Chromatographic separation of strontium in archaeological human and faunal enamel for Thermal Ionisation Mass Spectrometry (TIMS) analysis

If the dental elements are contaminated with soil, place the teeth in glass beaker and submerge with Milli-Q^TM (Millipore, resistivity >18 Ω/cm. Hereafter: Milli-Q). Clean the teeth ultrasonically for ca. 0h 10m 0s. Replace the Milli-Q if needed and repeat until the Milli-Q remains clean. Rinse the teeth with Milli-Q (>3 times) and dry on a hotplate or oven 0h 10m 0s at 50°C.

Next, place a weighing paper under the tooth. Remove the outer, and therefore possibly diagenetically altered layer of the enamel using a handheld drill (e.g., PROXXON or Dremel) equipped with a diamond tipped ball burr. Clean the tip of the burr after each sample with Milli-Q, 10% HCl, Milli-Q, and ethanol to avoid cross contamination. Discard the weighing paper with the (contaminated) enamel after each sample.

Clean the workspace with ethanol, or water and detergent. Place a sheet of aluminium foil on the workspace. Take a weighing paper and fold in two. Sample ca. 1-10mg of cleaned, crisp white dental enamel using an acid-cleaned diamond tipped ball burr and collect the sampled enamel on the weighing paper. If small (black) soil particles are present, remove these carefully from the weighing paper using your (gloved) hands or a tweezer. Carefully slide the sample in 2 ml glass vials with screw caps for subsampling, or (pre-weighted) acid-cleaned Eppendorf® centrifuge tubes (Safe-lock, 1.5 ml. See 2.1) for further analysis. Clean the burr tip following the steps outlined above, and discard the weighing paper.

Acid-cleaned Eppendorf® tubes: In a clean laboratory, place the Eppendorf® tubes in a clean jar and add 6-7M HCl (Sigma-Aldrich Company Ltd). Leave on a shaker for 5-7 days. Rinse 3 times with Milli-Q water and leave in an open container in a laminar flow hood for 2 days to dry.

If sampled in a glass vial, weigh 1-2mg of enamel powder into an acid-cleaned Eppendorf® centrifuge tube. If samples in a pre-weighted acid-cleaned Eppendorf® centrifuge tube, with the tube including the sample. Note down the weights in gram. Depending on the overall preservation and archaeological recovery conditions, you can either leach (see 3.1) first, or directly dissolve the sample (see 3.2).

Leach: Add 500µL 0.1Molarity (M) for ca.0h 10m 0s to allow the enamel to react. Next, centrifuge the samples for 0h 10m 0s at 12000rpm. Carefully pipette the 500µLHAc from the Eppendorf® tube. Use a new acid-cleaned pipette tip for each sample (see 4.2) , or wash the pipette tip with Milli-Q after each sample. Next, Add 500µL Milli-Q, vortex the sample, and centrifuge for 3 minutes at 12000rpm. Carefully pipette the 500µL Milli-Q from the Eppendorf® tube using new or Milli-Q washed pipette tips. Continue with 3.2 (dissolve).

Dissolve: Add 600µL of pro-analysis quality 3M HNO₃. Enamel powder will dissolve within seconds in Room temperature. If small fragments of enamel are sampled, place the Eppendorf® tube in an ultrasonic bath for ca. 0h 10m 0s to allow all enamel to dissolve. Next, centrifuge the samples for 0h 3m 0s at 12000rpm.

Separate Sr from the matrix using in-house made Sr columns made from 1.5 ml pipette tips and using a 3.5 mm PE frit (Angst and Pfister, h = 2 mm, porosity 35 μm).

The columns are stored in pipette tip boxes in ±3M HCl. Take out a column with a plastic tweezer, tap the HCL out of the pipette tip and rinse 3 times with Milli-Q. Place the column in the rack and carefully fill with Milli-Q. Add 80µL Sr resin (120µLin slurry (0.2M HNO₃), Eichrom Technologies, 100–150 μm mesh).

Clean the columns using the following steps:

1 CV ( c olumn v olume) 3M HNO₃

1 CV Milli-Q

1 CV 3M HNO₃

1 CV Milli-Q

Condition the columns by adding 500µL 3M HNO₃.

Next, load 500µL of sample. Use the remaining 100 µl for concentration measurements. Use a new, acid-cleaned pipette tip for every sample (see 4.2). Once the samples dripped through the columns, wash the samples twice with 900µL 3M HNO₃. Replace the waste beakers containing the pre-fraction with acid-cleaned 5 or 7 ml PFA (Savillex) beakers (see 4.1). To elute the Sr, wash with 900µL Milli-Q. Add 0.07 to 0.11 gram (1-3 drops) of ⁸⁴Sr spike to the blank(s). Add 1 drop 0.5% H₃PO₄ to the samples (and blanks and in-house standard if applicable).

Close the beakers and transfer them to a hotplate. Place the beakers and the caps on a hotplate at 120°C overnight. Once dry, nitrate with 4-6 drops of 14M (concentrated) HNO₃. Dry the samples (and blanks and in-house standards if applicable) at 120°C.

Cleaning Teflon® PFA (high-purity Perfluoroalkoxy resin) laboratory equipment: sub-boil in pro-analysis quality 3M HNO₃ and 6-7M HCl for 2 hours each in a fume cupboard. Rinse 3 times with Milli-Q between the baths. Add ca. 3 ml 6-7M HCl, close the caps and leave on a hotplate at 120°C for 2-5 days. Discard the acid, rinse 2 times with Milli-Q and store in a clean box.

Cleaning pipette tips: Fill a pipette box with pipette tips, leaving one slot empty. Fill the box with 500 ml of approximately 3M HCl and let it stand for about 5-10 days at Room temperature . Remove the acid and rinse three times with Milli-Q. Place on a hotplate at 60°C to dry.

Add 2µL 10% HNO₃to the dried samples. Place the outgassed single annealed rhenium filaments in the dedicated holders and increase the current to 1.5 mA. Create little 'dams' with parafilm. Reduce the current to 0 mA. Pipette 2µL of TaCl₅and 1µL of sample (50%, to allow for a rerun if needed) between the parafilm dams on the filament. Dry slowly at 1.0 mA. Once dry, gradually increase the current to 1.3 mA until the samples turn black, then to 1.6 mA to burn away the parafilm. Increase further to approximately 1.8-2.0 mA until the samples glow. Once they are bright red, immediately reduce the current to 0 mA and load the sample onto the turret.

Use a new, acid-cleaned pipette tip (see 4.2) for every sample.

Upload the ⁸⁷Sr/⁸⁶Sr data as a dataset on IsoArch.eu. IsoArcH is an open and collaborative database of georeferenced isotopic measures of bioarcheological samples from all time periods and all around the world. The IsoArcH initiative supports the CARE principles. In parallel, IsoArcH has adopted the FAIR practices to ensure that datasets are readily discoverable and compatible within the IsoArcH database.