Lipid Extraction Techniques for Stable Isotope Analysis and Ecological Assays

  • Kyle H. Elliott
  • James D. Roth
  • Kevin Crook
Part of the Methods in Molecular Biology book series (MIMB, volume 1609)


Lipid extraction is an important component of many ecological and ecotoxicological measurements. For instance, percent lipid is often used as a measure of body condition, under the assumption that those individuals with higher lipid reserves are healthier. Likewise, lipids are depleted in 13C compared with protein, and it is consequently a routine to remove lipids prior to measuring carbon isotopes in ecological studies so that variation in lipid content does not obscure variation in diet. We provide detailed methods for two different protocols for lipid extraction: Soxhlet apparatus and manual distillation. We also provide methods for polar and nonpolar solvents. Neutral (nonpolar) solvents remove some lipids but few non-lipid compounds, whereas polar solvents remove most lipids but also many non-lipid compounds. We discuss each of the methods and provide guidelines for best practices. We recommend that, for stable isotope analysis, researchers test for a relationship between the change in carbon stable isotope ratio and the amount of lipid extracted to see if the degree of extraction has an impact on isotope ratios. Stable isotope analysis is widely used by ecologists, and we provide a detailed methodology that minimizes known biases.

Key words

Lipid extraction Stable isotope analysis Polar lipids Neutral lipids Soxhlet apparatus Ecophysiology Ecotoxicology Diet reconstruction 



The protocol is based on a text developed by I. Burron and D. Mocker. Funding for sample preparation equipment was provided by the Canada Foundation for Innovation.

Supplementary material

371854_1_En_2_MOESM1_ESM.docx (48 kb)
Elliott-Elliot_Figure (DOCX 48 kb)


  1. 1.
    Hussey NE, Macneil MA, McMeans BC, Olin JA, Dudley SF, Cliff G, Wintner SP, Fennessy ST, Fisk AT (2014) Rescaling the trophic structure of marine food webs. Ecol Lett 17(2):239–250. doi: 10.1111/ele.12226 CrossRefPubMedGoogle Scholar
  2. 2.
    Inger R, Bearhop S (2008) Applications of stable isotope analyses to avian ecology. Ibis 150(3):447–461. doi: 10.1111/j.1474-919X.2008.00839.x CrossRefGoogle Scholar
  3. 3.
    Rubenstein DR, Hobson KA (2004) From birds to butterflies: animal movement patterns and stable isotopes. Trends Ecol Evol 19(5):256–263. doi: 10.1016/j.tree.2004.03.017 CrossRefPubMedGoogle Scholar
  4. 4.
    Braune BM, Gaston AJ, Hobson KA, Gilchrist HG, Mallory ML (2014) Changes in food web structure alter trends of mercury uptake at two seabird colonies in the Canadian Arctic. Environ Sci Technol 48(22):13246–13252. doi: 10.1021/es5036249 CrossRefPubMedGoogle Scholar
  5. 5.
    Braune BM, Gaston AJ, Hobson KA, Grant Gilchrist H, Mallory ML (2015) Changes in trophic position affect rates of contaminant decline at two seabird colonies in the Canadian Arctic. Ecotoxicol Environ Saf 115:7–13. doi: 10.1016/j.ecoenv.2015.01.027 CrossRefPubMedGoogle Scholar
  6. 6.
    Elliott JE, Elliott KH (2013) Environmental science. Tracking marine pollution. Science 340(6132):556–558. doi: 10.1126/science.1235197 CrossRefPubMedGoogle Scholar
  7. 7.
    DeNiro MJ, Epstein S (1977) Mechanism of carbon isotope fractionation associated with lipid synthesis. Science 197(4300):261–263CrossRefPubMedGoogle Scholar
  8. 8.
    Oppel S, Federer RN, O’Brien DM, Powell AN, Hollmén TE (2010) Effects of lipid extraction on stable isotope ratios in Avian egg yolk: is arithmetic correction a reliable alternative? The Auk 127(1):72–78. doi: 10.1525/auk.2009.09153 CrossRefGoogle Scholar
  9. 9.
    Post DM, Layman CA, Arrington DA, Takimoto G, Quattrochi J, Montana CG (2007) Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses. Oecologia 152(1):179–189. doi: 10.1007/s00442-006-0630-x CrossRefPubMedGoogle Scholar
  10. 10.
    Ricca MA, Miles AK, Anthony RG, Deng X, Hung SSO (2007) Effect of lipid extraction on analyses of stable carbon and stable nitrogen isotopes in coastal organisms of the Aleutian archipelago. Can J Zool 85(1):40–48. doi: 10.1139/z06-187 CrossRefGoogle Scholar
  11. 11.
    Hussey NE, Olin JA, Kinney MJ, McMeans BC, Fisk AT (2012) Lipid extraction effects on stable isotope values (δ13C and δ15N) of elasmobranch muscle tissue. J Exp Mar Biol Ecol 434–435:7–15. doi: 10.1016/j.jembe.2012.07.012 CrossRefGoogle Scholar
  12. 12.
    Kaufman TJ, Pajuelo M, Bjorndal KA, Bolten AB, Pfaller JB, Williams KL, Vander Zanden HB (2014) Mother-egg stable isotope conversions and effects of lipid extraction and ethanol preservation on loggerhead eggs. Conserv Physiol 2(1):cou049. doi: 10.1093/conphys/cou049 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kojadinovic J, Richard P, Le Corre M, Cosson RP, Bustamante P (2008) Effects of lipid extraction on δ13C and δ15N values in seabird muscle, liver and feathers. Waterbirds 31(2):169–178. doi:10.1675/1524-4695(2008)31[169:EOLEOC]2.0.CO;2CrossRefGoogle Scholar
  14. 14.
    Lesage V, Morin Y, Rioux È, Pomerleau C, Ferguson SH, Pelletier É (2010) Stable isotopes and trace elements as indicators of diet and habitat use in cetaceans: predicting errors related to preservation, lipid extraction, and lipid normalization. Mar Ecol Prog Ser 419:249–265CrossRefGoogle Scholar
  15. 15.
    Logan JM, Jardine TD, Miller TJ, Bunn SE, Cunjak RA, Lutcavage ME (2008) Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods. J Anim Ecol 77(4):838–846. doi: 10.1111/j.1365-2656.2008.01394.x CrossRefPubMedGoogle Scholar
  16. 16.
    Sotiropoulos MA, Tonn WM, Wassenaar LI (2004) Effects of lipid extraction on stable carbon and nitrogen isotope analyses of fish tissues: potential consequences for food web studies. Ecol Freshw Fish 13(3):155–160. doi: 10.1111/j.1600-0633.2004.00056.x CrossRefGoogle Scholar
  17. 17.
    Sweeting CJ, Polunin NV, Jennings S (2004) Tissue and fixative dependent shifts of delta13C and delta15N in preserved ecological material. Rapid Commun Mass Spectrom 18(21):2587–2592. doi: 10.1002/rcm.1661 CrossRefPubMedGoogle Scholar
  18. 18.
    Sweeting CJ, Polunin NV, Jennings S (2006) Effects of chemical lipid extraction and arithmetic lipid correction on stable isotope ratios of fish tissues. Rapid Commun Mass Spectrom 20(4):595–601. doi: 10.1002/rcm.2347 CrossRefPubMedGoogle Scholar
  19. 19.
    Tarroux A, Ehrich D, Lecomte N, Jardine TD, Bêty J, Berteaux D (2010) Sensitivity of stable isotope mixing models to variation in isotopic ratios: evaluating consequences of lipid extraction. Methods Ecol Evol 1(3):231–241. doi: 10.1111/j.2041-210X.2010.00033.x Google Scholar
  20. 20.
    Yurkowski DJ, Hussey NE, Semeniuk C, Ferguson SH, Fisk AT (2015) Effects of lipid extraction and the utility of lipid normalization models on δ13C and δ15N values in Arctic marine mammal tissues. Polar Biol 38(2):131–143. doi: 10.1007/s00300-014-1571-1 CrossRefGoogle Scholar
  21. 21.
    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917. doi: 10.1139/o59-099 CrossRefPubMedGoogle Scholar
  22. 22.
    Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1):497–509PubMedGoogle Scholar
  23. 23.
    Elliott KH, Elliott JE (2016) Lipid extraction techniques for stable isotope analysis of bird eggs: chloroform–methanol leads to more enriched 13C values than extraction via petroleum ether. J Exp Mar Biol Ecol 474:54–57. doi: 10.1016/j.jembe.2015.09.017 CrossRefGoogle Scholar
  24. 24.
    Dobush GR, Ankney CD, Krementz DG (1985) The effect of apparatus, extraction time, and solvent type on lipid extractions of snow geese. Can J Zool 63(8):1917–1920. doi: 10.1139/z85-285 CrossRefGoogle Scholar
  25. 25.
    Logan JM, Lutcavage ME (2008) A comparison of carbon and nitrogen stable isotope ratios of fish tissues following lipid extractions with non-polar and traditional chloroform/methanol solvent systems. Rapid Commun Mass Spectrom 22(7):1081–1086. doi: 10.1002/rcm.3471 CrossRefPubMedGoogle Scholar
  26. 26.
    Schlacher TA, Connolly RM (2014) Effects of acid treatment on carbon and nitrogen stable isotope ratios in ecological samples: a review and synthesis. Methods Ecol Evol 5(6):541–550. doi: 10.1111/2041-210X.12183 CrossRefGoogle Scholar
  27. 27.
    Lee-Thorp JA, Sealy JC, Van Der Merwe NJ (1989) Stable carbon isotope ratio differences between bone collagen and bone apatite, and their relationship to diet. J Archaeol Sci 16:585–599CrossRefGoogle Scholar
  28. 28.
    Paritte JM, Kelly JF (2009) Effect of cleaning regime on stable-isotope ratios of feathers in Japanese Quail (Coturnix Japonica). The Auk 126(1):165–174. doi: 10.1525/auk.2009.07187 CrossRefGoogle Scholar
  29. 29.
    Svensson E, Schouten S, Hopmans EC, Middelburg JJ, Sinninghe Damste JS (2016) Factors controlling the stable nitrogen isotopic composition (delta15N) of lipids in marine animals. PLoS One 11(1):e0146321. doi: 10.1371/journal.pone.0146321 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Natural Resource SciencesMcGill UniversityQuebecCanada
  2. 2.Department of Biological SciencesUniversity of ManitobaWinnipegCanada

Personalised recommendations