Isomer-Level Description of Complex Bio-Oils Enabled by gTIMS Hyphenated to 18 T FTICR MS

Isomer-Level Description of Complex Bio-Oils Enabled by gTIMS Hyphenated to 18 T FTICR MS

Importance of the topic

Bio-oils produced by thermochemical conversion of biomass are highly complex mixtures containing thousands of molecular formulas and numerous structural isomers. Conventional direct infusion FTICR MS provides exceptional elemental and mass resolving power but does not resolve isomeric complexity. Combining trapped ion mobility spectrometry (TIMS) with ultra-high-field FTICR MS enables separation of isomeric species prior to high-resolution mass analysis, improving molecular-level characterization critical for upgrading strategies, process optimization, and quality control in biofuel research and industrial applications.

Objectives and study overview

• Evaluate the analytical advantage of coupling a sweeping-ramp gated TIMS (gTIMS) to an 18 tesla FTICR MS for isomer-level characterization of bio-oils.
• Compare three types of bio-oils (fast pyrolysis, stabilized pyrolysis, and hydrothermal liquefaction) to determine whether identical molecular formulas correspond to different isomeric distributions depending on feedstock and upgrading.
• Demonstrate regions of interest (ROIs) and Gaussian-based isomer-profile fitting using single-isomer standards to guide interpretation of complex extracted ion mobilograms (EIMs).

Materials and methods

• Samples: Three bio-oils prepared from (1) fast pyrolysis, (2) stabilized fast pyrolysis (post-upgrading), and (3) hydrothermal liquefaction. Samples were dissolved in methanol at 10 mg mL-1.
• Ionization: Electrospray ionization in positive mode was employed for sample introduction.
• Ion mobility: A gated TIMS (sweeping ramp, gTIMS) was used to separate ions by reduced mobility (1/K0) and to generate extracted ion mobilograms for individual m/z values.
• Mass analysis: An 18 T FTICR MS provided ultrahigh mass resolving power (resolution ~1.2×10^6 at m/z 200) with a transient length of 1.3 s.
• Data processing: Gaussian approximations derived from single-isomer standards were used to fit mobility peaks and to estimate the number and width of isomer contributions in complex EIMs. Regions of interest were defined in gTIMS-FTICR heatmaps and correlated with van Krevelen plots to assign compound families.

Used instrumentation

• gTIMS (sweeping ramp mode) coupled to an 18 T FTICR mass spectrometer.
• Electrospray ionization source operated in positive-ion mode.
• Acquisition settings: transient length = 1.3 s; reported resolution ~1.2×10^6 at m/z 200.

Main results and discussion

• Isomer separation: gTIMS resolved distinct mobility features for identical molecular formulas, revealing different isomeric compositions between the three bio-oils. Some m/z values that appeared as single peaks by mass resolved into multiple mobility components.
• Use of standards: Single-isomer standards (e.g., monosaccharides, levoglucosan, representative polyphenols and linear compounds) provided reference mobility peak shapes. Gaussian fits based on these standards allowed deconvolution of complex EIMs and estimation of the number and relative arrival-time distributions of isomers present in bio-oil extracts.
• Compound family mapping: Defining regions of interest in gTIMS-FTICR heatmaps enabled separation of broad chemical families (carbohydrates, polyphenols, linear aliphatics, aromatic CH2 series) and facilitated the assignment of mobility-resolved molecular series across the van Krevelen space.
• Comparative profiles: For specific low-mass ions (examples at m/z 311.1254 and 243.0992) and carbohydrate-related ions (C6H12O6Na+, m/z 203.05261; C6H10O5Na+, m/z 185.04204), mobility profiles differed markedly between bio-oil 1 (fast pyrolysis), bio-oil 2 (stabilized), and bio-oil 3 (hydrothermal liquefaction). Levoglucosan and monosaccharide standards helped to attribute peaks in bio-oil 1 to discrete sugar-type isomers or dehydration products.
• Analytical performance: The combination of gTIMS separation and ultrahigh mass accuracy/resolution of 18 T FTICR MS provided a powerful two-dimensional separation (mobility + m/z) that increases confidence in compositional and structural inferences compared to mass-only analyses.

Benefits and practical applications of the method

• Isomer-level insight: Mobility separation reveals structural heterogeneity hidden in mass spectra, supporting better understanding of reactivity, stability, and upgradeability of bio-oil constituents.
• Process evaluation: Differences in isomeric distributions between raw and upgraded bio-oils can be directly monitored, aiding optimization of stabilization and upgrading procedures.
• Targeted analysis: ROIs and mobility-resolved peak deconvolution enable more selective monitoring of problematic species (e.g., oligomers, sugar derivatives, polymerization products) relevant for fouling or catalyst poisoning.
• Enhanced annotation: Coupling mobility fingerprints with ultrahigh mass accuracy reduces false assignments and supports structural hypothesis generation for complex mixtures.

Future trends and applications

• Quantitation and standardization: Developing calibrated mobility libraries and quantitative workflows will be needed to translate mobility-resolved fingerprints into routine QC metrics for biofuel production.
• Expanded ionization and polarity: Inclusion of negative-ion mode and multiple ionization techniques (e.g., APCI, APPI) will broaden coverage of nonpolar and acid species in bio-oils.
• Hyphenation with separative techniques: Combining gTIMS-FTICR with upstream LC or microfractionation can further reduce spectral congestion and enable targeted structural elucidation.
• Data science integration: Machine learning approaches for automated deconvolution of overlapping mobility peaks and for pattern recognition across production runs will improve throughput and interpretation.
• Industrial deployment: Streamlining instrument interfaces and acquisition speed to meet operational demands will facilitate adoption in refinery R&D and process monitoring.

Conclusion

Coupling gated TIMS operated in a sweeping-ramp mode to an 18 T FTICR MS substantially enhances molecular-level description of complex bio-oils by resolving isomeric complexity that is invisible to mass analysis alone. Using single-isomer standards for Gaussian-based fitting and defining regions of interest in mobility–mass heatmaps enables discrimination of compound families and demonstration that identical molecular formulas in different bio-oils often correspond to distinct isomer distributions. This combined mobility and ultrahigh-resolution mass approach offers valuable capabilities for biofuel research, upgrading evaluation, and future quality-control applications.

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.