Performance update for soil and sediment samples and their simultaneous analysis of

δ15N, δ13C, δ34S and NCS concentrations using an Elementar Vario Isotope EA and Isoprime

100 IRMS.

By Paul D. Brooks, Stefania Mambelli, Kari Finstad, Joey

Pakes and Todd E. Dawson. Univ. of California, Berkeley

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.

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. • Robin Sutka, formally of Elementar Americas Inc. • Everyone who has replied to questions on

Isogeochem and has attended ASITA or earlier CFIRMS conferences.

Instruments used

• All information in this presentation were generated using:

• An Elementar vario ISOTOPE cube interfaced to:

• A Isoprime 100 mass spectrometer.

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.

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.

sample name % Nug N in

capsule d 15N % Cmg C in capsule d 13C % S

ug S in capsule d 34S

Ephemerella maculata soap- 13.85 205 5.75 58.40 0.87 - 28.06 0.80 12 - 1.61Ephemerella maculata soap- 12.33 188 7.58 62.36 0.95 - 29.48 0.78 12 - 2.79Ephemerella maculata soap- 13.48 199 4.42 56.56 0.84 - 26.99 0.74 11 - 1.11Ephemerella maculata soap- 13.84 183 1.42 56.95 0.75 - 26.27 0.81 11 - 1.22Ephemerella maculata soap- 15.70 212 3.56 60.12 0.81 - 27.85 0.82 11 - 1.53

avg= 13.84 198 4.54 58.88 0.84 - 27.73 0.79 11 - 1.65stdev= 1.08 11 2.07 2.14 0.07 1.08 0.03 0 0.60

parasitic nematode 12.75 157 3.99 57.11 0.71 - 25.72 0.92 11 - 1.30Ephemerella maculata soap+ 12.10 208 1.20 54.21 0.93 - 22.25 0.70 12 - 0.78Ephemerella maculata soap+ 13.28 182 5.12 55.52 0.76 - 28.51 0.73 10 - 2.38Ephemerella maculata soap+ 12.60 178 1.61 59.61 0.84 - 25.70 0.74 10 - 0.61Ephemerella maculata soap+ 12.81 197 1.73 59.38 0.91 - 25.25 0.76 12 - 1.30Ephemerella maculata soap+ 13.11 173 8.17 58.11 0.77 - 27.59 0.79 10 - 1.55

avg= 12.78 187 3.57 57.37 0.84 - 25.86 0.74 11 - 1.32stdev= 0.41 13 2.70 2.15 0.07 2.17 0.03 1 0.63

Timpanoga_nymph 10.36 137 0.63 42.87 0.57 - 19.94 0.66 9 2.44Timpanoga_nymph 9.87 140 0.57 41.91 0.60 - 19.66 0.63 9 3.00Timpanoga_nymph 10.60 148 0.16 43.34 0.60 - 19.07 0.66 9 3.05

avg= 10.28 142 0.45 42.71 0.59 - 19.56 0.65 9 2.83stdev= 0.30 4 0.21 0.60 0.02 0.36 0.01 0 0.28

Timpanoga_subimago 11.82 160 1.34 55.44 0.75 - 16.17 0.82 11 2.37Timpanoga_subimago 11.87 183 0.96 55.23 0.85 - 17.06 0.81 12 2.91Timpanoga_subimago 10.92 181 1.51 60.21 1.00 - 16.92 0.70 12 2.88

avg= 11.54 175 1.27 56.96 0.87 - 16.72 0.78 12 2.72stdev= 0.44 10 0.23 2.30 0.10 0.39 0.05 1 0.25

Timpanoga_imago 11.89 183 1.23 58.16 0.90 - 17.50 0.82 13 2.46Timpanoga_imago 12.33 191 1.26 64.19 0.99 - 17.19 0.80 12 2.71

avg= 12.11 187 1.24 61.18 0.95 - 17.34 0.81 13 2.58

• Large number of samples required.

• Concentration required.• 34S may significantly improve

source identification.

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.

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 or

18O. 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).

0 50 100 150 200 250 30011121314151617

C/S

raw

d34

S

Silver sulfide with varying amounts of EDTA added to change C/S ratio with no quartz buffering tube.

Silver sulfide with varying amounts of EDTA added to change C/S ratio with quartz buffering tube.

O buffering of SO2 with quartz buffering tube. Magnesium perchlorate drying tube before quartz tube.

NOTE Y SCALES ARE DIFFERENT

On every analysis we measure a AgS2 standard with and without added sucrose with no difference in 34S.

Data from Robin Sutka.

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 SO2 be dissolving in a H2O film?

SO2 + H2O H2SO3

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

0 10 20 30 40 50 60-5.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00 S memory test

original delta

carryover cor-rected delta

Analysis Number

de

lta 3

4S

Water trap split to allow daily changes of magnesium perchlorate.

Split water trap in place over Cu column

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.)

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.

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.

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.

Preferred sample range 30-1000 µgN

0 200 400 600 800 1000-5

-3

-1

1

3

5

7

9

Isoprime 100 test of N range with NIST 1547 peach leaves and NIST 1577b bovine liver, 400 µAmp tuning

approx ug N in tin capsule

de

lta

15

N Peach leaves

Use dual mixing model to correct for small samples < 30 µgN

Bovine liver

TCDHeated quartz

SO2trap

CO2trap

Bypassvalves for SO2

P2O5trap

P2O5trap

P2O5trap

To MS

Mag

nesi

um p

erch

lora

te tr

ap

Cu re

ducti

on tu

be 8

80 °C

Tung

sten

oxi

de c

omb

tube

115

0°C

Qua

rtz

buffe

ring

tube

900

°C

Cu

WO

3 CuO

quartz

quartz

2nd Cu reduction tube 650°C

Final schematic for NCS isotope analysis

Large size CO2 trap.

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

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

TCD chromatogram

MS chromatogram

Is an added oxidant needed?

V2O5 melting temp 690 °C.Nb2O5 melting temp 1512 °C.WO3 melting temp 1473 °C.

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?

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).

%N d15

N %C d 13C %S d 34S

avg stdev avg stdev avg stdev avgstde

v avg stdev avg stdev

peach w/Nb2O5 3.02 0.02 1.92 0.03 49.76 0.72 -25.87 0.03 0.21 0.02 8.56 0.08

peach no Nb2O5 2.96 0.01 2.06 0.03 48.41 0.61 -25.86 3.00 0.20 0.01 8.10 0.06

Yolo soil w/Nb2O5 0.10 0.00 3.74 0.21 0.87 0.01 -25.43 0.11 0.02 0.00 -2.67 0.44

Yolo soil no Nb2O5 0.10 0.00 4.31 0.14 0.85 0.01 -25.57 0.02 0.02 0.00 -2.31 0.13

Joey Pakes data.

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.

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.

34S vs µ S for standard

2 4 6 8 10 12 14 16 187.00

7.20

7.40

7.60

7.80

8.00

8.20

8.40

8.60

8.80

9.00

rep dSy=ax2+xb+ccorractual

µS

del

ta 3

4S

reported avg = 7.98 stdev= 0.36

corrected avg = 8.10 stdev = 0.12

≈ 0.8 µg S

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.

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.

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.

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

Sample Target mg

Actual mg % N ug N δ 15N % C mg C δ 13C % S ug S Fry δ 34S

EM high organic B2151 5 5.08 0.69 35 4.42 9.12 0.46 -26.36 0.75 43 4.49EM high organic B2151 5 4.90 0.72 35 4.44 9.26 0.45 -26.31 0.76 42 4.50EM high organic B2151 10 10.12 0.67 68 4.56 9.33 0.94 -26.35 0.73 81 4.41EM high organic B2151 10 9.89 0.67 67 4.65 9.36 0.93 -26.42 0.73 79 4.25EM high organic B2151 10 10.15 0.67 69 4.61 9.39 0.95 -26.41 0.73 81 4.12

avg 0.68 4.54 9.29 -26.37 0.74 4.35std 0.02 0.10 0.11 0.05 0.02 0.16CERTIFIED VALUE 4.42 +_ .29 -26.27 +- 0.15 4.20

EM low organic B2153 70 70.94 0.14 98 6.95 1.55 1.10 -27.57 0.02 19 4.80EM low organic B2153 70 70.79 0.14 97 6.87 1.54 1.09 -27.50 0.02 18 4.66EM low organic B2153 70 70.27 0.14 97 6.91 1.56 1.09 -27.42 0.02 18 4.67EM low organic B2153 140 140.64 0.14 190 6.79 1.54 2.17 -27.39 0.02 36 4.64EM low organic B2153 140 139.93 0.14 189 6.91 1.53 2.15 -27.31 0.02 36 4.54EM low organic B2153 140 139.81 0.14 189 6.91 1.55 2.16 -27.25 0.02 36 4.49

avg 0.14 6.89 1.55 -27.41 0.02 4.63std 0.00 0.06 0.01 0.12 0.00 0.11CERTIFIED VALUE 6.7 +- .15 -27.46 +_ .11 4.94 +_ 1.4

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

SampleTarget

mgActual

mg% N ug N δ 15N % C mg C δ 13C % S ug S Fry δ 34S

Icacos soil 40 40.41 0.29 117 3.50 5.10 2.06 -28.25 0.05 24 15.38Icacos soil 40 41.70 0.29 120 3.41 5.05 2.11 -28.27 0.06 25 15.36Icacos soil 40 39.73 0.28 112 3.45 4.96 1.97 -28.24 0.06 25 15.57Icacos soil 60 59.17 0.29 170 3.49 5.13 3.03 -28.29 0.06 37 15.38Icacos soil 60 60.31 0.29 173 3.43 5.10 3.07 -28.24 0.06 38 15.20Icacos soil 60 60.81 0.29 174 3.50 5.10 3.10 -28.25 0.06 39 15.42Icacos soil 80 80.96 0.27 221 3.43 5.00 4.05 -28.09 0.05 50 15.96Icacos soil 80 80.88 0.27 219 3.44 4.98 4.03 -28.01 0.05 51 15.93Icacos soil 80 79.92 0.28 222 3.51 5.04 4.03 -28.07 0.05 51 15.95avg 0.28 3.46 5.05 -28.19 0.05 15.57std 0.01 0.04 0.06 0.10 0.00 0.29

Malachite lake Mud 5 5.19 2.12 110 -0.35 40.72 2.11 -27.08 0.21 12 8.78Malachite lake Mud 5 5.01 2.13 107 -0.31 40.71 2.04 -27.05 0.23 13 8.52Malachite lake Mud 5 4.98 2.05 102 -0.26 40.16 2.00 -26.99 0.23 12 8.63Malachite lake Mud 10 10.01 2.00 200 -0.16 40.47 4.05 -26.23 0.25 28 8.07Malachite lake Mud 10 10.09 2.02 204 -0.15 40.41 4.08 -26.29 0.26 29 7.96Malachite lake Mud 10 10.00 1.96 197 -0.22 40.12 4.01 -26.25 0.28 31 7.62

avg 2.05 -0.24 40.43 -26.65 0.24 8.26std 0.07 0.08 0.26 0.43 0.03 0.45Andrew Schauer vlaues 1.93 -0.37 39.48 -26.44 0.21 8.26

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

SampleTarget

mgActual

mg% N ug N δ 15N % C mg C δ 13C % S ug S Fry δ 34S

Yolo soil 50 50.16 0.11 55 4.48 0.88 0.44 -25.46 0.02 7 -3.41Yolo soil 50 50.10 0.11 56 4.36 0.88 0.44 -25.45 0.02 8 -3.60Yolo soil 50 50.02 0.11 56 4.50 0.88 0.44 -25.41 0.02 8 -3.30Yolo soil 70 70.04 0.11 80 4.46 0.87 0.61 -25.42 0.01 10 -3.37Yolo soil 70 70.07 0.11 76 4.41 0.87 0.61 -25.39 0.02 11 -3.60Yolo soil 70 70.04 0.11 79 4.46 0.88 0.61 -25.38 0.02 11 -3.76Yolo soil 100 100.03 0.11 108 4.54 0.98 0.98 -25.41 0.01 15 -3.93Yolo soil 100 100.13 0.11 109 4.50 0.98 0.98 -25.46 0.01 16 -3.52Yolo soil 100 100.13 0.11 110 4.51 0.98 0.98 -25.40 0.01 16 -3.56Yolo soil 120 120.04 0.11 129 4.56 0.88 1.05 -25.45 0.01 19 -4.19Yolo soil 120 120.16 0.11 134 4.53 0.88 1.05 -25.46 0.01 20 -3.63Yolo soil 120 120.08 0.11 135 4.40 0.84 1.01 -25.38 0.01 19 -3.26Yolo soil 140 140.09 0.10 145 4.54 0.84 1.17 -25.48 0.01 22 -3.67Yolo soil 140 140.07 0.10 144 4.45 0.84 1.17 -25.54 0.01 23 -3.55Yolo soil 140 140.03 0.10 144 4.51 0.83 1.17 -25.52 0.01 23 -3.38average 0.11 4.48 0.89 -25.44 0.01 -3.58std 0.00 0.06 0.05 0.05 0.00 0.24

SRM 1646a sediment appears to introduce a memory effect.

0 10 20 30 40 50 60 70

-20.00

-15.00

-10.00

-5.00

0.00

5.00

10.00

15.00

20.00 Memory Icacos soil alternate with SRM 1646a

run #

Del

ta 3

4S

Icacos soil28-32 µgS

SRM 1646a18-22 µgS

A 2 stage memory dual mixing model corrected the memory effect. But how would the analyst know what correction to

apply?

0 10 20 30 40 50 60 70

-20.00

-15.00

-10.00

-5.00

0.00

5.00

10.00

15.00

20.00memory correction 2 stage 0.8 µgS

run #

Dde

lta

34 S

SRM 1646a18-22 µgS

Icacos soil28-32 µgS

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

S pk ht δ 34Srun # name S pk ht should be δ 34S should be

104 anoxic sediment #1 16.01 14.48105 anoxic sediment #2 11.04 17.01106 anoxic sediment #3 10.77 16.84107 Blank 3.11 (0.08) 16.68108 bovliver 1.73 (4.07) 10.63 ( 7.6)109 var_bovliver 1.37 (2.78) 8.33 ( 7.6)110 blank 0.62 (0.08) 7.07111 blank 0.53 (0.08) 7.55112 blank 0.29 (0.08) 9.04113 blank 0.15 (0.08) 8.45114 blank 0.11 (0.08) 6.01

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.

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.

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.

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 34S analysis. 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.