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Hinrichs Lab - Isotope Ratio Mass Spectrometry (IRMS)

Introduction

The determination of stable elemental isotopic compositions of organic compounds is a strong tool for the study of various biogeochemical and hydrological processes in the environment. Today’s advanced isotope ratio mass spectrometers offer highly precise isotope measurements which are routinely analyzed on all kinds of organic compounds in the Hinrichs Lab.

Two dedicated on-line IRMS systems are available in our group: (1) a newly acquired IRMS system (Delta V Plus) equipped with the GC Isolink interface (GC-IRMS) and an elemental analyzer (EA-IRMS) and (2) an older system (Delta Plus XP) used either in connection with a GC combustion interface III or the liquid chromatography (LC) Isolink interface (LC-IRMS). The former system enables the determination of both C and H isotopes which are transformed after GC separation either via oxidation or high temperature pyrolysis to CO2 or H2, respectively. This new system can be also switched over to an EA thus giving the possibility to determine C and N isotope compositions of bulk organic matter and enriched or pure organic compounds. The latter IRMS system is dominantly used for the determination of C and H isotopes of natural gaseous compounds in the C1 to C6 range, e.g. methane, ethane, dimethyl sulfide, methyl amine etc., but is routinely switched over to the LC Isolink interface for carbon isotopic measurements of VFAs and other water-soluble organic compounds.

Gas chromatography coupled to isotope ratio mass spectrometry (GC-IRMS)

GC-IRMS is a highly specialized instrumental technique and is used in our lab to determine the relative ratio of light stable isotopes of hydrogen (2H/1H) and carbon (13C/12C) in individual compounds of complex sample fractions or mixtures. The stable isotopic composition of organic molecules in natural materials (sediment, soil, cell biomass etc.) varies slightly as a result of isotopic fractionation during physical, chemical and biological processes. These processes are controlled by kinetic or equillibrium isotope effects (KIE or EIE, respectively) with KIEs dominanting in biogeochemical research. In some cases, the relative isotopic ratio of specific compounds is highly diagnostic of key environmental processes (e.g. photosynthesis or methanogenesis). By the use of growth substrates artificially enriched in the heavier isotope and its uptake into living biomass, IRMS determinations aid in the deconvolution highly complex and often enigmatic biogeochemical pathways.

The primary prerequisite for GC-IRMS is that the compounds constituting the sample mixture are amenable to GC, i.e. they are suitably volatile and thermally stable. Polar compounds may require further chemical modification (derivatization) and in such cases the relative stable isotope ratio of the derivatization agent must also be determined. Just as for other GC techniques the sample solution is injected via the GC injector and separated on the capillary column. Single organic compounds eluting from the column then pass through an oxidizing combustion reactor fitted with an alumina tube maintained at 960ºC and containing Cu, Ni and Pt wires, resulting in the formation of CO2 and H2O. Water is then removed by passing the gas stream through a tube fitted with a water permeable Nafion membrane. For hydrogen isotope measurements, instead of combustion a high temperature thermal conversion reactor tube (open alumina) is operated at 1440°C, resulting in the formation of CO, elemental C and H2. The final analyte gas stream (CO2 or H2) is introduced into the ion source of the IRMS and ionization is achieved using electron ionisation (EI) at 150 eV. Ionized gas isotopes are separated in a single magnetic sector analyzer and detected by Faraday cups. Stable isotope ratios are calculated relative to standards of known isotopic composition and expressed using the dimensionless "per mil" notation against the Vienna Pee Dee Belemnite (VPDB) standard for carbon or Vienna Standard Mean Ocean Water (VSMOW) for hydrogen.

Elemental Analyzer coupled to isotope ratio mass spectrometry (EA-IRMS)

Our EA-IRMS instrument coupling is used for the simultaneous determination of bulk organic matter C and N isotopes (δ13C, δ15N) and of C and N concentrations in solid samples, i.e. sediment, soil and cell biomass.

The homogenized samples are weighed into tin capsules, tightly closed and introduced into the EA via an autosampler. The samples are combusted at 999°C in the oxidation oven containing chromium and other metal oxides. This leads to a conversion of sample organic matter into CO2, H2O and N2/NOx. NOx components are subsequently reduced to N2 with elemental copper in the reduction oven at 680°C. Water is eliminated using a chemical trap filled with magnesium perchlorate and target gases CO2 and N2 are separated from each other on a gas chromatographic column. Before entering the IRMS ion source, gas peak areas are automatically adjusted in the Conflo IV interface by helium dilution. Sample's preliminary isotope ratios are measured relative to reference gas pulses (CO2, N2) analyzed during each run with precision routinely checked by standard reference materials (i.e., IAEA-CH-6 and IAEA-N-2). In order to achive C and N concentrations, a daily calibration with a large-volume sediment laboratory standard is performed.

Liquid chromatography coupled to isotope ratio mass spectrometry (LC-IRMS)

In 2004, an on-line LC-IRMS system enabling reproducible and accurate determinations of stable carbon isotope ratios of water-soluble organic compounds was introduced (Krummen et al., 2004). Using the LC Isolink, all organic compounds eluting from a LC column are converted to carbon dioxide with sodium peroxodisulfate and phosphoric acid at high temperature (99.9°C) while maintaining the chromatographic resolution. Separation of gas from the liquid phase is performed in a membrane by counter-flow of helium. The gas is then moved in-line through two tubes fitted with water permeable Nafion membranes to the IRMS inlet where carbon isotopic determination is performed as described above.

The LC-IRMS technique opens a new field of carbon isotopic studies in biogeochemistry with target compounds of interest including for example volatile fatty acids (VFAs), amino acids, amino sugars, carbohydrates, and nucleotides. By appling this technique, we developed and validated a reversed-phase LC method that is suitable for highly-precise carbon isotope analysis of VFAs from sedimentary pore waters and other aqueous solutions (Heuer et al., 2006).

Thermo Scientific Delta V Plus with GC Isolink and EA

Our Thermo Scientific Delta V Plus is a newly acquired IRMS system equipped with a Trace GC ultra and a Flash 2000 EA interfaced via GC Isolink or Conflo IV, respectively. The Trace GC ultra is fitted with a large-volume S/SL and an on-column injector and a TriPlus autosampler. The dynamic signal range (up to 50 V) and its high ohmic resistors facilitate isotopic analysis of both natural abundance and isotopically labelled samples. It is dedicated to compound specfic stable isotope analysis for H and C using the GC Isolink interface and C and N using the Conflo IV interface. Full integration of the ISODAT 3.0 gas isotope data system enables easy set up, tuning and automatic operation.

Specifications:
Sensitivity (continuous flow mode): 1100 molecules CO2 per mass 44 ion
Isotope Ratio Linearity: 0.02 %/nA ion current (m/z 44)
Mass Range: 1-96 Daltons at 3 kV
Mass Resolution: m/Δm = 110 (10% valley)
System Stability: <10 ppm
H3+ Factor: <10 ppm/nA, stability <0.03 ppm/nA/h

Thermo Finnigan Delta Plus XP with Trace GC and LC Isolink

The Thermo Finnigan Delta Plus XP is an IRMS system equipped with a Trace GC ultra and a Thermo Surveyor LC interfaced via GC combustion III or the LC Isolink, respectively. The Trace GC ultra is fitted with a PTV and S/SL injector and an AS3000 autosampler. The Surveyor LC is equipped with an autosampler and PDA detector. The LC Isolink interface additionally harbours a direct injection port for the off-line analysis of water-soluble standards or dissolved organic matter. The IRMS dynamic signal range (up to 50 V) and its high ohmic resistors facilitate isotopic analysis of both natural abundance and isotopically labelled samples. It is dedicated to compound specfic stable isotope analysis for H and C using the GC combustion III interface and C using the LC Isolink interface. Full integration of the ISODAT 2.0 gas isotope data system enables easy set up, tuning and automatic operation.

Specifications:
Sensitivity (continuous flow mode): 1500 molecules CO2 per mass 44 ion
Isotope Ratio Linearity: 0.02 %/nA ion current (m/z 44)
Mass Range: 1-70 Daltons at 3 kV
Mass Resolution: CNOS: m/Δm = 95 (10% valley), H/D: m/Δm = 10 (10% valley)
System Stability: <10 ppm
H3+ Factor: <10 ppm/nA, stability <0.03 ppm/nA/h