Studies on the fragmentation mechanisms of deprotonated lignin model compounds and analysis of organosolv lignin mixtures by using mass spectrometry

The search for renewable resources of energy, chemicals, and materials is an active research area owing to the depletion of crude oil and deterioration of the global environment caused by the extensive usage of fossil fuels. Biofuels and/or value-added chemicals obtained via the conversion of renewable lignocellulosic biomass is a promising alternative to nonrenewable crude oil. However, the degradation of biomass usually generates very complex mixtures. The structures of the compounds in the biomass degradation mixtures remain largely unknown. The optimization of the further conversion processes requires a good understanding of the chemical compositions of the complex biomass degradation products.

High-performance liquid chromatography (HPLC) coupled to multi-stage tandem mass spectrometry (HPLC/MSⁿ) based on collision-activated dissociation (CAD) is an extremely powerful technique for the analysis of the complex lignin degradation mixtures. HPLC facilitates the separation of the analytes and MSⁿ provides structural information for the ionized unknown compounds in the mixtures. However, the application of HPLC/MSⁿ for the analysis of the unknown compounds in the above mixtures requires a fundamental understanding of how the ionized biomass-related compounds fragment upon CAD, which is still not well understood. In this research, the CAD mechanisms of the three major fragmentation pathways of deprotonated lignin model compounds with a β-O-4 linkage were probed by using deuterium labeling, synthesis of authentic compounds, and quantum chemical calculations. The structures of the key fragment ions of several pathways were determined by comparison of their CAD mass spectra to those measured for undeuterated and deuterated analogs and for deprotonated authentic compounds. Some of the proposed reaction mechanisms were examined by studying additional deprotonated synthetic model compounds. Quantum chemical calculations were used to delineate the most likely reaction pathways and reaction mechanisms. The quantum chemical calculations were performed by Wanru Li, Erlu Feng, and Dr. John Nash.

An HPLC/HRMSⁿ method was employed to partially separate unknown compounds in an organosolv poplar lignin mixture and to obtain structural information for the ionized unknown compounds. The structures of 62 unknown compounds were elucidated. Based on the proposed structures, compounds in the organosolv lignin sample contain β-O-4, 5-5, β-5, and possibly also 4-O-5 linkages.

The severity of the organosolv treatment affects the chemical compositions of the generated complex mixtures. Understanding the effects of the process severity on the types and relative abundances of the individual compounds in the mixtures is of great importance for the optimization of the treatment and the development of downstream conversion processes. In this study, seven organosolv switchgrass lignin samples prepared at different reaction temperatures and using different acid concentrations and reaction times were initially analyzed by using (-)ESI HRMS and fast pyrolysis coupled with (-)APCI HRMS (py/(-)APCI HRMS). HPLC/(-)ESI HRMSⁿ based on CAD was employed to obtain structural information for the most abundant compounds as well as a β-O-4 dimer with a relatively low abundance. The effects of the process severity on the relative abundances of some abundant compounds, including lignin-carbohydrate complexes, lignin monomers, lignin dimers with a 5-5 and/or a β-O-4 linkage, were probed. The relative abundances of lignin-carbohydrate complexes were found to be high under mild organosolv treatment conditions but become low under moderate and harsh treatment conditions. As lignin compounds with β-O-4 linkages are not stable under acidic conditions, the relative abundances of these compounds were found to be very low. The relative amounts of lignin monomers decreased as the treatment severity increased while the relative abundances of lignin dimers, trimers, and bigger oligomers increased, possibly due to repolymerization reactions under the harsher treatment conditions.