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Methods & Procedures

Samples submitted to Aeon are subjected to a series of physical and chemical processes that isolate the pure elemental carbon from the desired portion of a sample for radiocarbon dating. These processes, collectively called pretreatment, separate contaminant carbon from the desired material. The specific course of pretreatment steps depends on the type and amount of material and its degree of preservation.

Physical selection

Our research scientists carefully examine each sample under magnification and make note of samples that could potentially contain carbon from multiple sources. If necessary, we will contact you to verify the portion of the sample that will yield the best results based on your research objectives. This is especially important when submitting "bulk" samples that may contain a mixture of charcoal, fragments of wood, soil, plant debris, etc., which could yield different ages.

For all samples, extraneous materials (rootlets and other debris) are removed with clean forceps under magnification. Samples are often crushed with a mortar and pestle to increase surface area or sieved to isolate a particular size fraction. A portion of the sample is then selected for chemical treatment, based on appearance.

Chemical isolation

Contamination can come in many forms, from the addition of secondary organic molecules to atmospheric gases that sorb onto the sample carbon. Below we summarize the primary chemical pretreatments that we employ at Aeon. By no means is this list exhaustive. Our procedures are standardized to ensure quality control, but we remain flexible to account for variance or unexpected discoveries in samples that are submitted to our laboratory.

Acid-base-acid (ABA). The ABA treatment of organic materials (plant remains, charcoal, peat, wood) is employed by every radiocarbon laboratory in the world. At Aeon, the ABA treatment consists of the following steps:

Acid only. The acid-only method consists of the first step of the ABA method. This method can be applied to organic materials (plant remains, charcoal, peat, wood) and yields the 14C age of the total organic content of the sample.

Wood cellulose extraction. Cellulose is the most stable chemical component of wood. Other major components are lignins, resins and hemicellulose. Numerous minor compounds are present as well. These less complex molecules are more mobile than the higher-molecular weight cellulose, which once formed, remains in place within the wood structure and does not exchange carbon with neighboring molecules or the atmosphere. Degraded wood samples or those which have been subjected to wet alkaline or other severe conditions can be dated more reliably if only the cellulose is analyzed.

Cellulose extraction is accomplished by additional processing steps before and after ABA pretreatment. Before the first acid wash, the sample is finely shaved or cryogenically pulverized to a powder and soaked in a strong, hot alkali solution. This disrupts the wood structure and renders the various compounds more accessible to the subsequent chemical treatments. Following ABA, the material is bleached in a hot sodium chlorite solution to isolate holocellulose, a mixture of cellulose and some of the heavier molecular weight hemicelluloses. In extreme cases of contamination concern, the holocellulose is further purified to α-cellulose with additional base and acid steps. Finally, the extracted cellulose is rinsed in ultrapure water until neutral and dried.

Acid-base-wet oxidation (ABOX). Charcoal and graphite samples may be treated using a wet oxidation technique developed by Michael Bird and colleagues at Australian National University. This is an aggressive chemical pretreatment technique which consists of the same initial acid and base steps that are used in the ABA treatment, followed by immersion of the base-insoluble fraction in a strong oxidizing solution (0.1M K2Cr2O7 in 2N H2SO4) for as long as the material can survive. This acid-base-wet oxidation technique, known as ABOX, removes more contaminant carbon than the ABA method, but is limited to materials composed of elemental carbon.

Bone pretreatment. This pretreatment is applied to bone, tooth, antler and similar samples. A preliminary selection is made and mechanically cleaned. Any visible surface contamination is removed by scraping or grinding, and the selection is reduced to <0.4 mm powder.

Before further pretreatment is undertaken, elemental analysis is conducted on about 5 mg of the powder to determine the suitability of the sample for radiocarbon dating. Samples with a low nitrogen content (%N < 0.1) may not have enough residual collagen protein for analysis. Those with a high carbon-nitrogen ratio (C:N > 11) may have significant inseparable contaminant carbon. Samples with both figures "out of bounds" are recommended for discontinuation.

The preliminary elemental analysis also allows a good estimate of the sample quantity required to provide sufficient gelatin for dating and the supplemental analyses that are performed for supporting data. If necessary, further material is selected, cleaned and pulverized as above. If past conservation efforts included penetrating consolidants or preservatives, solvent extractions may be required; if so, this is usually done before pulverizing.

The clean bone powder is de-mineralized in dilute HCl. The resultant insoluble protein residue (commonly, "collagen") is isolated by centrifugation and rinsed in UPW until the pH is greater than four. Base-soluble organics, humates, are removed in a bath of 0.1 N NaOH. The collagen is then denatured into gelatin in 0.01 N HCl at a precisely-controlled temperature of 58 to 60 °C. This step usually takes about 40 hours. The hot, liquefied gelatin is then filtered at 0.22 µm. Additional purification steps may include ultrafiltration (30 kDa MWCO) or column chromatography through a bed of polymeric adsorbent resin, depending on the particular characteristics of each sample. The purified gelatin is dried for analysis.

Carbonate preparation. Shells of gastropods, other molluscs, or foraminifera ("forams") are examined closely for detritus and other contaminants that are often hidden inside the whorls. Long-stemmed shells are broken and inspected as sediments tend to become trapped in the spire. The sample is repeatedly sonicated in ultrapure water until all visible extraneous matter has been washed away. Following this, the carbonate is soaked for several hours in a hot H2O2 solution to destroy persistent organic residues. The bleached material is thoroughly rinsed. Secondary carbonates are dissolved using dilute HCl, and finally the clean sample is returned to neutral pH and dried for analysis.

Solvent extraction. Samples contaminated with oil, glue or preservatives usually require extended treatment in various solvents to remove the unwanted substance. This type of chemical extraction typically involves many hours of solvent reflux using a Soxhlet apparatus. The solvents are selected based on the chemistry of the contamination, and they may be employed alone, in a sequence or as a mixture. Solvent extraction may also be employed to collect specific fractions of a sample, such as certain proteins or groups, for analysis.

CO2 conversion, graphitization, and measurement

Once the sample has been chemically isolated, it is dried and weighed. The carbon content is estimated based on the sample mass and type. An appropriately-sized portion is selected for carbon extraction. If the total carbon content is expected to be very low, we will contact you for instruction before extraction.

The sample is preheated under vacuum to remove adhering atmospheric gases, including CO2. Carbonate samples are converted to CO2 with H3PO4. Organic samples are combusted online in the presence of excess high-purity oxygen. Excess oxygen is removed and the CO2 is purified. Water and other contaminants (including SOx, NOx, and halide species) are removed using a combination of cryogenic and high-temperature fine wire copper and silver wool traps. The resulting pure CO2 gas is measured manometrically.

Small sample protocol. The ideal amount of carbon for routine AMS radiocarbon measurement is about 1 milligram. As the sample size decreases, measurement uncertainty increases. Below a certain point, additional steps are required to obtain a reliable radiocarbon age. Samples containing less than about 100 micrograms of carbon (down to about 10 µg C) can be dated using a special protocol. A known quantity of tightly controlled, precisely characterized dilution carbon is added to the carbon extracted from the sample material. The additional carbon provides the mass needed to enable a reliable AMS radiocarbon measurement. The measurement obtained for the mixture is then corrected to compensate for the known quantity and activity of the dilution carbon, yielding the activity and radiocarbon age of the original test sample. Because these steps also unavoidably increase the measurement uncertainty, the procedure is recommended only when that increase is less than what otherwise would result.

Upon request, a tiny amount of the CO2 gas is taken for independent stable isotope (δ13C) analysis by IRMS. The remaining CO2 is converted to graphite over an Fe catalyst in the presence of excess hydrogen. The resulting graphite powder is pressed into a target cartridge and the 14C activity is measured by AMS.

 
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