Detailed molecular composition of wood pyrolysis bio-oils revealed by HPLC-FT-ICR MS

Abstract

The highly complex nature of wood pyrolysis bio-oils, which contain thousands of distinct molecular species with varying ionization efficiencies, poses a significant challenge for characterization by direct-infusion high-resolution mass spectrometry. This study presents a novel method combining high-performance liquid chromatography (HPLC) with 21 Tesla Fourier transform ion cyclotron resonance mass spectrometry (21T FT-ICR MS) for detailed molecular characterization of bio-oils within the scope of negative-ion ESI. The HPLC method is optimized to separate polyfunctional oxygen-containing molecules using a polymeric stationary phase with dimethylaminopropyl functionalities, and a methanol-water eluent with dimethylamine. The acidic compounds in bio-oils equilibrate between the DEA-containing mobile phase and the stationary phase, facilitating efficient gradient elution of oxygen-rich species. Coupling online HPLC with 21T FT-ICR MS revealed similar to 3,000 additional monoisotopic O x molecular formulas compared to direct-infusion FT-ICR MS. Newly detected compounds exhibited higher H/C ratios and a wider range of oxygen content, characteristic of low-molecular-weight carbohydrates and species with a composition that resembles biomass. The method enabled the detection of carbohydrate-like species (O/C approximate to 1, H/C approximate to 2) and highly aromatic compounds (H/C < 0.6, O/C < 0.3) that were undetectable via direct infusion. Early eluting, methanol-soluble species showed higher H/C ratios (similar to 1.5 to 2.0) and oxygen content consistent with lignin oligomers, while later-eluting compounds exhibited increased aromaticity, with compositions typical of condensed aromatic species. Advanced data processing using a Python-based, PyC2MC, software package further revealed compositional trends aligned with the solubility of bio-oils. Despite the overlap between LC-MS and direct infusion MS, single ion chromatograms revealed distinct elution patterns for identical molecular formulas, providing insights into potential isomeric diversity that are not accessible through direct infusion analyses. These findings demonstrate the enhanced molecular-level characterization achieved by HPLC-FT-ICR MS, providing key insights into the intricate composition of bio-oils and their potential for energy applications. The proposed approach provides a unique perspective on isomeric diversity and the distribution of functional groups, laying the groundwork for understanding the molecular basis of reactivity and upgrading potential in bio-oils. As the developed method targets the separation and characterization of polyfunctional oxygen-containing species, it can also be applied to dissolved/natural organic matter, photo-oxidation products, and emerging contaminants, e.g., water-soluble species leaching from materials like asphalt and petroleum-based road sealants.