High-Resolution Characterization of Bio-Oils Using Reversed-Phase LC Coupled to an 18 T FT-ICR Mass Spectrometer

Significance of the topic

High-resolution molecular-level characterization of bio-oils is essential for understanding upgrading pathways, detecting isomeric complexity, and guiding process optimization in biomass valorization. Combining reversed-phase liquid chromatography (RP-LC) with an ultrahigh‑field 18 T Fourier transform ion cyclotron resonance mass spectrometer (FT‑ICR MS) reduces ionization competition, increases observable species, and enables robust mass accuracy across varying ion currents—capabilities critical for accurate chemical family mapping (e.g., lignin derivatives, carbohydrates, lipids) and for assessing upgrading efficiency in complex bio-oil matrices.

Objectives and study overview

The study aimed to demonstrate the analytical advantages of coupling reversed‑phase LC to an 18 T FT‑ICR MS for high‑resolution characterization of upgraded bio‑oils. Specific goals included:

• Comparing LC‑FT‑ICR MS to direct infusion FT‑ICR MS in terms of peak detection, isomer separation, and ionization effects;
• Assessing mass accuracy and attribution error across chromatographic runs and after scan‑by‑scan calibration;
• Characterizing elution patterns and chemical family distributions using van Krevelen analysis;
• Providing proof‑of‑concept for isomer profiling via extracted ion chromatograms (XICs).

Methodology

The workflow combined reversed‑phase liquid chromatography with electrospray ionization in positive mode (ESI(+)) and ultrahigh‑resolution FT‑ICR MS detection. Method development steps included optimization of transient/FID length to balance resolving power and scan rate, ensuring sufficient resolving power at m/z 200 for detailed compositional analysis. Calibration was performed scan‑by‑scan using PyC2MC software to minimize attribution error across the chromatogram. Performance was evaluated by direct comparison between LC‑coupled and direct infusion datasets using van Krevelen diagrams, base peak chromatograms (BPC) of standards, extracted ion chromatograms, and heatmap/average spectrum visualizations for multiple bio‑oil samples.

Used instrumentation

• 18 tesla FT‑ICR mass spectrometer (superconducting magnet) providing extreme resolving power (reported R at m/z 200 up to ~1,300,000 and alternative settings ~680,000).
• Reversed‑phase liquid chromatography system interfaced to ESI(+) source.
• Data processing and calibration with PyC2MC software (scan‑by‑scan calibration to control attribution error).

Main results and discussion

Key findings and their interpretation:

• Substantial gain in detected components: LC‑FT‑ICR MS revealed nearly 1,000 additional peaks relative to direct infusion, attributed to reduced ionization competition when analytes are separated prior to ionization.
• Isomer separation and profiling: Extracted ion chromatograms (examples at m/z 381.13086 and m/z 227.05261) revealed multiple isomeric peaks with distinct elution profiles; putative structures were proposed for resolved isomers, demonstrating that LC adds crucial isomeric information absent from direct infusion.
• Robust mass accuracy across chromatogram: Measured mass shifts for selected ions (e.g., m/z 383.14648) remained very low (<0.2 ppm) over large changes in total ion current, attributed to the 18 T magnet mitigating space‑charge effects in the ICR cell.
• Improved attribution error after calibration: Scan‑by‑scan calibration with PyC2MC reduced assignment errors across retention time; median and interquartile ranges of errors were tracked and lowered compared to pre‑calibration traces.
• Elution trends reflect chemical families: Van Krevelen analysis along chromatographic time revealed consistent elution order—early eluting polar species such as carbohydrates, followed by lignin derivatives, unsaturated hydrocarbons, and later eluting lipids—matching retention behavior observed for a panel of 21 ESI(+) standards whose elution correlated with logP values.
• Spectral visualization and heatmaps: Chromatography‑resolved average spectra and heatmaps clarified compositional shifts across retention time windows and highlighted coelution regions that would obscure minor components in direct infusion.

Benefits and practical applications

Coupling RP‑LC to 18 T FT‑ICR MS delivers several practical advantages for bio‑oil analysis:

• Enhanced detection of low‑abundance components through reduction of ion suppression and competition.
• Resolution of isomers that inform mechanistic interpretation of pyrolysis/upgrading reactions and product selectivity.
• High and stable mass accuracy across chromatographic runs, enabling confident molecular formula assignment in complex matrices.
• Ability to map chemical families by retention time and elemental composition, supporting targeted and non‑targeted monitoring in upgrade process development and QA/QC.

Future trends and potential uses

Potential developments and broader applications include:

• Integration of orthogonal chromatographic modes (e.g., HILIC, normal phase) or multidimensional LC to further enhance isomer separation and coverage of polar and nonpolar fractions.
• Routine use of ultrahigh‑field FT‑ICR MS (≥18 T) in industrial research to provide robust mass accuracy and reduce space‑charge artifacts for quantitative and non‑targeted workflows.
• Application of advanced data processing, machine learning for pattern recognition across chromatographic‑FT‑ICR datasets, and automated structural annotation tools to accelerate interpretation.
• Use in comparative studies to evaluate upgrading catalysts and conditions by tracking molecular class distributions and isomeric shifts.

Conclusion

Reversed‑phase LC coupled to an 18 T FT‑ICR MS significantly improves molecular characterization of bio‑oils compared with direct infusion. The approach increases detectable species, separates isomers, maintains sub‑ppm mass accuracy across variable ion currents, and enables retention‑resolved chemical family mapping. These capabilities provide powerful analytical support for bio‑oil upgrading research, process optimization, and advanced non‑targeted compositional studies.

References

1. Mase C. et al. Journal of Analytical and Applied Pyrolysis 2024, 177.
2. Wootton C. et al. Analytical Chemistry 2024, 96 (28), 11343–11352.