Sediment preparation for measurements of diatom microfossil-bound 15N/14N

Physical Separation

1. Place ~2 g sediment in centrifuge tube (50 mL conical-bottom, polypropylene)

2. Remove CaCO3 with ~20 mL 25% HCl (i.e., 3 parts H2O, 1 part concentrated HCl)

3. Centrifuge samples for 20 minutes at 4000 rpm; pour off HCl

4. Add ~40 mL of 2% Na-polyPO4 solution

5. Apply sonic probe for 1 minute or place in sonic bath for ~20 minutes

6. Sieve off >150 micron fraction

7. Pour sample into standard 200 mL beaker, fill w/ Na-polyPO4 to 200 mL mark

8. Allow to settle 1.5 hours

9. Repeat steps 7 and 8 w/ H2O until liquid overlying sediment appears clear

10. Wash samples back into centrifuge tubes; centrifuge and pour off liquid

11. Dry sediments in oven overnight

12. Add 40 mL of Na-polytungstate diluted to density of 2.15 g/cm3

13. Apply sonic probe for 2 minutes

14. Centrifuge for 20 minutes at 4000 rpm

15. Pour upper column of Na-polytungstate through 8.0 micron filter

16. Rinse filter w/ 500 mL of H2O

17. Place filter in clean centrifuge tube

18. Add 40 mL of H2O; vortex and remove filter

19. Centrifuge samples and pour off water

Chemical Cleaning

1. Add ~15 mL dithionite-citric acid to sample  
Dithionite-citric acid
Stock solution: 500 mL H2O + 31 g Sodium Citrate (Na3C6H5O7*2H2O) + 5 g Sodium Bicarbonate (NaHCO3)
Working solution: Add 0.5 g Sodium Dithionite (Na2S2O4) per 10 mL of stock solution

2. Let sit for 1 hour in 80 degrees C water bath

3. Dilute solution with H2O to 40 mL; centrifuge and pour-off

4. Rinse samples with another 40 mL of H2O, centrifuge and pour off

5. Add ~25 mL 30% H2O2 to sample and cap (use punctured caps)

6. Place samples in boiling water bath for 1 hour

7. Let samples cool, then centrifuge and decant

8. Transfer samples to 40 mL glass tubes 

9. Add ~20 mL 70% perchloric acid and cap

10. Let samples sit for ~2 hours

11. Loosen caps to allow release of evolved CO2 and then retighten 

12. Place samples in boiling water bath for 2 hours

13. Let samples cool, then pour off most of the perchloric acid

14. Wash samples back into centrifuge tubes, and dilute with H2O to 40 mL mark

15. Rinse three times with 40 mL H2O

16. After final pour-off, place in oven overnight