Collagen Extraction

This procedure is performed on bones (not incinerated nor burnt), teeth, ivory, and antler. The Longin method (Longin Reference Longin1971) is used with additional steps. Before starting the procedure for collagen extraction, it is necessary to clean mechanically the samples with a Dremel® rotary tool equipped with a diamond cut-off wheel. It involves removing the surface to eliminate the porous parts, which can be source of contamination. For bones, the cortical part is preferably selected because it is often less porous (less contaminant can be absorbed). For teeth, only the part where dentine is present is taken for collagen extraction. Only the dentine part of the tooth contains collagen whereas enamel does not.

Between 0.5 and 1 g of sample is required which is broken into pieces measuring a few millimeters in length and width to increase the treatment surface and to fit inside round-bottom plastic tubes (16 × 100 mm) in order to use Ezee™ syringe filters (polypropylene with a polyethylene filter and a 60–90 µm pore size) for the demineralization process. Beforehand, the Ezee™ syringe filters are cleaned three times with Milli-Q™ water. The pieces are immersed in a 2.4 M HCl solution for 15 min and the HCl is removed using Ezee™ syringe filters. Then Milli-Q™ water is added and removed using Ezee™ syringe filters, this is repeated three times to rinse the sample thoroughly. This step also helps in eliminating some organic contaminants (like fulvic acids) and breaking some collagen hydrogen bonds for the further solubilization in water (Longin Reference Longin1971). To eliminate any other contaminants such as humic acids (Arslanov and Svezhentsev Reference Arslanov and Svezhentsev1993), the sample pieces are placed into a 0.25 M NaOH solution for 15 min and again rinsed with Milli-Q™ water and Ezee™ syringe filters. The pieces are then submerged for a second time in HCl at lower concentration (0.3 M) for 5 min to remove atmospheric CO₂ (which could have been absorbed during the previous step), and to neutralize the base if still present, and then rinsed with Milli-Q™ water. The treated bone fragments are transferred into a Duran® glass tube containing a pH3 HCl solution and left at 90°C for 10 hr. Afterwards, the solution is filtered with a Büchner funnel and a Millipore® glass fiber filter (7 µm pore size, i.e., about 525 kDa threshold) and freeze dried overnight.

The C:N ratio, the collagen yield and the carbon and nitrogen contents are checked, as well as the color and texture of the extracted collagen, to control the collagen quality (Van Klinken Reference Van Klinken1999). Commonly, well-preserved collagen is white and fluffy while degraded collagen appears brown in color and crystalline (Boudin et al. Reference Boudin, Bonafini, Van den Brande and Berghe2017). For modern bones, the percentage of collagen is around 20 %wt and after burial, the collagen content drops. At RICH, bones containing less than 2% collagen are considered as poorly preserved. Nevertheless, we date all collagen samples having a C:N ratio between 2.9 and 3.6 as C:N is used to indicate contamination (Boudin et al. Reference Boudin, Bonafini, Van den Brande and Berghe2017). The degree of preservation is also followed by the carbon and nitrogen contents. They should range between 15.3 to 47 %wt and 5.5 to 17.3 %wt respectively (Ambrose Reference Ambrose1990). All these parameters are taken into account to ensure a reliable date. This method has shown good agreement in inter-laboratory AMS ¹⁴C dating (Kuzmin et al. Reference Kuzmin, Fiedel and Street2018) and for example, in dating skeletons from the same layers (Lull et al. Reference Lull, Micó, Rihuete-Herrada, Risch, Van Strydonck and Boudin2016).

Solvent Procedure

Solvents are used in the case of animal textile (wool, silk), leather, hair, rhino horn and when chemical contaminants such as consolidants and preservatives are present on objects. It is necessary to start by the most apolar solvent. The modus operandi consists of immersing the sample placed in a beaker into an ultrasonic bath during 15 min in n-hexane (twice), then in acetone (twice), then in absolute ethanol (twice) and finally, once in Milli-Q™ water. The sample is finally dried in the oven at 50°C. If the solution is still opaque after a step, it is repeated until the solution is limpid.

In the case of consolidants, it is better to know the composition to find out which solvent would be the best to dissolve it. The customer may know which product was used or it can be determined using Fourier transformed infrared spectroscopy or py-GCMS. In the case of bees wax or unknown varnish, chloroform (stabilized with 0.6% ethanol) is used for 30 min in ultrasonic bath, which can be repeated for 15 min if the solution is opaque, followed by the solvent procedure (as explained previously). When the consolidate is unknown, toluene can also be used twice for 15 min in ultrasonic bath also followed by the solvent procedure.

Further treatment is sometimes required, for samples such as hair (if there is enough material), if the soil matrix contained a lot of organic compounds (e.g. humic acids). A treatment with 0.25 M NaOH (15 min) followed by 0.3 M HCl (15 min) at room temperature can be applied.

As for collagen, following pretreatment, the C:N and the carbon and nitrogen contents are measured to test for remaining contamination and the preservation state. For uncontaminated wool and hair, the C:N ratio is between 3.4 and 3.8 and the carbon and nitrogen contents are respectively around 45 %wt and 15 %wt (Benfer et al. Reference Benfer, Typpo, Graf and Pickett1978; Boudin et al. Reference Boudin, Bonafini, Berghe and Maquoi2016). The silk C:N ratio ranges between 2.9 and 3.4 and the carbon and nitrogen contents between 44.3 ± 2.6 %wt and 16.7 ± 0.7 %wt, respectively (Boudin et al. Reference Boudin, Boeckx, Vandenabeele and Van Strydonck2014). The reliability of the method was shown by the good agreement of the ¹⁴C dates with the presumed historical dates on archaeological silk and wool (Boudin et al. Reference Boudin, Bonafini, Berghe and Maquoi2016).

In the case of felt-tip pen applied on fragile pieces, the labels are removed with a scalpel and the samples are placed in acetone for 15 min in ultrasonic bath. The treatment is repeated if the liquid is not limpid. The sample is then rinsed with ethanol and with Milli-Q™ water.

Nanofiltration

¹⁴C dating of bones is usually performed on the collagen fraction. However, this collagen can contain exogenous molecules, including humic substances and/or other soil components that may have a different age than the bone. Incomplete removal can result in biased ¹⁴C dates. Ultrafiltration of collagen, dissolved as gelatin (molecular weight ~100 kDalton), has received considerable attention to obtain more reliable dates (Brown et al. Reference Brown, Nelson, Vogel and Southon1988; Ramsey et al. Reference Ramsey, Higham, Bowles and Hedges2004). Ultrafiltration is an effective method of removal of low-molecular (<10–15 kDalton depending on the chosen molecular weight cut off of the filter) weight contaminants from bone collagen but it does not remove high-molecular weight contaminants (>10–15 kDalton), such as cross-linked humic collagen complexes. However, comparative dating studies have raised the question whether this cleaning step itself may introduce contamination with carbon from the filters used (Hüls et al. Reference Hüls, Grootes and Nadeau2007, Reference Hüls, Grootes and Nadeau2009). A nanofiltration method was developed at RICH using a ceramic filter to avoid a possible extraneous carbon contamination introduced by the filter. This method is applicable to various contaminated protein materials e.g. collagen, silk, wool, hair. Protein material is considered contaminated if they don’t fall within the following C:N range after the pretreatments explained previously (collagen extraction for bone and solvent procedure and AAA for hair, silk and wool):

• Collagen: C:N ratio between 2.9 and 3.6 (DeNiro Reference DeNiro1985; Ambrose Reference Ambrose1990);

• Wool: C:N between 3.4 and 3.8 (Boudin et al. Reference Boudin, Bonafini, Berghe and Maquoi2016);

• Silk: C:N between 2.9 and 3.4 (Boudin et al. Reference Boudin, Bonafini, Berghe and Maquoi2016).

The first step of this method is releasing the amino acids by hot acid hydrolysis of the contaminated protein. Hereafter, the cross-flow nanofiltration is performed and the molecular weight cut off of the ceramic filter is 368 Dalton. That enables the separation of the amino acids, all smaller than 368 Dalton, and the contaminants that are larger than 368 Dalton e.g. humic substances. The amino acids are collected in the filtrate (permeate) and the contaminants are collected in the retentate. Finally, the collected amino acids after nanofiltration are radiocarbon dated. To verify if the sample quality is improved after cross-flow nanofiltration C:N analysis of the filtrate (permeate) is performed. If the C:N ratio falls within the C:N ranges as described above, the sample is considered uncontaminated and a more accurate ¹⁴C date is obtained. The method is described in detail in Boudin et al. (Reference Boudin, Boeckx, Vandenabeele and Van Strydonck2013, Reference Boudin, Bonafini, Van den Brande and Berghe2017).

Shells (Marine, Freshwater, and Land Snails)

The pretreatment consists of removing the surface with HCl 0.3 or 2.4 M during a few minutes, the time and concentration depending on sample size, weight and state. It is necessary to assess the shell species since it has been claimed, for example, that some burrowing mollusks living in calcareous rocks can absorb old carbon as their local environment has a lower ¹⁴C activity than the surrounding ocean, but in most cases, the ¹⁴C activity in shells is close to the activity of the water where the shells live (Mangerud Reference Mangerud1972). Most of dating problems that arise from shell dating are mostly linked to hard water effect in marine or lacustrine environment for aquatic shells and from ingestion of limestone for terrestrial gastropods (Goodfriend and Stipp Reference Goodfriend and Stipp1983; Douka et al. Reference Douka, Higham and Hedges2010b). Moreover, diagenetic processes can lead to the replacement of the original crystal structure of shell, which often involves carbonate dissolution, recrystallization, and carbon exchange with the burial environment (Brock et al. Reference Brock, Higham, Ditchfield and Ramsey2010; Douka et al. Reference Douka, Hedges and Higham2010a). X-ray diffraction can be performed to assess the calcium carbonate polymorph (calcite and/or aragonite) and whether recrystallization has occurred. Shells made of aragonite can undergo recrystallization processes to form calcite. Knowing the composition of the shell species and determining if recrystallization happened can help prevent dating problematic shells.

Cremated Bones

Cremated bones no longer contain collagen due to the high temperature of burning, since organic matter disappears completely at temperatures higher than 600°C. The mineral part of the bone is dated with the few remaining carbonates. These were certainly subjected to carbon substitution with the wood during the cremation (Van Strydonck et al. Reference Van Strydonck, Boudin and De Mulder2010). However, it has been proven that reliable dates can be measured from cremated bones, since in most cases the wood has a similar age to the cremated individual (Major et al. Reference Major, Dani and Kiss2019).

For the pretreatment, it is first necessary to clean the samples with a soft or steel brush to remove any visible contaminants such as sediments present on the surface. Charred parts (black in color) are also removed if present. The sample is then weighed. If it weighs more than 1.5 g, the sample may be placed in a 2.4 M HCl solution between 1 and 10 min. This step is carried out to remove the surface of the bone where it is more likely that carbon substitution happens (Van Strydonck et al. Reference Van Strydonck, Boudin and De Mulder2009). It also helps remove sediments trapped in the bone. The duration of this first step depends on the sample state. The sample is removed from the solution when approximately one third of its volume is dissolved (the sample is weighed before and after this step). If the sample weighs around 1 g but exhibits a high density, it is possible to place it for less than a minute in the 2.4 M HCl solution or in a 0.3 M HCl solution. After this step, the sample is thoroughly rinsed with Milli-Q™ water and dried at 90°C for 30 min to 1 hr (or overnight at lower temperature). When dry, the sample is weighed and reduced to a fine powder in a ceramic mortar. Then, it is placed in a 0.17 M acetic acid solution for 24 hr, this treatment is performed in order to remove the secondary carbonates possibly present. It is again thoroughly rinsed with Milli-Q™ water and dried between 70 and 100°C during ~1 hr (or overnight). During the rinsing step, around 15% of the sample weight is lost (Rose et al. Reference Rose, Meadows, Palstra, Hamann, Boudin and Huels2019). Therefore, if the sample weighs less than 1 g, it will be ground after the acetic acid treatment to avoid mass loss. The results of radiocarbon dating using this method are in good agreement with the ones from other laboratories (Naysmith et al. Reference Naysmith, Scott, Cook, Heinemeier, van der Plicht, Van Strydonck, Bronk Ramsey, Grootes and Freeman2007; Rose et al. Reference Rose, Meadows, Palstra, Hamann, Boudin and Huels2019).

Bone Apatite

Certain burial environments are not suitable for collagen preservation. In these cases, the apatite part of the bone can be dated. Arid and semi-arid environments are the best conditions for the preservation of apatite (Zazzo and Saliège Reference Zazzo and Saliège2011). The pretreatment performed at RICH consists of grinding the bone (with mortar and pestle) and immersing the bone sample (after mechanically cleaning the surface as explained in the collagen extraction section) into a 0.17 M acetic acid solution at room temperature during 24 hr.

Chitin-Containing Materials

For insects remains, they are placed for 15 min in a beaker containing acetone, in ultrasonic bath and rinsed thoroughly with Milli-Q™ water. Afterwards, they are submerged by a 0.3 M HCl solution for 24 hr at room temperature and rinsed again with Milli-Q™ water. This method was adapted from the one proposed by Tripp et al. (Reference Tripp, Higham and Hedges2004).

Mortar and Lime Burial

In the case of mortars and lime burials, the whitest and softest pieces are carefully chosen to avoid sand, agglomerates and unburnt limestone. The selected pieces are then crushed with a stainless-steel pestle in a ceramic mortar to obtain a fine powder. This process breaks up the porous, soft mortar carbonate while leaving the harder limestone particles intact. The powder is placed in a metal sifter for sieving (succession of 0.5, 0.25, 0.18, 0.1 mm and a collector) and placed in the shaker (EML200 – Haver and Boeker) at intensity 10 for 10 min. The smallest grains of mortar carbonate fragments pass through the coarser sieves and are thus separated from the larger aggregate grains, including the calcite crystals of the unburned limestone. Therefore, the grain size fraction < 0.1 mm is used for dating.