On-line two-dimensional liquid chromatography hyphenated to mass spectrometry and ion mobility-mass spectrometry for the separation of carbohydrates from lignocellulosic biomass.

Lignocellulosic biomass is a promising resource of renewable energy. Its transformation to ethanol requires efficient pretreatment leading to complex liquid mixtures made of hundreds of oxygenated analytes. A large part of the released compounds belong to the carbohydrates family. To overcome the complexity of such samples, a comprehensive on-line two-dimensional reversed-phase liquid chromatography hyphenated to high-resolution mass spectrometry (RPLC × RPLC-HRMS) was dedicated to the separation of carbohydrates and more specifically oligomers coming from pretreated lignocellulosic biomass. The first part of this study consisted in the optimization of such hyphenation (i.e. selection of stationary phases, mobile phases, sampling time, etc.). Then, the analytical method was applied to an industrial aqueous biomass product coming from the sulfuric acid-based pretreatment of a wheat straw. Around 70 well-resolved chromatographic peaks corresponding to oligomers were obtained. Occupation of the separation space between each chromatographic dimension was estimated to 75%. In the last part of this study, the interest of ion mobility-mass spectrometry in addition to RPLC × RPLC was discussed. Some examples highlighted the additional separation that can bring ion mobility to RPLC × RPLC-IMS-HRMS method. Using this four-dimensional hyphenation method, each analyte was described by two retention times, the collisional cross section and the molecular formula allowing to reach a level of detail never seen for biomass sample compositions.

Input of an Off-Line, Comprehensive, Three-Dimensional Method (CPC×SFC/HRMS) to Quantify Polycyclic Aromatic Hydrocarbons in Vacuum Gas Oils.

Heavy polycyclic aromatic hydrocarbons (HPAHs) are known to cause undesirable effects in petroleum hydrocracking processes by deactivating the catalysts and accumulating in the downstream of reactors. Polycyclic aromatic hydrocarbons with less than seven rings (PAHs) naturally contained in vacuum gas oils (VGOs) act as precursors in the HPAHs formation. However, getting a detailed quantitative characterization of such polycyclic hydrocarbons has never been done until now, because of the high chemical complexity of VGOs. Thus, an off-line, comprehensive, three-dimensional methodology was required to achieve a quantitative analysis: centrifugal partition chromatography (CPC) as the first dimension of separation, supercritical fluid chromatography (SFC) as the second dimension hyphenated to Fourier transform ion cyclotron resonance mass spectrometry as the third dimension. In this study, we demonstrated that the developed CPC method fractionated samples according to the hydrocarbons' alkylation degree, whereas our SFC method provided an elution order according to their double bond equivalent. Finally, high-resolution mass spectrometry (HRMS) brought crucial information on the identity of analytes and proved to be essential in the event of unresolved peaks from CPC and SFC chromatograms. To assess the ability of the three-dimensional method for quantification purposes, matrix effects were evaluated by spiking VGO samples with deuterated pyrene. A strong ion suppression phenomenon was highlighted when using only SFC/HRMS, whereas no significant matrix effect was observed with the CPC×SFC/HRMS approach. These experiments revealed the great potential of this innovative methodology to quantify both PAH and HPAH in VGOs for the first time.

Characterization of liquid-liquid extraction fractions from lignocellulosic biomass by high performance liquid chromatography hyphenated to tandem high-resolution mass spectrometry.

The conversion of lignocellulosic biomass is a major challenge in the field of renewable energies and bio-based chemicals. The diversity of biomasses and processes leads to complex products having a wide range of polarities and molecular weights. Nowadays, the molecular description of these oxygenated matrices is still largely incomplete and new analytical strategies are required to have a better understanding of biomass products properties. The present study proposes a reliable protocol based on successive liquid-liquid extractions prior to high performance liquid chromatography hyphenated to high-resolution tandem mass spectrometry (HPLC/MSn) using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ/FT-ICR). The protocol allowed to fractionate an industrial sample coming from the sulfuric acid-based pretreatment of a wheat straw into four key chemical families: carbohydrates, organic acids, phenols and neutral compounds. Each fraction was separately analyzed, which limited matrix effects during mass spectrometry ionization step. Electrospray and atmospheric pressure chemical ionization sources were used in both positive and negative modes in order to ionize and detect a maximum of compounds. Thanks to HPLC/MSn, structures of heavy lignin-carbohydrate complexes (LCC) were elucidated (up to 600 g/mol) as well as carbohydrate oligomers having acid functionalities. Mono, di, tri and tetra-aromatic compounds coming from lignin were also detected. The results reported in this paper demonstrate the complexity of pretreated biomass samples and propose an analytical approach from sample simplification to data treatment in order to describe the biomass composition.

A rational strategy based on experimental designs to optimize parameters of a liquid chromatography-mass spectrometry analysis of complex matrices.

A high number of factors controlled by the experimenter has to be optimized to successfully separate, ionize and detect compounds when analyzing complex matrices by liquid chromatography hyphenated to high resolution mass spectrometry (LC-UV/MS). Key steps to manage such hyphenation are focused on desolvation and ionization processes. In this study, a design of experiments approach was used to optimize decisive parameters (i.e. nebulising, drying and sweep gas flow rates, ion transfer capillary voltage and temperature) for electrospray ionization and atmospheric pressure chemical ionization sources both in positive and negative modes. Central composite designs including 131 experiments each were built to cover rationally a sufficiently wide range of operating conditions. Each run was repeated three times to insure stable conditions of ionization and thus a satisfactory repeatability. Extracted ion chromatograms of twelve model oxygenated compounds were integrated and used as responses for experiment designs. Quadratic models for each standard allowed to take into account interactions between factors. Then responses were simultaneously maximized to achieve optimized factors. To illustrate the methodology relevance, optimal conditions were applied to a lignocellulosic biomass fast pyrolysis oil. Thanks to our high sensitivity method, a large number of molecular formulae was identified, as for instance in negative-ion mode electrospray with more than 5500 identified molecular formulae whereas analysis of the same sample by mass spectrometry without any prior chromatographic separation provided less than 2000 molecular formulae. In short, this study proposed a rational methodology to optimize ionization efficiency for LC-UV/MS analysis of complex mixtures.