By Paul D. Brooks, Stefania Mambelli , Kari Finstad , Joey Pakes and Todd E. Dawson .

1. Performance update for soil and sediment samples and their simultaneous analysis of δ15N, δ13C, δ34S and NCS concentrations using an ElementarVario Isotope EA and Isoprime 100 IRMS. By Paul D. Brooks, StefaniaMambelli, Kari Finstad, Joey Pakes and Todd E. Dawson. Univ. of California, Berkeley

2. Disclaimer • The product names used in this presentation are for information only and do not constitute a promotion or endorsement by the University of California, university affiliates or employees.

3. Acknowledgements • The authors would like to thank: • Dr. Brian Fry, formerly Univ. of Hawaii. • Dr. Andreas Rossmann, Isolab Germany. • Steve Silva, USGS Menlo Park, Ca. • Scott Hughes, Elementar Americas Inc. • RobinSutka, formally of ElementarAmericas Inc. • Everyone who has replied to questions on Isogeochem and has attended ASITA or earlier CFIRMS conferences.

4. Instruments used • All information in this presentation were generated using: • An Elementarvario ISOTOPE cube interfaced to: • A Isoprime 100 mass spectrometer.

5. Why analyze NCS isotopes in one sample? • Analysis of food web can greatly benefit from the addition of 34S by adding an additional dimension to the analysis. Food web studies usually require a large number of samples to reduce the noise level of the data. • Analysis for NCS concentration, then weighing out individual aliquots of each sample for separate N, C, S isotope analysis reduces the number of field samples that can be analyzed. • Many samples are so small that it is impractical to subsample them into two different aliquots for analysis by two different methods. • Combining samples results in the loss of the field sample noise which is usually critical to answering the experimental hypothesis in Ecosystem sciences.

6. Example of NCS data for individuals from stream population. Note noise level in populations and change in S ratio when N and C do not change. Samples from student Hiromi Uno.

7. Large number of samples required. • Concentration required. • 34S may significantly improve source identification.

8. To be useful, the NCS isotope analysis must meet these requirements • Be capable of a high throughput of over 60 unknown samples per day in order to analyze many field samples and reduce field noise level. • Costs, sample preparation and ease of analysis should not be excessively higher than 15N 13C analysis. • The analysis system must be able to analyze a wide sample range with different concentrations range of N, C and S. • Precision and accuracy must be similar to conventional NC and S methods.

9. Problems solved for high throughput NCS isotope analysis • S analysis usually uses one combined combustion reduction column with short lifetime. Solution: Use separate combustion and reduction columns connected with a heated quartz bridge. Only fill 110 mm center of reduction tube with Cu and heat to 880 °C. • Variable 18 O in samples interferes with SO2 mass 66 as 66 can be due to 34S or18O. Solution: Use a magnesium perchlorate water trap immediately after the 1st reduction tube followed by a 900 °C quartz buffering tube with CuO at center to buffer O. (Fry et al. 2002).

10. O buffering of SO2 with quartz buffering tube. Magnesium perchlorate drying tube before quartz tube. Silver sulfide with varying amounts of EDTA added to change C/S ratio with noquartz buffering tube. NOTE Y SCALES ARE DIFFERENT Silver sulfide with varying amounts of EDTA added to change C/S ratio with quartz buffering tube. On every analysis we measure a AgS2 standard with and without added sucrose with no difference in  34S. Data from Robin Sutka.

11. Mitigation S memory • Memory effects for S. Mitigation: Use a drying tube immediately after the 1st Cu tube to trap water. • Hypothesis, this may be due to SO2 and H2O being in equilibrium with H2SO3. Keeping the water trap hot may prevent H2O from condensing. • This may be why a combined comb/red column or heated connection between separate combustion and copper tube is necessary for SO2. • Could SO2be dissolving in a H2O film? SO2 + H2O H2SO3

12. Test of various standards for memory effect. Currently 0.11-0.15 µgS on UCB system.

13. Water trap split to allow daily changes of magnesium perchlorate.

14. Split water trap in place over Cu column

15. Prevent SO2 trailing, fully reduce NOx • Problem: SO2 begins to trail as ash build up in combustion tube. • Solution: Trap SO2 and release after all SO2 is collected. • Problem: NOx is not fully reduced in 880 °C Cu reduction column needed to pass SO2. • Solution: Use a second 650 °C Cu reduction column after the SO2 trap. (Brian Fry, personal communication.)

16. Analyze N 29/28 and S 66/64 on same triple collector. • Problem: As the N 29/28 ratio is much smaller than S 66/64, careful sample size selection based on prior knowledge of the N and S concentration is necessary to avoid saturating S on mass 66 or insufficient N on mass 28 with 10 volt AD converters. • Solution: Use an IRMS with 100 volt AD converters for wide dynamic range.

17. Get accurate concentrations for NCS • Concentration of N, C and S is not as precise using the IRMS as from the EA. • Solution: Interface the MS and EA software so sample names and weights are input automatically into the EA software and the TCD concentration and IRMS isotope results are combined in one final Excel file.

18. Calibration requires a large number of standards. • Preferred range of sample size is 30-1000 µgN, 0.2-5 mgC (adjustable with different dilution) and 10-140 µgS in a capsule. • Calibration of all three isotopes requires a large number of standards. • Solution: Use 120-place auto-sampler, a 10 minute per analysis method, and analyze 133 capsules with 46 standards per analysis and 81 unknowns, 3 standards and 3 blanks at beginning to stabilize system. • 133 total capsules takes ≈22.2 hours. • There is potential for reducing the number of standards required.

19. Preferred sample range 30-1000 µgN Bovine liver

20. Final schematic for NCS isotope analysis 2nd Cu reduction tube 650°C Large size CO2 trap. P2O5 trap CO2 trap SO2 trap quartz Cu reduction tube 880 °C Cu Bypass valves for SO2 Tungsten oxide comb tube 1150°C Magnesium perchlorate trap Quartz buffering tube 900°C P2O5 trap P2O5 trap CuO WO3 quartz TCD Heated quartz To MS

21. Combustion tube, 1st reduction tube, and magnesium perchlorate water trap.

22. Use tungsten oxide in long ash finger to mitigate long term memory andIncrease combustion tube life.

24. TCD chromatogram

25. MS chromatogram

26. Is an added oxidant needed? V2O5 is very toxic and we do not allow its use by our undergraduates who weigh most of our samples and standards. Nb2O5 or WO3 are used as substitutes but do not seem to work as well (Steve Silva personal communication).Could this be because of melting temperature? V2O5 melting temp 690 °C. Nb2O5 melting temp 1512 °C. WO3 melting temp 1473 °C.

27. Is an extra oxidant needed? • Oxidants seem to be added to help mitigate trailing problems with SO2. This may not be necessary if the SO2 is trapped, but depends on material (see later slides). Joey Pakes data.

28. 15N and 13C results are the same in NC and NCS mode • Since the second Cu reduction tube was added 15N results have been the same as in NC mode. • 13C results have always been the same.

29. S is more challenging (difficult). • Mass 66 saturates at about 140 ug S with current system, potential exists to gain shift and increase the range. • If the samples are all similarly small size then sample less than 4 µgS are feasible. • The memory effect of the current system models at about 0.11-0.15 µg S as estimated by fitting a dual mixing model to the data. This may limit the precision and accuracy of small samples with big differences in isotope ratio. • There is a phantom blank effect equivalent to about 0.8 µg S.

30. 34S vs µ S for standard

32. ≈ 0.8 µg S

33. Standardization procedure • Use a calibration standard of 3.8-4.2 mg (32 µgS) bovine liver every 12 samples to correct for drift, large size minimizes carryover. • Put a variable weight bovine liver after the calibration standard to use for QC. • Put in 10 variable weight standards each of fishmeal and spirulina to check carryover, adjust linearity and normalize isotope values.

34. Post analysis calculation • Drift correct between calibration standards using peak to peak correction. • Check S carryover using variable weight fishmeal and spirulina standards. • Summarize different standards data and move to dual mixing model spreadsheet for linearity and normalization correction. • Check blank correction for S using fishmeal and spirulina.

35. Soils and sediment analysis. • Soil analyze well for N and C, but S may be difficult for some soils and sediments. • For example, SRM 1646a appears to have a slow release of S resulting in a big memory effect. • This effect may in turn affect S analysis of later samples.

36. Soils analysis for NCS shows no bias with size and without V2O5 show good agreement with other analysis.

37. Soils analysis for NCS shows no bias with size and without V2O5 show good agreement with other analysis.

38. Note analysis works well up to 140 mg of soil, and possibly higher.

39. SRM 1646a sediment appears to introduce a memory effect.

40. A 2 stage memory dual mixing model corrected the memory effect. But how would the analyst know what correction to apply? Icacos soil 28-32 µgS

41. Anoxic sediment had a sever carryover and even appears to adsorb S from the next sample. N and C results looked good.

42. How to further improve NCS isotope analysis (and current NC analysis?) • Provide at least 3 standards with all NCS values either heavy, light, and one to use as a QC in between. • Treat soils for S analysis carefully, especially anoxic sediments.

43. Conclusions 1 • The system can analyze 133 total capsules (samples including standards) in 22.2 hours. • NCS mode requires additional standards, so 81 unknowns can be analyzed in 22.2 hours. • Precision in a size range of 30-1000 µgN, 0.2-5 mgC and 10-140 µgS in a capsule compares well with separate NC and S analysis. • The only significant additional maintenance compared to NCS is the changing of the 1st Cu reduction tube and small water trap daily. • S analysis is improved with capability to analyze 10 variable weight samples of 3 different 34S isotope standards for a total of 30 normalization and QC standards.

44. Conclusions 2 • S analysis should be over 10 µgS and better over 20 µgS which minimizes problems with blank correction and carryover. • This is not difficult to achieve as the Vario Isotope Cube is easily capable of burning samples weighing of at least 10 mg. The upper limit on sample size has not been explored. • Soil samples up to at least 140 mg can be analyzed. • Some soil or sediment samples do not analyze well for S even though results for N and C are good. • We have not tried to measure these problem sediment samples adding V2O5 or other accelerants such as ammonium nitrate. • We hypothesize that anaerobic sediments are problematic for S analysis as they have a large carryover.

45. References • Fry, Brian. 2007. Coupled N, C and S stable isotope measurements using a dual-column gas chromatograph system. Rapid Communications in Mass Spectrometry. 21:750-756. • Fry, B., et al. 2002. Oxygen isotope corrections for online 34Sanalysis. Rapid Communications in Mass Spectrometry. 16:854-858. • Sieper, Hans-Peter et al. 2006. A measuring system for the fast simultaneous isotope ratio and elemental analysis of carbon, hydrogen, nitrogen and sulfur in food commodities and other biological material. Rapid Communications in Mass Spectrometry. 20:2521-2527. • Hansen, T. et al. 2009.Simultaneous 15N, 13C and 34S measurements of low biomass samples using a technically advanced high sensitivity elemental analyzer connected to an isotope ratio mass spectrometer. Rapid Communications in Mass Spectrometry. 23:2521-2527.