes » partly dissolved during gelatinization and basic washes

From : (van Klinken, 1995)



Collagen diagenesis

Collagen denaturation

The mineral matrix may protect collagen from degradation in the burial environment BUT

Hydrolysis with preferential loss of some amino acids

Collagen polypeptide chains

Breaking of weak bonds

Breaking of peptide bonds

Free amino acids(possibly modified e.g. deamidated asparagine or glutamine)

Degradation process favoured in hot and humid environments



Quality control of extracted collagen

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Yield

Carbon content and C/N ratio

Is the extracted collagen suitable for 14C measurement ?

- Weigh the cleaned bone before extraction- Weigh the "collagen" extracted after drying. -Calculate the ratio of the two

< 1 % : unsuitable !

%C <30 % or %C >50% of the weight of the collagen : unsuitable !C/N <2,9 : too degraded or C/N > 3,5 : contaminated : unsuitable !

Isotopic ratio mass spectrometry (IRMS)



Quality control of extracted collagen

Proteomics

Py – GCxGC /MS

Is the extracted collagen suitable for 14C measurement ?

Soft ionization mass spectrometry techniques (MALDI-MS or LC-MS/MS)

If no endogenouspeptides detected:

unsuitable !

Pyrolysis 2-dimensional gas chromatography coupledwith mass spectrometry If contaminants

identified/ if collagen fingerprint

is missing : unsuitable !



Upstream quantity control

ATR- FTIR

FTIR : Fourier TransformInfra-Red (spectroscopy)

ATR : Attenuated Total Reflectance

Estimation of the preservation of the organic phaseFrom 1 mg of crushed bone sample !

From : (Lebon, 2016) and (Rofes, 2020)

Mineralband

Organicband

amide I/PO4 α % collagen in the bone !



Upstream quantity control

Collagen preservation (%)

1 mg of C

Estimation of the minimum quantity of bone to be sampled

From : (Rofes, 2020)

Well-preservedBadly

preserved

Data processing


Measurements presentation

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To allow further comparison and quality control, please provide on publication/reports :

• Yield and C/N ratio

• Collagen amount and carbon mass

• Raw result = 14C age (BP) : to allow the date to be recalibrated in the future

with a more accurate calibration curve

• Lab code = traceability which allows to go back to details about pretreatment

Data processing


Calibration

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Result of the measurement in year "Before Present" (BP = before 1950) with an associated margin of imprecision (±).

Exemple 1Measure : 22 920 ± 80 BP

After calibration : the age of the dated mammoth has a 95.4% chance of being

between 25 521 and 25 116 B.C.

OxCal program can be found here : https://c14.arch.ox.ac.uk/oxcal.html

Calibration curve Calibrated date

Plateau

Exemple 2Measure : 2500 ± 30 BP

After calibration : the age of the datedhuman has a 95,4% chance of being

between 788 and 537 B.C.

Thanks for yourattention !

Listening your questions

ReferencesStuiver, M. (1961), Variations in radiocarbon concentration and sunspot activity, J. Geophys. Res., 66( 1), 273– 276

Suess, H.E. (1955). Radiocarbon Concentration in Modern Wood. Science, 122(3166), 415-417.

Reimer, P., et al. (2013). IntCal13 and Marine13 Radiocarbon Age Calibration Curves 0–50,000 Years cal BP. Radiocarbon, 55(4), 1869-1887.

Waterbolk, H.T. (1971). Working with radiocarbon dates. Proc. Prehist. Soc. 37, 15e33.

Wood, R. (2015). From revolution to convention: the past, present and future of radiocarbon dating. JAS, 56, 61-72.

Klinken, G. J. Van et al. (1995) Experiments on Collagen-Humic Interactions: Speed of Humic Uptake, and Effects of Diverse Chemical Treatments. J. Archaeol. Sci., 22, 263–270

Cersoy, S., et al. (2018). Pyrolysis comprehensive gas chromatography and mass spectrometry: A new tool to assessthe purity of ancient collagen prior to radiocarbon dating. Analytica chimica acta, 1041, 131–145.

Stevenson, F.J. 1982. Humus chemistry genesis, composition, reactions. Willey Inter science, New York.

Zazzo, A., Saliège, J.-F. (2011). Radiocarbon dating of biological apatites: A review. Pal., Pal., Pal., 310 (1–2), 52-61.

Fresnais, M. et al. (2015). Recent advances in the characterization of hair of mummies from the Chilean Andean coast. Forensic Science International, 249, 25-34.

Johnson, J.S. (1994). Consolidation of Archaeological Bone: A Conservation Perspective. Journal of Field Archaeology, 21(2), 221-233.

Huls, C., et al.(2009). Ultrafiltration: Boon or Bane?. Radiocarbon, 51(2), 613-625.

References

Brock, F., et al. (2010). Current Pretreatment Methods for AMS Radiocarbon Dating at the Oxford RadiocarbonAccelerator Unit (Orau). Radiocarbon, 52(1), 103-112.

Richardin, P., et al. (2011). A new protocol for radiocarbon dating of hair and keratin type samples—application to an Andean mummy from the National Museum of Natural History in Paris. Archaeol Anthropol Sci 3, 379–384.

Cersoy, S., et al.(2017). Collagen Extraction and Stable Isotope Analysis of Small Vertebrate Bones: A Comparative Approach. Radiocarbon, 59(3), 679-694.

Cersoy, S., et al. (2017) Radiocarbon dating minute amounts of bone (3–60 mg) with ECHoMICADAS. Sci Rep 7, 7141.

Fewlass, H., et al. (2019). Pretreatment and gaseous radiocarbon dating of 40–100 mg archaeological bone. Sci Rep9, 5342 (2019)

Brock, F., et al. (2013). Analysis of Bone “Collagen” Extraction Products for Radiocarbon Dating. Radiocarbon, 55(2), 445-463.

van Klinken, GJ. (1999). Bone collagen quality indicators for palaeodietary and radiocarbon measurements. JAS26(6):687–95.

Lebon, M., et al. (2016). Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy. Radiocarbon, 58(1), 131-145.

Rofes, J., et al. (2020), Detecting stratigraphical issues using direct radiocarbon dating from small‐mammal remains. J. Quaternary Sci, 35: 505-513.

Questions about quality assurance procedures

• Organised by SUERC/Glasgow University • Long-running program of inter-laboratory quality

control exercises undertaken by the 14C community (e.g. TIRI, FIRI, VIRI, SIRI in 2003; 2010 and 2017)

• Liquid scintillation (LS) and gas counting (GC) laboratories, as well as AMS laboratories

• Results analysed and published by E. Marian Scott (School of Mathematics and Statistics, University of Glasgow) and colleagues

International intercalibration studies

MNHN procedure

• Including know-age references samples in batches : same material, sameage, if possible international intercalibration samples, prepared the sameway as the real samples

• Including blank (14C free) in the batches : e.g. Phtalic anhydride• Including standard (modern 14C) in the batches : e.g. oxalic acid II NIST• Handle blanks and standards with dedicated tools (no contact with samples

or solvents)• Treatment of samples, weighing and handling on clean benches, covered

with aluminum foil like glasswares

Reference bone samples from the FifthRadiocarbon Inter-comparison (VIRI) :

Pleistocene mammoth bone from the Yukon Territory, Canada (VIRI E, 38772± 2532 BP). Consensus values for the ages made on 40

measurements (42 laboratories)

treatment methods applied to sample preparations for

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