Compound-specific isotope analysis

Compound-specific isotope analysis (CSIA). Application to archaeology, biomedical sciences, biosynthesis, environment, extraterrestrial chemistry, food science, forensic science, humic substances, microbiology, organic geochemistry, soil science and sport. Eric LICHTFOUSE Soil and Environment Laboratories, INRA-ENSAIA/INPL, BP 172, 54505 Vandoeuvre-les-Nancy, France. Eric.Lichtfouse@ensaia.inpl-nancy.fr

ABSTRACT The isotopic composition, e.g. 14C/12C, 13C/12C, 2H/1H, 15N/14N and 18O/16O, of matter elements is heterogeneous. It is ruled by physical, chemical and biological mechanisms. Isotopes thus be enable to follow the fate of mineral and organic compounds during biogeochemical transformations. The determination of the isotopic composition of organic substances occurring at trace level into very complex mixtures such as sediments, soils and blood, has been made possible during the last 20 years due to the rapid development of molecular level isotopic techniques. After a brief glance at pioneer studies revealing isotopic breakthroughs the molecular and intramolecular levels, this paper reviews selected applications of compound-specific isotope analysis in various scientific fields.

INTRODUCTION Most stable isotopic research relies on two concepts. First, natural and artificial chemical reactions fractionate isotopes, thus leading to the occurrence of various organic and inorganic materials having different isotopic compositions. Isotopes can thus record biogeochemical changes. Second, isotopes can be used as natural or artificial tracers to follow the behaviour of organic molecules in complex media such as living organisms and ecosystems. The determination of 13C/12C ratios1 of natural organic matter has been restricted for a long time to the analysis of bulk samples2. Then, the development of gas chromatography coupled to a combustion furnace then to an isotope ratio mass spectrometer3-5 (GC-C-IRMS, Figure) has allowed the analysis of individual substances occurring at trace levels in very complex mixtures. It thus opened new research fields in various scientific areas6-8 such as organic geochemistry9,10, food science11, medecine12, nutrition13, pharmacy14, sport15, phytochemistry16,17, archaeology18, soil science19,20, environment21-25, humic substances26-28, extraterrestrial science29 and forensic science30,31. 15N/14N and 2H/1H ratios of individual molecules have also been measured using a GC coupled to an IRMS32-35. Besides, since GC is limited to the study of volatile substances, another hyphenated technique has been developed, coupling liquid chromatography to an isotopic ratio mass spectrometer via a combustion furnace (LC-C-IRMS), thus enabling high-molecular, polar, or thermally sensible molecules to be analysed36,37. Further, determination of 2H/1H and 13C/12C ratios of each atomic

2 site of a pure substance can be performed by site-specific natural isotope fractionation nuclear magnetic resonance (SNIF-NMR)38-40. 14C dating of individual substances occurring in complex mixtures has been recently achieved using preparative-GC to isolate pure lipids41. Hereafter, in the Early Days section, several examples will show how some pioneers have been able to determine 13C/12C ratios of individual substances, and even of individual atomic sites, before GC-C-IRMS has been available. Then, selected applications of GC-C-IRMS in various scientific fields will be given.

3 CuO 850°C Water trap

• 100°C CO2 m/z = 44, 45, 46 Gas Chromatography Combustion Isotope Ratio
  Mass Spectrometry Time -22‰ -36‰ -15‰ GC-C-IRMS Relative Intensity m/z = 44 ion current FIGURE

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EARLY DAYS Before the advent of hyphenated techniques allowing the on-line purification of complex mixtures, off-line isotopic analysis relied on the availability of a pure substance in reasonable amounts, from about 10µg to 10mg, for quartz tube CuO combustion followed by CO2 transfer into the mass spectrometer. Therefore the analysis of individual compounds occurring in complex media such as living organisms and sediments required careful analytical fractionation to yield pure substances. Here, a milestone has been set by Abelson and Hoering42 who isolated individual amino acids from algal and bacterial cultures by several analytical steps including ion-exchange chromatography. They found that amino acids and lipids were respectively 13C-enriched and 13C-depleted relative to the total organic carbon. Moreover, after ninhydrin decarboxylation of amino acids, they showed that carboxyl groups are strongly enriched in carbon 13 relative to the whole molecule, thus making 13C investigations a promising tool to study biosynthesis. This non-statistical isotopic composition at each atomic site has also been shown by NMR43 for algal amino acids grown on 13C-CO2.

Parker measured 13C/12C ratios of in

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