Chromatographic separation of strontium in archaeological cremated remains for Thermal Ionisation Mass Spectrometry (TIMS) analysis

Mechanically clean the cremated bone samples using a handheld drill (e.g., PROXXON or Dremel) to remove the outer layer of the bone, so all soil residue is removed. If present, also remove all of the trabecular bone (but see De Coster et al. 2024 paragraph 4.1).

Next, following the protocol described in Snoeck et al., 2015,2018 transfer the cleaned bone fragments to glass vials and leach them with circa 1.0mL(10:1 ratio) 1Molarity (M) ultrasonically for 0h 3m 0s- 0h 10m 0s, followed by two Milli-Q^TM rinses, and a 0h 10m 0s Milli-Q^TM ultrasonic wash. Repeat these steps until the Milli-Q^TM in the glass vials are clear or show a white cloudy color. Dry the samples on a hotplate at 50°C 0h 10m 0s.

If Pars petrosae were sampled, cut them midmodiolarly using a Buehler IsoMet 1000 precision saw (Veselka et al. 2021). Locate the otic capsule and subsample in acid-cleaned Eppendorf^® tubes approximately 10mg of bone powder with a handheld drill (e.g., PROXXON or Dremel) and transfer the samples to a clean laboratory. Here, follow the protocol described in step 1.

After drying the samples on the hotplates the samples, subsample ca. 10-20mg using a handheld drill (e.g., PROXXON or Dremel) equipped with a diamond tipped ball burr. The bone should be fully white after the mechanical cleaning. Any grey or black bones are not suitable for Sr isotope analysis. Therefore, exclude the discolored samples from further analysis. However, if only parts of the bone are not fully calcined/white, specifically target the white surfaces.

Weigh 2-10mg of cremated bone powder into an acid-cleaned Eppendorf® centrifuge tube. Note down the weights in gram. Next, add 600µL of pro-analysis quality 3M HNO₃. Bone powder will dissolve within seconds in Room temperature. If small fragments of bone 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. The remaining 100 µl can be used 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.