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Carbonate Preparation for Enamel Apatite Samples



Caveat:The procedure below represents a basic outline for preparing apatite samples for isotopic analysis by acid digestion. Modification of this procedure may be required for samples of different histories.



Recommended Carbonate Preparation for Tooth Enamel Samples

1.Weigh out 5-10 mg of powdered enamel into a microcentrifuge tube.
2. Add 1 ml of 30% H2O2 (currently located in the lab refrigerator) for 10 mg of sample (scale according to the sample weight).
3. Seal the microcentrifuge tube and agitate (vortex genie) each sample for 30-60 seconds.
4. Loosen the lids (so that gas can escape) and allow samples to sit and react for 24 hours (agitate often).
5. Centrifuge the samples and aspirate the H2O2 away taking care to leave the powder behind.
6. Rinse the sample by adding 1 ml of MilliQ water and agitating. Then centrifuge and repeat for a total of 5 rinses.
7. Once samples have been rinsed 5 times, add 2 ml of 1M acetic acid buffered with calcium acetate to pH of ~5* for 25 mg of sample (scale according to the sample weight). Currently the acetic acid buffered with calcium acetate is found in the flammables cabinet.
8. Agitate samples and allow them to react for 24 hours**.
9. Centrifuge the samples and aspirate the acetic acid buffer solution away taking care to leave the powder behind.
10. Rinse the sample by adding 1 ml of MilliQ water and agitating. Then centrifuge and aspirate as in step 9. Repeat four additional times leaving the powder only (no water) after the final aspiration.
11. Uncap each sample and cover with aluminum foil. Use a sharp to make a small hole in the foil covering each sample tube.
12. Freeze the samples for ~25 minutes or longer.
13. Place samples on a freeze dryer overnight.
14. Weigh out between 0.5 and 1 mg for a Kiel run into sample boats.
15. Vacuum roast the samples for ~ 1 hour at 65°C before running them on a mass spectrometer.

*Buffer solution should be made by mixing a 1M solution of acetic acid with a 1M solution of calcium acetate. Using the Henderson-Hasslebach equation this yields a solution of ~pH 5. Confirm this pH periodically. Do not use buffer solution if pH falls below 4.5.

**Both temperature and time of reaction can affect the C isotope composition of the sample. It is, therefore, imperative to leave samples in solution for exactly 24 hours. This value is not approximate. The reaction time was formerly 12 hours (and may still be 12 hours for enamel) but C isotope composition of dentine carbonate changes rapidly within the first 12 hours, thus 24 hours should give more consistent results.

Recommended Carbonate Preparation for Dentine and Bone Apatite Samples

1.Weigh out 10-15 mg of powdered dentine or bone into a microcentrifuge tube.
2. Add 0.5 ml of 30% H2O2 (currently located in the lab refrigerator) for 20 mg of sample (scale according to the sample weight).
3. Seal the microcentrifuge tube and agitate (vortex genie) each sample for 30-60 seconds.
4. Loosen the lids (so that gas can escape) and allow samples to sit and react for 24-48 hours (agitate often).
5. Centrifuge the samples and aspirate the H2O2 away taking care to leave the powder behind.
6. Repeat steps 2 and 3 and go to step 7.
7. Leave samples react until the total time of reaction gets to 72 h (if you left the samples 24h in step 4, you´ll need to leave them now for 48h).
8. Centrifuge the samples and aspirate the H2O2 away taking care to leave the powder behind.
9. Rinse the sample by adding 1 ml of MilliQ water and agitating. Then centrifuge and repeat for a total of 5 rinses.
10. Once samples have been rinsed 5 times, add 0.5 ml of 1M acetic acid buffered with calcium acetate to pH of ~5* for 20 mg of sample (scale according to the sample weight). CurrentIy acetic acid buffered with calcium acetate is found in the flammables cabinet.
11. Agitate samples and allow them to react for 24 hours**.
12. Centrifuge the samples and aspirate the acetic acid buffer solution away taking care to leave the powder behind.
13. Rinse the sample by adding 1 ml of MilliQ water and agitating. Then centrifuge and aspirate as in step 9. Repeat four additional times leaving the powder only (no water) after the final aspiration.
14. Uncap each sample and cover with aluminum foil. Use a sharp to make a small hole in the foil covering each sample tube.
13. Freeze the samples for ~25 minutes or longer.
14. Place samples on a freeze dryer overnight.
15. Weigh out between 1 mg for a Kiel run on metal boats
16. Vacuum roast the samples for ~ 1 hour at 65°C before running them on a mass spectrometer.

*Buffer solution should be made by mixing a 1M solution of acetic acid with a 1M solution of calcium acetate. Using the Henderson-Hasslebach equation this yields a solution of ~pH 5. Confirm this pH periodically. Do not use buffer solution if pH falls below 4.5.

**Both temperature and time of reaction can affect the C isotope composition of the sample. It is, therefore, imperative to leave samples in solution for exactly 24 hours. This value is not approximate. The reaction time was formerly 12 hours (and may still be 12 hours for enamel) but C isotope composition of dentine carbonate changes rapidly within the first 12 hours, thus 24 hours should give more consistent results.