CHAPTER 1: Introduction to Compound-specific Isotope Analysis

Since the 1940s,¹  stable isotope analysis (SIA) has found widespread application in various branches of science. First applications of SIA were mainly geochemical in nature and focused on fundamental aspects such as isotope variations caused by differences in properties of isotopes relating to (i) thermodynamics, (ii) chemical kinetics, (iii) their masses such as in diffusion processes, and (iv) the forces between atoms (thermal diffusion processes).²  Also during that time, the theoretical underpinning of these processes was developed by Urey, Bigeleisen and others.^3–5  Furthermore, the precision of isotope abundance determinations was gradually improved¹  by changes to isotope ratio mass spectrometers (IRMS), including multiple Faraday cup collector systems and better amplifier electronics.^6,7  Groundbreaking applications of SIA during this time include a paleotemperature scale based on ¹⁸O/¹⁶O isotope ratio measurements of fossil carbonate.^1,8,9  The observation by Nier and Gulbransen in 1939 that carbon in nature varies in isotope composition, with living organisms and their remains such as coal, natural gas and petroleum containing less ¹³C than carbonate of limestone, opened the way to using isotopes to study biogeochemical processes and interactions.^10,11  The isotope abundances of nitrogen, oxygen, hydrogen and sulfur caused by exchange processes gave fundamental insights into the evolution of the Earth's crust and atmosphere as well as the history and origin of life.²  It was also recognized early on that variations in natural abundance of the stable isotopes caused by isotope fractionation processes could be utilized as nonradioactive natural or artificial tracers to follow complex geochemical and biological processes in geological cycles, ecosystems, organisms and chemical reactions.^1,12 

Nowadays, a variety of techniques and instrumentation using mass spectrometry or spectroscopic methods for the determination of isotope ratios are available. Mass spectrometry is still the most important technique, both for heavy and light elements.¹³ 

While in general all mass spectrometers are able to measure isotope abundances, dedicated mass spectrometers with precisions in the order of 10⁻⁴ to 10⁻⁶ are mandatory for isotope ratio determination at natural abundance level.^13,14  Such high precision can be obtained by magnetic sector field instruments with Faraday cups enabling simultaneous detection of ion currents from the different mass-to-charge ratios for isotope ratio determination. For heavier elements, thermal ionization¹⁵  and, more recently, inductively coupled plasmas are used as ion sources (thermal ionization mass spectrometry, TIMS,¹⁵  multicollector inductively coupled plasma mass spectrometry, MC-ICP-MS).^16–18  In this book, we mainly focus on the lighter elements carbon (¹³C/¹²C), nitrogen (¹⁵N/¹⁴N), oxygen (¹⁸O/¹⁶O), sulfur (³⁴S/³²S) and hydrogen (²H/¹H), which represent the main elements in biological systems. These elements are typically introduced into an IRMS after conversion to low molecular weight gases such as CO₂, N₂, CO, SO₂ and H₂, which are ionized by electron impact in a tight gas source (see Chapter 3). This fundamental IRMS design was developed by Alfred Nier (see Figure 1.1) and co-workers in the 1940s and is, in principle, still the basis for all modern instruments.^6,19,20  An interesting biographical review about Alfred Nier and his mass spectrometer developments was written by De Laeter and Kunz.²¹  A more detailed discussion of the mass spectrometer instrumentation is given in Chapter 3.

The conversion of analytes to gaseous form was traditionally carried out ‘off-line’; for organic matter, this typically involved combustion or reduction in sealed quartz tubes (tube combustion), with the products cryogenically purified in vacuum lines and transferred to the IRMS via the ‘dual viscous flow inlet system’ or ‘dual inlet’.²²  The dual-inlet was originally introduced by Murphey for thermal gas diffusion investigations.²³  Its incorporation in the IRMS by McKinney and co-workers²⁰  can be considered as the birth of high precision IRMS.¹⁴  With modern dual-inlet systems, as they will be described in Chapter 3, relative ratios at highest precisions (<0.1‰) for the biologically relevant elements discussed here can be achieved.¹⁴  Until the mid-1970s, isotope ratio analysis was carried out exclusively by gas IRMS using such dual-inlet systems.

Today one can distinguish between the dual-inlet or viscous flow inlet system in which the pure sample gas is introduced into the IRMS and ‘continuous flow’-IRMS (CF-IRMS) in which the sample gas is introduced via an inert helium carrier gas stream into the IRMS ion source.

SIA techniques can be classified according to the kind of sample (bulk sample or individual compounds), of which the isotope ratio is analysed (see Figure 1.2).

In ‘bulk stable isotope ratio analysis’ (BSIA), the isotope ratio of the bulk sample is measured. If isotope ratios of individual compounds are analysed, the technique is referred to as ‘compound-specific isotope analysis’ (CSIA). Another interesting application of stable isotope analysis is the intramolecular measurement of isotope ratios of certain atoms within a molecule. Following the precedent set by CSIA, this technique is termed ‘position-specific isotope analysis’ (PSIA).¹⁴ 

It has to be mentioned that a variety of sample preparation devices for special purposes have been developed. Because the focus of this book is on gas chromatography (GC) and liquid chromatography (LC) in combination with IRMS we will mention only a few techniques and refer to the literature^24–26  for more detailed information on special devices, for example, for atmospheric gases, carbonates and water. The bulk analysis of water is carried out with equilibrium devices for controlled oxygen and hydrogen exchange²⁴  or with temperature conversion of water and organic materials on glassy carbon at temperatures >1400°C.^27,28 

For the analysis of trace gases, pre-concentration devices²⁹  as well as membrane inlet devices (MIMS-IRMS)^30,31  and membrane permeation gas chromatography isotope ratio mass spectrometry (MP-GC-IRMS)³²  are used. The measurement of spatially narrow sample compartments, such as tree rings is nowadays carried out by laser ablation prior to GC-IRMS (LA-GC-IRMS).³³ 

Continuous flow-IRMS can be hyphenated with gas chromatographs or, more recently, liquid chromatographs in order to analyse isotope ratios of individual compounds in a complex mixture. In analogy to ‘CSIA’, the term ‘bulk stable isotope ratio analysis’ (BSIA) was coined by K. Habfast.^24,34,35  In Figure 1.3, the fundamental difference between BSIA by EA-IRMS and CSIA by GC-IRMS is illustrated. In BSIA, the compounds are converted to low molecular weight gases (CO₂, N₂, CO, SO₂ and H₂) prior to separation by GC, thus allowing the analysis of isotope ratios of multiple elements, but only of the entire sample,³⁶  in contrast to CSIA, in which compounds in complex mixtures are first separated by GC or LC prior to conversion to low molecular weight gases and subsequent introduction into an IRMS. CSIA allows the differentiation of isotope ratios of individual compounds, but the isotope ratio of only one element at a time can be measured for the separated compounds.

An advantage of CSIA by GC- or LC-IRMS over BSIA is the ease and efficiency of on-line sample preparation and transfer, the ability to measure all compounds in a complex sample mixture in a single data acquisition run, and the significantly reduced sample size requirements.³⁷ 

In the literature, various terms for hyphenation of GC or LC with IRMS are used, which can be confusing for users unfamiliar with this nomenclature. Here, reviews by Douthitt³⁶  and Sessions²²  provide insights into the nomenclature of CSIA instrumentation. Matthews and Hayes coined the term ‘isotope ratio-monitoring GC-MS’ (irm-GC-MS),^22,36  by analogy with the molecular GC-MS technique of selected-ion-monitoring, ‘SIM’.¹⁴  Although often used, we will not use this term in this book since it could easily be mistaken by non-specialists as just another detection mode of an organic mass spectrometer, thus clouding the fundamental differences with an IRMS and the requirements for high precision measurements at natural isotope abundance.

The term ‘GC-IRMS’ was introduced in the 1980s²²  and relates to the coupling of gas chromatography with an IRMS as detector. Other acronyms used today describe the nature of the post GC chemical conversion, including ‘C’, for combustion (GC-C-IRMS), ‘P’ or ‘py’ for pyrolysis (GC-P-IRMS, GC-py-IRMS) or, for direct introduction without modification, GP for ‘general purpose’ (GC-GP-IRMS). Thermo Fisher Scientific uses ‘thermochemolysis’ and ‘thermal conversion’ (TC) in the names of their commercial products (GC-TC-IRMS, TC/EA)²²  as a more accurate description of the chemistry often described as pyrolysis or (incorrectly) as ‘chromium pyrolysis’. To prevent misunderstandings we will generally use GC- or LC-IRMS within this book and mention explicitly the elements measured.

The term ‘compound-specific isotope analysis’ (CSIA) was proposed by Martin Schoell in the title of a workshop at the 203rd ACS meeting in San Francisco in 1992.^38,39  However, Hayes et al. had already used this term in the title of the publication ‘Compound-specific isotopic analyses: A novel tool for reconstruction of ancient biogeochemical processes’ in 1990.⁴⁰  Although CSIA, in principle, relates to all methods for determination of isotope ratios of single compounds, including off-line isolation of compounds from mixtures⁴¹  or bulk analysis of pure substances, it is most frequently restricted to isotope analysis of individual compounds separated by chromatographic methods coupled on-line to an IRMS. In this book we will follow that terminology and focus on CSIA in this sense.

Stimulated by the advances of gas chromatography coupled to organic mass spectrometry (GC-MS) in the second half of the 20th century, attempts were made to combine separation techniques such as GC and LC with an IRMS.

In 1976, Sano et al. studied volatile metabolites after administration of 100ng ¹³C-labelled aspirin after a 24h urine collection. To that end, the metabolites were separated by GC and combusted to CO₂ using an on-line combustion reactor. Isotope ratios of m/z ⁴⁴CO₂ and ⁴⁵CO₂ were measured by quadrupole-MS alternately in 0.5s intervals.^42,43 

Two years later, Matthews and Hayes coupled a conventional single-collector sector field mass spectrometer with a GC. They used a copper-oxide-packed combustion furnace at 750°C inserted between the gas chromatographic column outlet and a GC-MS interface attached to a computer-controlled beam-switching isotope ratio mass spectrometer.⁴⁴  By monitoring relevant ion currents of N₂ and CO₂ for continuous measurement of ¹⁵N/¹⁴N or ¹³C/¹²C ratios, directly comparable isotope ratios for all eluting compounds were obtained, regardless of composition and mass spectrometric behaviour of the parent compounds. Carbon and nitrogen isotope ratios could be measured with a precision of 5‰ or better with 20nmol of CO₂ or 100nmol of N₂. The obtained precisions approached the required precisions for natural abundance variation studies.

In 1984, Barrie from VG and Bricout and Koziet from the research laboratories of Pernod-Ricard in Paris coupled the first on-line combustion interface (a quartz furnace filled with Co₃O₄ at 700°C) between a capillary GC and a dual collector isotope ratio mass spectrometer,⁴⁵  thus permitting continuous recording of isotope ratios by detecting two successive masses at the same time,^43,46  measuring carbon isotope ratios from 8nmol CO₂ to precisions <1‰. A vent valve at the end of the GC column acted as backflush to prevent solvent peaks from entering the combustion oven.⁴⁷  The aim of this coupling was to generate data useful for the authenticity control of flavour compounds and ethanol.⁴⁷  GC-IRMS instrumentation has been commercially available since 1988, when devices for coupling GC with an IRMS were introduced at the 11th International Mass Spectrometry Conference in Bordeaux.⁴⁷ 

Two systems that demonstrated GC-IRMS determination of nitrogen isotope ratios for derivatized amino acids were introduced in 1992.²²  Preston and Slater presented a system with a combustion furnace, liquid nitrogen cold trap for trapping interfering CO₂ and water, as well as a porous layer open tubular ‘PLOT’ column to resolve N₂ from any CO formed by poor conversion.⁴⁸  Merritt and Hayes presented a comparable system but with an additional reduction furnace, loaded with Cu wires maintained at 600°C, to reduce N-oxides to N₂. They reported a precision of 0.2‰ for a sample size of 2nmol of an amino acid.⁴⁹ 

In 1994, Brand et al. introduced a GC-IRMS system for CSIA of oxygen by converting oxygen-containing organic compounds on-line to CO by a pyrolytic reaction (the so called ‘Unterzaucher reaction’) in a high temperature micro-furnace.³⁸  An interface for oxygen isotope ratio measurements with CSIA has been commercially available since 1996.

Compound-specific isotope ratio analysis of hydrogen by GC-IRMS posed a number of analytical challenges, discussed in detail in Chapter 3. Tobias and Brenna demonstrated CSIA for hydrogen by using an on-line combustion micro-reactor filled with CuO and held at 850°C followed by a reduction reactor filled with nickel metal held at 950°C.⁵⁰  Burgoyne and Hayes showed that quantitative pyrolysis can be achieved without metal reductants by using a carbon-lined non-porous alumina tube reactor heated to temperatures >1440°C,⁵¹  a method that was commercialized in 1998.²² 

Apart from chlorine and bromine isotope ratio determination by GC-IRMS,^52–54  recently, GC-quadrupole-MS was used for the determination of chlorine isotope ratios at natural abundance level with acceptable levels of precision for environmental degradation studies.^55–58  Additionally, GC-MC-ICP-MS has been applied to determine chlorine⁵⁹  and bromine^60,61  isotope ratios of organic compounds. So far, no CSIA of sulfur by GC-IRMS is available, although ³⁴S-CSIA by GC-MC-ICP-MS has been reported.^62–64  A more detailed discussion can be found in Chapter 7.

Gas chromatography is restricted to GC-compatible compounds, which can be transferred into the gas phase without thermal degradation. For other compounds, derivatization into a GC-amenable form is necessary. For large molecules, or if isotope fractionation by kinetic isotope effects during derivatization is unavoidable, LC is the method of choice for separation. Several attempts were made over the last decades to combine LC with IRMS, including a chemical reaction interface⁶⁵  and a moving wire^66–69  to remove the water and/or organic molecules in the mobile phase, but these were not commercialized. The first commercially available instrument was introduced in 2004 and is based on wet chemical oxidation (peroxodisulfate and concentrated phosphoric acid) for the conversion of carbon in organic molecules to CO₂ after their elution from the LC column.⁷⁰  This method is restricted to an aqueous mobile phase without organic modifiers or solvents and can only be used for carbon isotope ratio measurements.^13,71 

The interface can also be used without a chromatographic column to measure isotopic signatures of water-soluble pure substances by flow injection analysis (FIA-IRMS).⁷⁰  A detailed discussion of LC-IRMS is given in Chapter 3.

A widely applied method to determine isotope ratios of pure liquid substances (for example, ethanol from wine samples) is site-specific natural isotope fractionation-nuclear magnetic resonance spectroscopy (SNIF-NMR).^72,73  So far, on-line coupling of SNIF-NMR with chromatographic techniques has not been realized and is beyond the scope of this book.

Recently, laser spectroscopy for isotope ratios is an emerging field of investigation and instrumental development. The near future will show which role these methods will play apart from already established isotope ratio measurements of water and carbon dioxide.^74–76  However, these spectroscopic methods are also beyond the scope of this book.

The Scripps Center for Metabolomics and Mass Spectrometry

The home page offers an interactive view of the history of mass spectrometry with links to key publications.

http://masspec.scripps.edu

National Academy of Sciences

Here one can find a detailed biography of Alfred O. C. Nier by John H. Reynolds.

http://www.nap.edu/readingroom.php?book=biomems&page=anier.html

The Official Web Site of the Nobel Prize

The site provides a biography of Harold C. Urey and a link to the Nobel Prize lecture of the year 1934.

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1934/urey-bio.html