Analysis of Lipids and Polycyclic Aromatic Hydrocarbons as Indicators of Past and Present (Micro)Biological Activity

  • Guido L. B. WiesenbergEmail author
  • Martina I. Gocke
Part of the Springer Protocols Handbooks book series (SPH)


Analysis of lipids and hydrocarbons is performed frequently in recent and ancient plant tissues, soils, sediments, peat deposits, oil, rocks, anthropogenic artifacts (archeological samples), and other materials to trace the contribution of different biological and anthropogenic sources of organic matter as well as environmental changes and the fate of organic matter like degradation. The approaches for the analysis of lipids and hydrocarbons strongly vary from traditional methodologies like thin-layer chromatography to universal approaches like pyrolysis, whereas the preparative separation of lipid fractions based on their polarity enables gas-chromatographic analyses of single fractions and compound-specific analysis of stable (2H/1H, 13C/12C) and radioactive (14C) isotope compositions. Often, lipid extraction operationally defines a subfraction of total lipids. On the one hand, free extractable lipids are obtained by extraction with organic solvents, whereas on the other hand, total samples or extraction residues are extracted for more polar lipid fractions using highly polar organic solvents and water, to release bound lipids. Procedures for extraction of free extractable lipids are diverse and mainly defined by the target of research and availability of instrumentation. In the current protocol, state-of-the-art techniques for the investigation of free extractable lipids in various materials are explained, which can be applied even in laboratory environments with limited technical equipment. The protocols cover sample preparation, extraction, purification, analysis, as well as a brief overview of the data evaluation using lipid molecular proxies and compound-specific isotopes.


Alkanes Biomarkers Fatty acids Gas chromatography Lipid extraction Lipid fraction Molecular proxies Preparative separation Solid-phase extraction 


  1. 1.
    Liebig J, Merck E, Mohr F (1837) Das aetherische Oel der Getraide. Ann Pharmacother 24:248–251Google Scholar
  2. 2.
    Eglinton TI, Eglinton G (2008) Molecular proxies for paleoclimatology. Earth Planet Sci Lett 275:1–16CrossRefGoogle Scholar
  3. 3.
    Eglinton G et al (1962) Hydrocarbon constituents of the wax coatings of plant leaves: a taxonomic survey. Phytochemistry 1:89–102CrossRefGoogle Scholar
  4. 4.
    van Mourik JM, Jansen B (2013) The added value of biomarker analysis in palaeopedology; reconstruction of the vegetation during stable periods in a polycyclic driftsand sequence in SE-Netherlands. Quat Int 306:14–23CrossRefGoogle Scholar
  5. 5.
    Jansen B et al (2008) Characteristic straight-chain lipid ratios as a quick method to assess past forest-páramo transitions in the Ecuadorian Andes. Palaeogeogr Palaeoclimatol Palaeoecol 262:129–139CrossRefGoogle Scholar
  6. 6.
    Huang Y et al (1996) Isotope and molecular evidence for the diverse origins of carboxylic acids in leaf fossils and sediments from the Miocene Lake Clarkia deposit, Idaho, U.S.A. Org Geochem 24:289–299CrossRefGoogle Scholar
  7. 7.
    Bush RT, McInerney FA (2013) Leaf wax n-alkane distributions in and across modern plants: implications for paleoecology and chemotaxonomy. Geochim Cosmochim Acta 117:161–179CrossRefGoogle Scholar
  8. 8.
    Schwark L, Zink K, Lechterbeck J (2002) Reconstruction of postglacial to early Holocene vegetation history in terrestrial Central Europe via cuticular lipid biomarkers and pollen records from lake sediments. Geology 30:463–466CrossRefGoogle Scholar
  9. 9.
    Zheng Y et al (2007) Lipid biomarkers in the Zoigê-Hongyuan peat deposit: indicators of Holocene climate changes in West China. Org Geochem 38:1927–1940CrossRefGoogle Scholar
  10. 10.
    Zink K et al (2001) Temperature dependency of long-chain alkenone distributions in recent to fossil limnic sediments and in lake waters. Geochim Cosmochim Acta 65:253–265CrossRefGoogle Scholar
  11. 11.
    Grice K et al (2003) Structural and isotopic analysis of kerogens in sediments rich in free sulfurised Botryococcus braunii biomarkers. Org Geochem 34:471–482CrossRefGoogle Scholar
  12. 12.
    Schouten S et al (2007) Archaeal and bacterial glycerol dialkyl glycerol tetraether lipids in hot springs of Yellowstone National Park. Appl Environ Microbiol 73:6181–6191CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Schwark L, Empt P (2006) Sterane biomarkers as indicators of palaeozoic algal evolution and extinction events. Palaeogeogr Palaeoclimatol Palaeoecol 240:225–236CrossRefGoogle Scholar
  14. 14.
    Yunker MB et al (2002) PAHs in the Fraser River basin: a critical appraisal of PAH ratios as indicators of PAH source and composition. Org Geochem 33:489–515CrossRefGoogle Scholar
  15. 15.
    Eckmeier E, Wiesenberg GLB (2009) Short-chain n-alkanes (C16–20) in ancient soil are useful molecular markers for prehistoric biomass burning. J Archaeol Sci 36:1590–1596CrossRefGoogle Scholar
  16. 16.
    Leythaeuser D, Schwark L, Keuser C (2000) Geological conditions and geochemical effects of secondary petroleum migration and accumulation. Mar Pet Geol 17:857–859CrossRefGoogle Scholar
  17. 17.
    Hallmann COE et al (2007) Temporal resolution of an oil charging history – a case study of residual oil benzocarbazoles from the Gidgealpa Field. Org Geochem 38:1516–1536CrossRefGoogle Scholar
  18. 18.
    Weijers JWH et al (2010) Carbon isotopic composition of branched tetraether membrane lipids in soils suggest a rapid turnover and a heterotrophic life style of their source organism(s). Biogeoscience 7:2959–2973CrossRefGoogle Scholar
  19. 19.
    Wiesenberg GLB et al (2004) Source and turnover of organic matter in agricultural soils derived from n-alkane/n-carboxylic acid compositions and C-isotope signatures. Org Geochem 35:1371–1393CrossRefGoogle Scholar
  20. 20.
    Sachse D, Radke J, Gleixner G (2004) Hydrogen isotope ratios of recent lacustrine sedimentary n-alkanes record modern climate variability. Geochim Cosmochim Acta 68:4877–4889CrossRefGoogle Scholar
  21. 21.
    Jambu P et al (1991) Incorporation of natural hydrocarbons from plant residues into an hydromorphic humic podzol following afforestation and fertilization. J Soil Sci 42:629–636CrossRefGoogle Scholar
  22. 22.
    Cranwell PA (1991) Paleolimnological studies using sequential lipid extraction from recent lacustrine sediment – recognition of source organisms from biomarkers. Hydrobiology 214:293–303CrossRefGoogle Scholar
  23. 23.
    Herbin GA, Robins PA (1968) Studies on plant cuticular waxes –I. The chemotaxonomy of alkanes and alkenes of genus Aloe (Liliaceae). Phytochemistry 7:239–255CrossRefGoogle Scholar
  24. 24.
    Collister JW et al (1992) An isotopic biogeochemical study of the Green River oil shale. Org Geochem 19:265–276CrossRefPubMedGoogle Scholar
  25. 25.
    Ralph J, Hatfield RD (1991) Pyrolysis-GC-MS characterization of forage materials. J Agric Food Chem 39:1426–1437CrossRefGoogle Scholar
  26. 26.
    Beyer L (1996) Soil organic matter composition of spodic horizons in Podzols of the Northwest German Lower Plain. Sci Total Environ 181:167–180CrossRefGoogle Scholar
  27. 27.
    Wiesenberg GLB, Schwark L, Schmidt MWI (2004) Improved automated extraction and separation procedure for soil lipid analyses. Eur J Soil Sci 55:349–356CrossRefGoogle Scholar
  28. 28.
    Baas M et al (2000) A comparative study of lipids in Sphagnum species. Org Geochem 31:535–541CrossRefGoogle Scholar
  29. 29.
    Gülz PG (1968) Normale und verzweigte Alkane in Chloroplastenpräparaten und Blättern von Antirrhinum majus. Phytochemistry 7:1009–1017CrossRefGoogle Scholar
  30. 30.
    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917CrossRefPubMedGoogle Scholar
  31. 31.
    Peters KE, Walters CC, Moldowan JM (2005) The biomarker guide. Cambridge University Press, CambridgeGoogle Scholar
  32. 32.
    van Bergen PF et al (1997) Organic geochemical studies of soils from the Rothamsted classical experiments – I. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk wilderness. Org Geochem 26:117–135CrossRefGoogle Scholar
  33. 33.
    McCarthy RD, Duthie AH (1962) A rapid quantitative method for the separation of free fatty acids from other lipids. J Lipid Res 3:117–119Google Scholar
  34. 34.
    Radke M, Willsch H, Welte DH (1980) Preparative hydrocarbon group type determination by automated medium pressure liquid chromatography. Anal Chem 52:406–411CrossRefGoogle Scholar
  35. 35.
    Bianchi G, Corbellini M (1977) Epicuticular wax of Triticum aestivum DEMAR 4. Phytochemistry 16:943–945CrossRefGoogle Scholar
  36. 36.
    Jetter R, Schäffer S (2001) Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development. Plant Physiol 126:1725–1737CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Luque de Castro MD, García-Ayuso LE (1998) Soxhlet extraction of solid materials: an outdated technique with a promising innovative future. Anal Chim Acta 369:1–10CrossRefGoogle Scholar
  38. 38.
    Jansen B et al (2006) The applicability of accelerated solvent extraction (ASE) to extract lipid biomarkers from soils. Appl Geochem 21:1006–1015CrossRefGoogle Scholar
  39. 39.
    Lüniger G, Schwark L (2002) Characterisation of sedimentary organic matter by bulk and molecular geochemical proxies: an example from Oligocene maar-type Lake Enspel, Germany. Sediment Geol 148:275–288CrossRefGoogle Scholar
  40. 40.
    Shantha NC, Napolitano GE (1992) Gas chromatography of fatty acids. J Chromatogr A 624:37–51CrossRefGoogle Scholar
  41. 41.
    Wang Z, Yang C, Kelly-Hooper F, Hollebone BP, Peng X, Brown CE, Landriault M, Sun J, Yang Z (2009) Forensic differentiation of biogenic compounds from petroleum hydrocarbons in biogenic and petrogenic compounds cross-contaminated soils and sediments. J Chromatogr A 1216:1174–1191CrossRefPubMedGoogle Scholar
  42. 42.
    Wakeham SG, McNichol AP (2014) Transfer of organic carbon through marine water columns to sediments – insights from stable and radiocarbon isotopes of lipid biomarkers. Biogeoscience 11:6895–6914CrossRefGoogle Scholar
  43. 43.
    Maffei M (1996) Chemotaxonomic significance of leaf wax alkanes in the Gramineae. Biochem Syst Ecol 24:53–64CrossRefGoogle Scholar
  44. 44.
    Volkman JK et al (1998) Microalgal biomarkers: a review of recent research developments. Org Geochem 29:1163–1179CrossRefGoogle Scholar
  45. 45.
    Lockheart MJ, van Bergen PF, Evershed RP (2000) Chemotaxonomic classification of fossil leaves from the Miocene Clarkia lake deposit, Idaho, USA based on n-alkyl lipid distributions and principal component analyses. Org Geochem 31:1223–1246CrossRefGoogle Scholar
  46. 46.
    Rommerskirchen F et al (2006) Chemotaxonomic significance of distribution and stable carbon isotopic composition of long-chain alkanes and alkan-1-ols in C4 grass waxes. Org Geochem 37:1303–1332CrossRefGoogle Scholar
  47. 47.
    Harwood JL, Russell NJ (1984) Lipids in plants and microbes. Allen and Unwin, LondonCrossRefGoogle Scholar
  48. 48.
    Maffei M, Badino S, Bossi S (2004) Chemotaxonomic significance of leaf wax n-alkanes in the Pinales (Coniferales). J Biol Res 1:3–19Google Scholar
  49. 49.
    Wiesenberg GLB, Schwark L (2006) Carboxylic acid distribution patterns of temperate C3 and C4 crops. Org Geochem 37:1973–1982CrossRefGoogle Scholar
  50. 50.
    Vogts A et al (2009) Distribution patterns and stable carbon isotopic composition of alkanes and alkan-1-ols from plant waxes of African rain forest and savanna C3 species. Org Geochem 40:1037–1054CrossRefGoogle Scholar
  51. 51.
    Sinninghe Damsté JS et al (2000) Newly discovered non-isoprenoid glycerol dialkyl glycerol tetraether lipids in sediments. Chem Commun 1683–1684Google Scholar
  52. 52.
    Ries-Kautt M, Albrecht P (1989) Hopane-derived triterpenoids in soils. Chem Geol 76:143–151CrossRefGoogle Scholar
  53. 53.
    Didyk BM et al (1978) Organic geochemical indicators of palaeoenvironmental conditions of sedimentation. Nature 272:216–222CrossRefGoogle Scholar
  54. 54.
    Weijers JWH et al (2006) Occurrence and distribution of tetraether membrane lipids in soils: implications for the use of the TEX86 proxy and the BIT index. Org Geochem 37:1680–1693CrossRefGoogle Scholar
  55. 55.
    Garcin Y et al (2012) Hydrogen isotope ratios of lacustrine sedimentary n-alkanes as proxies of tropical African hydrology: insights from a calibration transect across Cameroon. Geochim Cosmochim Acta 79:106–126CrossRefGoogle Scholar
  56. 56.
    Huang Y et al (2004) Hydrogen isotope ratios of individual lipids in lake sediments as novel tracers of climatic and environmental change: a surface sediment test. J Paleolimnol 31:363–375CrossRefGoogle Scholar
  57. 57.
    Chikaraishi Y, Naraoka H (2007) δ13C and δD relationships among three n-alkyl compound classes (n-alkanoic acid, n-alkane and n-alkanol) of terrestrial higher plants. Org Geochem 38:198–215CrossRefGoogle Scholar
  58. 58.
    Cayet C, Lichtfouse É (2001) δ13C of plant-derived n-alkanes in soil particle-size fractions. Org Geochem 32:253–258CrossRefGoogle Scholar
  59. 59.
    Lichtfouse É et al (1997) Molecular, 13C, and 14C evidence for the allochthonous and ancient origin of C16-C18 n-alkanes in modern soils. Geochim Cosmochim Acta 61:1891–1898CrossRefGoogle Scholar
  60. 60.
    Rethemeyer J et al (2004) Complexity of soil organic matter: AMS 14C analysis of soil lipid fractions and individual compounds. Radiocarbon 46:465–473CrossRefGoogle Scholar
  61. 61.
    Bol R et al (1996) The 14C age and residence time of organic matter and its lipid constituents in a stagnohumic gley soil. Eur J Soil Sci 47:215–222CrossRefGoogle Scholar
  62. 62.
    Jenkins BM et al (1996) Emission factors for polycyclic aromatic hydrocarbons from biomass burning. Environ Sci Technol 30:2462–2469CrossRefGoogle Scholar
  63. 63.
    Simoneit BRT, Elias VO (2000) Organic tracers from biomass burning in atmospheric particulate matter over the ocean. Mar Chem 69:301–312CrossRefGoogle Scholar
  64. 64.
    Birk JJ et al (2012) Combined quantification of faecal sterols, stanols, stanones and bile acids in soils and terrestrial sediments by gas chromatography–mass spectrometry. J Chromatogr A 1242:1–10CrossRefPubMedGoogle Scholar
  65. 65.
    Evershed RP (2008) Organic residue analysis in archaeology: the archaeological biomarker revolution. Archaeometry 50:895–924CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of GeographyUniversity of ZurichZurichSwitzerland

Personalised recommendations