Elemental Analyser Isotope Ratio Mass Spectrometry (EA-IRMS)

Sample Submission

For the cheapest and fastest turnaround: Submit samples weighed into 3x5 mm tin capsules, ready for analysis and stored in plastic 96-well plate/culture tray with lids. We would be happy to analyze (for free) 2-4 samples materials (at ~0.3 – 0.4mg) to help pinpoint an accurate target weight for you before weighing all your samples. Inverts and animal tissues generally fall around that 0.3-0.4 mg point (we can test to make sure) but algae, benthic matter, plants and sediments can vary greatly.

If we are to weigh the samples: The material (100-500 mg) must be dried and ground to a powder and submitted in small glass vials. Sample homogeneity is very important as only a small amount is required for analysis. The best control over your research is to oversee sample material manipulation and treatments directly (i.e. fraction separation, acidification, lipid extraction etc.), to ensure exactly what you want analyzed, gets analyzed – it is a bulk analysis; i.e. all sources of nitrogen and carbon get converted to N2 and CO2 and analyzed for δ13C+δ15N.

If you do need us to take on some of this sample preparation work, it comes at additional cost and time as it is labour intensive and we are primarily an analytical facility. We would require a sub-sampled portion of the material you wish analyzed (~500 mg), frozen if we are to freeze-dry it as well.

View more information about material treatments and preparation options.

Template for EA Sample Tray Submission (XLSX)

Material Requirements for Carbon

Analysis Analysis Requirement Amount Requested Precision
δ13C 0.01 mg C

~10 mg dry/ground (tissue)
~100-500 mg d/g (soil, plant)

2σ = 0.2 ‰ VPDB
Any materials that you suspect may have inorganic carbon contamination, like aquatic sourced materials (plants, inverts, algae POM etc. as well as sediments or soils) you may need to consider an acid wash to have it removed. We suggest a mild 5-10% HCl wash (90 min @ 50-60°C) then rinsed to neutral pH 3-4 times with nano-pure water.

Material Requirements for Nitrogen

Analysis Analysis Requirement Amount Requested Precision
δ15N 0.02 mg N ~10 mg d/g (tissue)
~100-500 mg d/g (soil, plant)
2σ = 0.3 ‰ Air-N2

Material Requirements for Sulphur

Analysis Analysis Requirement Amount Requested Precision
δ34S 0.03 mg S ~5-10 mg dry BaSO4
~10 mg d/g (soil, tissue)
2σ = 0.3 ‰ VCDT

Material Requirements for Oxygen

Analysis Analysis Requirement Amount Requested Precision
δ18O 0.03 mg O ~5 mg dry BaSO4
~10 mg d/g (purified/tree-ring cellulose)
2σ = 0.3 ‰ VSMOW

Please note the amount requested does not pertain to pre-weighed encapsulated samples. All materials must be dry, homogeneous (finely ground) in small, well-labelled glass (preferably) vials.

Visit our pricing page for cost of analysis or contact us with any questions.

Additional Information for Sample Submission

13C/15N Analysis in Solid Samples

Solid Sample Submission

Pre-weighed for the cheapest and fastest turnaround:

Submit samples weighed out in tin capsules (Elemental Microanalysis D1002 5x3.5mm), ready for analysis and stored in solid plastic 96-well plate/culture tray w/lids. The tin capsule should be closed and (void of air) and rolled / squeezed into a compact ball or cube; no flat pancake or sprawling shapes as they jam the autosampler. These pre-weighed samples are charged at a lesser '€˜per drop’ cost so sample repeats must be planned for by the researcher.

Generally, invertebrates and animal tissues fall around that 0.35-0.45mg range however other materials like algae, benthic matter, plants and sediments can vary greatly. Please contact EIL for further information. We are happy to analyze (at no charge) 2-4 test sample materials (at ~0.5mg) to help pinpoint an accurate target weight for you before proceeding to weigh all your samples; improperly weighed samples will be charged for if analysis is attempted.

Note - sometimes the C/N ratio is too great so that samples must be done twice, once at an appropriate amount for a carbon value and then a second larger weight for the nitrogen.

EIL weighed samples:

The material (100-500mg) must be dried and ground to a powder and submitted in a small glass vial. Sample homogeneity is very important as only a small amount is required for analysis.

The best control over your research project is to oversee sample material manipulation and treatments directly, to insure exactly what you want analyzed, gets analyzed – this is a 'bulk’ analysis; all sources of weighed material will be converted to N2 and CO2 and analyzed.

If you do need to pass off some of this work, it comes at additional cost and time as it is labor intensive and we are primarily an analytical facility. We would require a sub-sampled portion of the material you wish analyzed (~500mg), frozen if we are to freeze-dry it as well.

Any materials that you suspect may have inorganic carbon contamination, like aquatic sourced materials (plants, inverts, algae POM etc. as well as sediments or soils) you may need to consider an acid wash to have it removed with a mild acid rinse.

EIL can help with some of these sample prep processes with additional cost(s) – please contact for further details.

Template for EA Sample Tray Submission (XLSX)

Equipment

The analysis of solid materials for 13C and 15N isotope measurements is determined through combustion conversion of sample material to gas through a 4010 Elemental Analyzer (Costech Instruments, Italy) coupled to a Delta Plus XL (Thermo-Finnigan, Germany) continuous flow isotope ratio mass spectrometer (CFIRMS).

General Precision details

The δ13C data (δ13C IRMEA / VPDB column) is the corrected delta value, reported in per mil (‰) units, against the primary reference scale of Vienna Pee Dee Belemnite (VPDB).

The δ15N data (δ15N IRMEA / AIR column) is the corrected delta value, reported in per mil (‰) units, against the primary reference scale of Atmospheric AIR.

Data quality control is monitored and corrections made using an array of international reference material and in-house EIL standards that are calibrated using certified international reference materials (i.e. IAEA-N1 + N2, IAEA-CH3 + CH6, USGS-40 + 41) with values provided through CIAAW. A mix of EIL in-house (i.e. EIL-72, EIL-3 and JSEC-01) and international materials are analyzed in each run.

A 'like’ material is also used when possible; usually a NIST material like NIST-1577b (Bovine Liver) is used when tissues or invertebrates are analyzed and NIST 2704 (River sediment) is used when analyzing soils and sediments. This provides a post-correction check throughout the analysis run for both isotopic and elemental percent data.

Reference Materials - International

   
 

Material #

Material Type

13C Value

15N Value

IAEA-N1

8547

ammonium sulfate

 

+0.43 ‰

IAEA-N2

8548

ammonium sulfate

 

+20.35 ‰

IAEA-CH-6

8542

sucrose

-10.45 ‰

 

IAEA-CH-3

 

cellulose

-24.72 ‰

 

USGS40

8573

L-glutamic acid

-26.39 ‰

-4.52 ‰

USGS41

8574

L-glutamic acid

+37.63 ‰

+47.57 ‰

In-House Standard Materials - corrected to Internationals

 

Material #

Material Type

13C Value

15N Value

EIL-72

 

cellulose

-25.47 ‰

 

EIL-3

 

ammonium sulfate

 

+0.77 ‰

JSEC-01

 

ammonium sulfate

 

+20.21 ‰

NIST-1577b

 

Bovine Liver

-21.60 ‰

+7.72 ‰

NIST-2704

 

Sediment

-19.78 ‰

+3.82 ‰

http://www.ciaaw.org/vendors.htm

Quality Assurance / Quality Control (QA/QC)

Sample repeats are included every 8 to 10 sapmles (depending on the file size) and of the total sample number dropped in an analytical run, no less than 20% are Std/Ref materials. These Std/Ref measurements are used in data normalization and to ensure daily mass spec precision and accuracy; also to assess linearity issues or mass spec drift throughout the duration of the run. With these QA/QC checks, an error of 0.2‰ δ13C and 0.3‰ δ15N are required for reportable data.

Minimum Concentration

For '€˜regular’ sample analysis the minimum the mass spec requires falls ~ 0.08% C + 0.02% N (C analysis is diluted with Helium carrier) or 0.008% C only (non-diluted analysis).
Based on up to a maximum 80-100 mg of sample – larger amounts lead to poor changing combustion properties, with possible poor chromatography and bad data reproducibility.

References

FRY B., Brand, W., Mersch, F.J., Tholke, K., and Garrittl, R. Automated analysis system for coupled δ13C and δ15N measurements. Analytical Chemistry. 1992;64:288 -291.

34S Analysis in Solid Samples

Solid Sample Submission

The bulk of the sulphur analysis at EIL is in the form of BaSO4 from groundwater, however we also analyze mineral sulphides and a few organic materials (fish tissues). Materials (particularly organics and some sediments) having sulphur content below 0.5% become a combustion problem, which may increase the ± error further until any reliable analysis is impossible (i.e. materials below 0.25%S).

Pre-weighed for the cheapest and fastest turnaround;

We currently accept limited client pre-weighed sample submission for sulphur analysis, as the addition of WO3 is required. Contact us for further information.

EIL weighed samples;

Most samples for sulphur analysis are weighed here at EIL for a set cost.

The material (10-100mg) must be dried and ground to a powder and submitted in a small glass vial. Sample homogeneity is very important as only a small amount is required for analysis.

Sulphate extraction from water is a separate cost – contact us for details.

Template for EA Sample Tray Submission (XLSX)

Equipment

The analysis of solid materials for 34S isotope measurements is determined through combustion conversion of sample material to gas through an 4010 Elemental Analyzer (Costech Instruments, Italy) coupled to a Isochrom (Micromass UK) continuous flow isotope ratio mass spectrometer (CFIRMS).

The δ34S data (δ34S RESULT / VCDT column) is the corrected delta value, reported in per mil (‰) units, against the primary reference scale of Vienna-Canyon Diablo Troilite meteorite (VCDT).

Data quality control is monitored and corrections made using an array of international reference material and in-house EIL standards (i.e. EIL-40 + 41 + 42) that are calibrated using certified international reference materials (IAEA-SO-5, IAEA-SO-6, NBS-127, NBS-123, IAEA-S1 to-S3) with values provided through CIAAW. Whenever possible we try to run like-against-like materials; sulphide Std/Ref materials with sulphide samples, sulphate Std/Ref materials with sulphate samples.

Sample and Standard materials are weighed into tin capsules with a few mg of ground WO3 added to help in the conversion to SO2 gas.

Reference Materials - International

 
 

Material #

Material Type

34S Value

IAEA-S-1

8554

silver sulphide

-0.30 ‰

IAEA-S-2

8555

silver sulphide

+22.62 ‰

IAEA-S-3

 

silver sulphide

-32.49 ‰

IAEA-SO-5

 

barium sulphate

+0.49 ‰

IAEA-SO-6

 

barium sulphate

-34.05 ‰

NBS-127

8557

barium sulphate

+21.12 ‰

In-House Standard Materials - corrected to Internationals

 

Material #

Material Type

34S Value

EIL-40

 

copper sulphide

-0.23 ‰

EIL-41

 

lead sulphate

+11.00‰

EIL-42

 

barium sulphate

-4.70‰

EIL-43

 

zinc sulphide

+21.00 ‰

NIST-1577b

 

bovine liver

+7.60 ‰

NIST-2976

 

mussel

+18.70 ‰

http://www.ciaaw.org/vendors.htm

Quality Assurance / Quality Control (QA/QC)

Of the total sample number dropped in an analytical run, in the order of 100-160 depending on the material type, every 5th or 6th sample is repeated and no less than 20% are Std/Ref materials. These Std/Ref measurements are used in data normalization and to ensure daily mass spec precision and accuracy; also to assess linearity issues or mass spec drift throughout the duration of the run. With these QA/QC checks an error of 0.3‰ δ34S for standards are required for reportable data.

Minimum Concentration

For δ34S analysis, the mass spectrometer requires ~0.03 mg of sulphur whether ~0.1 mg sulphide or 0.2 mg BaSO4. Materials having sulphur content below 0.5% become a combustion issue which may increase the ± error further until any reliable analysis is impossible (materials below 0.25%).

References

REES, C. E. The isotopic analysis of sulphur and oxygen. In: McMaster University. Isotopic, Nuclear and Geochemical Studies Group. 1984;139.

MORRISON, J., Fallick, T., Donelly, T., Leossen, M., St. Jean, G. and Drimmie, R. J. d34S measurements of standards from several laboratories by continuous flow isotope ratio mass spectrometry (CF-IRMS). Micromass UK Ltd. Technical Note 309. 1996.

18O Analysis in Solid Samples

Solid Sample Submission

The bulk of the solid oxygen analysis at EIL is in the form of BaSO4 from groundwater.

Pre-weighed for the cheapest and fastest turnaround:

We currently accept only limited client pre-weighed sample submission for client pre-weighed sample submission for oxygen analysis. Contact us for further information.

EIL weighed samples:

Most samples for solid oxygen analysis are weighed here at EIL for a set cost.

The material (10-100mg) must be dried and ground to a powder and submitted in small glass vial. Sample homogeneity is very important as only a small amount is required for analysis.

Sulphate extraction from water is a separate cost – contact us for details.

Template for EA Sample Tray Submission (XLSX)

Equipment:

The analysis of solid materials for 18O isotope measurements is determined through high temperature (1450OC) pyrolysis combustion conversion of sample material to CO gas. This is accomplished using an Elementar Vario Pyro Cube elemental analyzer coupled to an Isoprime (GV Instruments) continuous flow isotope ratio mass spectrometer (CFIRMS).

General Precision details

The δ18O data (δ18O RESULT / VSMOW column) is the corrected delta value, reported in per mil (‰) units, relative to the primary reference scale of VSMOW water.

Data quality control is monitored and corrections made using an array of international reference material and in-house EIL standards that are calibrated using certified international reference materials (IAEA-SO-5 + SO-6, NBS-127, IAEA-600, IAEA-601 + 602), with values provided through CIAAW. Whenever possible, we find it best to run like-against-like materials i.e. sulphate Std /Ref materials with sulphate samples. For cellulose analysis (tree ring, sediment cellulose), isotopic values have been determined for cellulose materials (IAEA-CH3, EIL-52 and EIL-54) and are used for data correction of these sample materials.

Reference Materials - International

 
 

Material #

Material Type

18O Value

IAEA-SO-5

 

barium sulphate

+12.13 ‰

IAEA-SO-6

 

barium sulphate

-11.35 ‰

NBS-127

8557

barium sulphate

+8.59 ‰

In-House Standard Materials - corrected to Internationals

 

Material #

Material Type

18O Value

EIL-42

 

barium sulphate

+11.63‰

EIL-46

 

barium sulphate

-5.47‰

http://www.ciaaw.org/vendors.htm

Quality Assurance / Quality Control (QA/QC)

Sample materials are repeated every 4-5 sample. Of the total sample number dropped in an analytical run, no less than 20% are Std/Ref materials. These Std/Ref measurements are used in data normalization and to ensure daily mass spec precision and accuracy; also to assess linearity issues or mass spec drift throughout the duration of the run. With these QA/QC checks, an error of 0.3‰ δ18O are required for reportable data.

Minimum Concentration

For 18O analysis, the mass spec requires ~ 0.03mg of oxygen, which relates to ~ 0.1mg BaSO4 or pure cellulose.

References

FALCONE, M. D., Mark, W. A., Heemskerk, A. R., and Wolfe, B. B. 18O/16O analysis in cellulose using EA-Pyrolysis-IRMS. Environmental Isotope Laboratory Technical Report. 2005;10.1:1-8.

Material Treatments and Other Information

The best control over your research is to oversee sample material manipulation and treatments directly, to insure exactly what you want analyzed, will get analyzed. Work on the EA is a 'bulk’ analysis; all Carbon, Nitrogen, Sulphur and/or Oxygen sources in the material weighed for analysis will get converted to N2, CO2, SO2 and CO analyzed.

13C and/or 15N in Bulk Gas or Pure Liquid Samples

Headspace gas or pure liquid compound samples may be manually injected into the Costech Instruments Elemental Analyzer (CHNS-O ECS 4010 / Milan, Italy) for δ13C and/or δ15N isotopic analyses. Note that EA analysis is a 'bulk’ analysis and all N containing compounds will be converted to N2 and all C containing compounds to CO2, so this analysis type would be for pure compounds only.

Maple Syrup

The Environmental Isotope Lab procedure follows the Association of Official Analytical Chemists (AOAC) standard method 984.23 - Corn Syrup and Cane Sugar in Maple Syrup, Carbon Ratio Mass Spectrometric Method (1988). Samples are run for Stable Carbon Isotope composition on a Carlo Erba Elemental Analyzer (CHNS-O EA1108 - Italy) coupled to a DeltaPlusXL Continuous Flow Stable Isotope Ratio Mass Spectrometer (Thermo Finnigan / Bremen-Germany). Analysis consists of approximately 15 reference material analyses, one of which is a certified pure Maple Syrup from the Ontario Ministry of Agriculture and Foods 1987 and at least duplicate of the sample.

CARRO, O., Hillaire-Marcel, C., et al. Detection of adulterated maple products by stable carbon isotope ratio. Journal Association Official Analytical Chemists. 1980;63(4):840-843.

MORSELLI, M. F. and Baggett, K. L. Mass spectrometric determination of cane sugar and corn syrup in maple syrup by use of 13C/12C ratio: Collaborative study. Journal Association Official Analytical Chemists 1984;67(1):22-24

Assistance with Sample Prep;

If you do need assistance with some of the sample prep work, EIL can help with some aspects at additional cost; there are limitations as some methods can be time as it is labour intensive and we are primarily an analytical facility. We would require a sub-sampled portion of the material you wish analyzed (~500 mg), frozen.

Contact us for more details.

Some Common Pre-Treatment Methods

Any materials suspect of inorganic carbon contamination, (i.e. aquatic plants, inverts, algae, POM as well as sediments or soil profiles) may require an acid wash to have it removed.

Removing Carbonate from BaSO4 for 18O and 34S analysis

  1. Put sample in 20 ml vial filled with 10 ml of de-ionized water.
  2. Reduce pH to 4 but not less than 3, with 10% HCl.
  3. Heat to 95-100°C on a hot plate and wait 30 minutes.
  4. Remove from heat and allow to settle or centrifuge.
  5. Aspirate supernatant and refill with de-ionized H2O.
  6. Allow to settle and again centrifuge/aspirate supernatant. Wash precipitate at least three times with de-ionized water.
  7. Centrifuge/aspirate excess water and dry at no higher than 80C. Store in small vial until analyzed.

Irwin H., Curtis C., Coleman M., 1977. Isotopic evidence for source of diagenetic carbonates formed during burial of organic-rich sediments. Nature 269,209-213

Land L.S., Lang J.C., Barnes D.J., 1977. On the stable carbon and oxygen isotopic composition of some shallow water, ahermatypic, scleractinian coral skeletons. Geochemica et Cosmochemica Acta, 41,169-172

Removing Carbonate from organic samples for 15N and 13C Analysis

This is a gentler touch to minimize possible effects on the organics and Nitrogen in particular

  1. Put sample in 20 ml vial.
  2. Wash with 2% HCl.
  3. Heat to ~50C on a hot plate and wait 90 minutes.
  4. Remove from heat and allow to settle or centrifuge.
  5. Check pH and sample has reached neutral, aspirate supernatant and re-apply fresh acid to assure carbonate has been removed.
  6. Allow to settle and again centrifuge/aspirate supernatant. Wash precipitate at least three times with nano-pure water to neutral.
  7. Centrifuge/aspirate excess water and dry at no higher than 60C. Store in vial until ready to be weighed and analyzed.

Lipid Extraction (Simplified – Modified Combined Folch/Bligh)

  1. Freeze-dry a sample portion (~30 mg) and grind.
  2. Wash with 600 µl 2:1 Chloroform:Methanol (v/v) and let sit for 30 minutes.
  3. Centrifuge for 5 minutes to separate tissue and solvent (containing lipid) and decant or suction liquid to waste.
  4. Repeat washing and centrifuging steps 2 more times.
  5. Oven dry @ 80C and grind tissue pellet in mortar or ball mill as material dictates.

BLIGH, E.G., and Dyer, W.JA rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology. 1959;37:911-917.

FOLCH, J., Lees, M., and Sloane Stanley, G.HA simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry. 1957;226:497-509.

Alternate Lipid Extraction (modified Bligh&Dyer extraction protocol)

Adapted from Summons et al. (1994) and is based on the Kates modification of the original Bligh and Dyer extraction (Bligh and Dyer, 1959; Kates, 1975).

Buffer Solutions

  • 50mM Phosphate buffer: dissolve 8,7g K2HPO4 (or 18,0g Na2HPO4 x 12 H2O) in 1 L MilliQ water and add 3,5ml of a 6N HCl solution (final pH of the buffer should be 7.4), (6N HCl solution: mix 294ml of concentrated HCl (37%) with 206ml of MilliQ water)
  • 50mM TCA buffer: dissolve 50g of trichloroacetic acid in 1 L MilliQ water.

Bligh and Dyer procedure:

  • Measure out X grams of freeze dried and ground sediment into a 100ml centrifuge glass
  • Add 5µl of an appropriate Recovery Standard (RS) to the sample (for example: 1,0mg/ml solution of xx).
  • Add Bligh and Dyer extraction mix in the following proportions (For every 1 gram of sediment):
    • 1 ml DCM
    • 2 ml MeOH
    • 0.8 ml Phosphate buffer (aq.)
  1. Sonicate for 10 minutes in an ultrasonic bath, and centrifuge for 10 minutes at 2000rpm.
  2. Transfer/decant the supernatant to a separatory funnel.
  3. Repeat steps 3 to 6 one more time with the phosphate buffer.
  4. Now, use the TCA-buffer and repeat steps 3 to 6 twice (DCM:MeOH:Buffer, 1:2:0.8)
  5. Add the same amount of DCM you added during the first four extractions and the same amount of water to the separatory funnel to separate the organic phase from the aqueous phase.
  6. Mix phases and wait for layers to separate - they can be cloudy.
  7. Draw off organic layer which is the bottom layer in your funnel. (The best way to get a full separation is to draw off the two layers and put them in the freezer overnight. Then return the organic layer to the separation funnel, draw off the now clear organic fraction.
  8. Add the aqueous layer (from the fridge) to the water left in the separation funnel and draw off the small organic layer that came out of the aqueous fraction.
  9. Extract aqueous fraction 3 times with DCM and combine DCM extracts with DCM layer (discard aqueous fraction).
  10. Return organic fraction to the separation funnel and wash it 3 times with water (discard water) collecting the organic layer in a glass container.
  11. Transfer the extract from the glass container to a 200ml TurboVap vial through a funnel filled with Na2SO4 (place some glass wool at the bottom of the funnel to stop Na2SO4). This step is aimed to dry the extract from remaining water.
  12. Clean the glass container a couple of times with DCM and add it to the content of the TurboVap vial.
  13. Evaporate DCM from the total lipid extract under a flux of nitrogen in the TurboVap evaporator at no more than 37°C.

This methodology was adapted from that of Summons et al. (1994) and is based on the Kates modification of the original Bligh and Dyer extraction (Bligh and Dyer, 1959; Kates, 1975).

BLIGH, E.G., and Dyer, W.JA rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology. 1959;37:911-917.

KATES, M., Work, T.S., Work, E. eds. 1975. Laboratory Techniques in Biochemistry and Molecular Biology: Techniques of lipidology. Amsterdam, Netherlands: Elsevier; 1975.

SUMMONS, R.E., Jahnke, L.L., Roksandic, Z. Carbon isotopic fractionation in lipids from methanotrophic bacteria: Relevance for interpretation of the geochemical record of biomarkers. Geochimica et Cosmochimica Acta. 1994;58:2853-2863.

WHITE, D.C., Ringelberg D. B. In: Burlage RS, Atlas R, Stahl D, Geesey G, Sayler G, eds. Techniques in Microbial Ecology. New York, USA: Oxford University Press; 1998: 255-259.

Collagen Extraction

  1. Clean/degrease in 3:1 Chloroform: methanol to remove lipid and water contamination.
  2. Grind in ball mill or pulverize 1 gm of sample and place in 40 ml glass vial.
  3. Clean mill with DI and ethyl alcohol after each sample.
  4. Pour in 30 ml of 1N HCl (1:10 stock HCl:nano-pure H2O) for 24 hours.
  5. Wash with 30 ml aliquots of nano-pure H2O until pH 3.
  6. Adjust to pH 3 with small drops of HCl if required.
  7. Seal vial and heat to 90°C for 24 hours (34 on Precision Oven control in EIT 5022).
  8. Filter through 0.45µ cellulose Nitrate filter into clean 40 ml vial.
  9. In fume hood, heat to dryness on hotplate.

CORMIE, A.B., and Schwarcz, H., 1996. Effects of climate on deer bone 15N and 13C: lack of precipitation effects on 15N for animals consuming low amounts of C4 plants. Geochemica et Cosmochimica Acta 1996;60(21):4161-4166.