Laboratory Techniques

SPE coupled FT-ICR MS - characterizing the molecular-level composition of marine organic matter

Solid Phase Extraction (SPE) coupled with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) is a powerful, ultrahigh-resolution technique for characterizing organic matter, particularly in marine environments. This approach enables the identification of molecular sources, transformation pathways, and the persistence of specific compounds, providing critical insights into the marine biological carbon pump.

Laboratory with scientific equipment, computer monitors, and control panels surrounding a large industrial machine labeled 'Bruker 21T FT-ICR'.

21-Tesla FT-ICR MS, National High Magnetic Field Laboratory, Tallahassee, Florida

SPE coupled with FT-ICR MS is a widely applied method for isolating and characterizing marine organic matter (e.g., Kujawinski, 2002; Fleurs et al., 2012; Lechtenfeld et al., 2014; Seidel et al., 2022). SPE is used to concentrate, desalinate, and isolate organic matter from seawater. Typically, samples are acidified (pH ~2) and passed through a sorbent cartridge (most commonly using PPL for marine applications) which retains a broad range of organic compounds, from highly polar to non-polar (Dittmar et al., 2008). After rinsing with acidified ultrapure water to remove salts, the retained organic matter is eluted using an organic solvent, usually methanol. The methanol extract is then directly infused into an FT-ICR MS, typically using negative-mode electrospray ionization (ESI) for marine organic matter. FT-ICR MS provides ultrahigh mass resolution and accuracy, enabling the detection and assignment of thousands of distinct molecular formulae in complex environmental samples. Mass spectra are processed to assign molecular formulae based on mass-to-charge ratios (m/z), which are then categorized into compound classes using elemental ratios such as H/C and O/C.

References:

Kujawinski, E. B. (2002). Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS): characterization of complex environmental mixtures. Environmental Forensics, 3(3-4), 207-216.

Flerus, R., Lechtenfeld, O. J., Koch, B. P., McCallister, S. L., Schmitt-Kopplin, P., Benner, R., ... & Kattner, G. (2012). A molecular perspective on the ageing of marine dissolved organic matter. Biogeosciences, 9(6), 1935-1955.

Lechtenfeld, O. J., Kattner, G., Flerus, R., McCallister, S. L., Schmitt-Kopplin, P., & Koch, B. P. (2014). Molecular transformation and degradation of refractory dissolved organic matter in the Atlantic and Southern Ocean. Geochimica et Cosmochimica Acta, 126, 321-337.

Seidel, M., Vemulapalli, S. P. B., Mathieu, D., & Dittmar, T. (2022). Marine dissolved organic matter shares thousands of molecular formulae yet differs structurally across major water masses. Environmental Science & Technology, 56(6), 3758-3769.

Dittmar, T., Koch, B., Hertkorn, N., & Kattner, G. (2008). A simple and efficient method for the solid‐phase extraction of dissolved organic matter (SPE‐DOM) from seawater. Limnology and Oceanography: Methods, 6(6), 230-235.

“Denitrifier method” - measuring marine nitrogen isotopes

Nitrogen isotopes offer powerful insights into the ocean’s nitrogen cycle, revealing key biogeochemical processes and ecosystem dynamics that enable the evaluation of marine biological carbon pump functionality.

Laboratory setup with scientific equipment, including a Thermo Scientific Delta V Advantage mass spectrometer, on a gray floor with a white wall background.

ThermoFisher Delta V Advantage IRMS interfaced with a Gasbench II, Florida State University

To quantify the nitrogen isotope composition (δ¹⁵N) of nitrate (NO₃⁻) and organic nitrogen pools in marine systems, the denitrifier method is widely employed. This technique enables the conversion of dissolved inorganic nitrogen species (NO₃⁻ + NO₂⁻) into nitrous oxide (N₂O) gas for analysis by isotope ratio mass spectrometry (IRMS) (Sigman et al., 2001; Casciotti et al., 2002; McIlvin & Casciotti, 2011). Relying on a denitrifying bacterial strain that lacks nitrous oxide reductase activity, the denitrifier method quantitatively reduces NO₃⁻ and NO₂⁻ to N₂O without further conversion to dinitrogen (N₂). When the reaction proceeds to completion, the δ¹⁵N of the produced N₂O accurately reflects the isotopic composition of the original NO₃⁻ + NO₂⁻ substrate (Mariotti et al., 1981; Barford et al., 1999).

References:

Sigman, D. M., Casciotti, K. L., Andreani, M., Barford, C., Galanter, M. B. J. K., & Böhlke, J. K. (2001). A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater. Analytical chemistry, 73(17), 4145-4153.

Casciotti, K. L., Sigman, D. M., Hastings, M. G., Böhlke, J. K., & Hilkert, A. (2002). Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method. Analytical chemistry, 74(19), 4905-4912.

McIlvin, M. R., & Casciotti, K. L. (2011). Technical updates to the bacterial method for nitrate isotopic analyses. Analytical Chemistry, 83(5), 1850-1856.

Mariotti, A., Germon, J. C., Hubert, P., Kaiser, P., Letolle, R., Tardieux, A., & Tardieux, P. (1981). Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes. Plant and soil, 62(3), 413-430.

Barford, C. C., Montoya, J. P., Altabet, M. A., & Mitchell, R. (1999). Steady-state nitrogen isotope effects of N2 and N2O production in Paracoccus denitrificans. Applied and Environmental Microbiology, 65(3), 989-994.