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.

Off-line comprehensive size exclusion chromatography × reversed-phase liquid chromatography coupled to high resolution mass spectrometry for the analysis of lignocellulosic biomass products.

Biochemical and thermochemical processes are two pathways to convert lignocellulosic biomass into fuels and chemicals. Both conversion types produce aqueous complex samples containing many oxygenated chemical functions over a wide range of masses. Nowadays, composition of these biomass products is still largely unknown, especially their nonvolatile part (300-1000 Da) mostly made of carbohydrates and their derivatives. In the present study, size exclusion chromatography (SEC) was investigated and applied on water soluble phase of a fast pyrolysis bio-oil (thermochemical conversion) and on aqueous phase of pretreated wheat straw (biochemical conversion). An optimization of mobile phase composition using model molecules was necessary to limit non-steric interactions and elute all chemical families. At the end, separation of carbohydrates, heterosides and aromatic species was performed. The chemical organization of SEC chromatograms was confirmed by coupling SEC with a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) using electrospray ionization (ESI) in the negative mode. On-line SEC-UV/FT-ICR MS hyphenation was a powerful tool to provide exact mass distribution of samples and get molecular formulae classed by chemical family. To go further, the complementarity of SEC with reversed-phase liquid chromatography (RPLC) was established with an off-line comprehensive 2D-LC analysis of the two samples. First, 140 fractions were collected physically from SEC separation for each sample, then each fraction was analyzed by RPLC hyphenated to an Ion Trap - Time of Flight mass spectrometer (SEC × RPLC-UV/IT-TOF MS) using ESI in both positive and negative modes. This comprehensive approach combining 2D-LC and high resolution mass spectrometry nearly doubled the number of peaks detected in comparison with 1D RPLC analysis and thus offered well resolved 2D contour plots, considered as relevant analytical fingerprints of the aqueous phase of biomass samples.

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.

Centrifugal partition chromatography as a fractionation tool for the analysis of lignocellulosic biomass products by liquid chromatography coupled to mass spectrometry.

The conversion of lignocellulosic biomass into biofuels and bio-products leads to oxygenated matrices having a wide range of polarities and molecular weights. A complete analytical characterization of these complex mixtures is necessary to improve conversion processes. In this study, an innovative centrifugal partition chromatography (CPC) protocol was developed to fractionate aqueous biomass samples with a MTBE-water solvent system, by mixing elution, displacement and extrusion modes in the same run. This new protocol was validated on model molecules and applied to the water soluble phase of a fast pyrolysis bio-oil. It demonstrated a promising separation with a relevant selectivity on the most significant chemical families of biomass samples: carbohydrates, furans, carboxylic acids and phenols. CPC fractions of the sample were collected and analyzed comprehensively by HPLC-UV/MS (with ESI negative and positive ionization modes). This CPC x LC approach allowed more accurate attributions on the 217 peaks detected. The use of different detection modes gave a complete view of the water soluble phase of a fast pyrolysis bio-oil through 2D maps. Molecular characterization was enhanced by independent information: CPC retention time, LC retention time, UV and MS spectra. Concomitance of these different chemical information is of precious help for unambiguous identification.

Centrifugal partition chromatography a first dimension for biomass fast pyrolysis oil analysis.

Biomass fast pyrolysis oils contain molecules having a large variety of chemical functions and a wide range of molecular weights (from several tens to several thousand grams per mole). The good knowledge of their complex composition is essential for optimizing the conversion of bio-oils to biofuels, thereby requiring powerful separation techniques. In this work, we investigate the interest of centrifugal partition chromatography (CPC) as a first dimension for the analysis of a bio-oil. A CPC method is proposed to separate oxygen containing compounds according to their partition coefficients in the solvent system. This approach is a powerful and easy-to-use technique that enables fractionation of a bio-oil at a semi-preparative scale, without any sample loss related to adsorption on the stationary phase. Collected fractions are then injected in liquid chromatography as a second dimension of separation. Contour plot representations of the CPC × LC separation are established to discuss the potential of this approach. These representations can be used as a veritable fingerprint in the comparison of different samples or samples at different steps of a conversion process but also as a powerful tool to identify new compounds and describe the entire composition of the bio-oil.

Ultra-high performance supercritical fluid chromatography hyphenated to atmospheric pressure chemical ionization high resolution mass spectrometry for the characterization of fast pyrolysis bio-oils.

Extensive characterization of complex mixtures requires the combination of powerful analytical techniques. A Supercritical Fluid Chromatography (SFC) method was previously developed, for the specific case of fast pyrolysis bio oils, as an alternative to gas chromatography (GC and GC × GC) or liquid chromatography (LC and LC × LC), both separation methods being generally used prior to mass spectrometry (MS) for the characterization of such complex matrices. In this study we investigated the potential of SFC hyphenated to high resolution mass spectrometry (SFC-HRMS) for this characterization using Negative ion Atmospheric Pressure Chemical ionization ((-)APCI) for the ionization source. The interface between SFC and (-)APCI/HRMS was optimized from a mix of model compounds with the objective of maximizing the signal to noise ratio. The main studied parameters included both make-up flow-rate and make-up composition. A methodology for the treatment of APCI/HRMS data is proposed. This latter allowed for the identification of molecular formulae. Both SFC-APCI/HRMS method and data processing method were applied to a mixture of 36 model compounds, first analyzed alone and then spiked in a bio-oil. In both cases, 19 compounds could be detected. Among them 9 could be detected in a fast pyrolysis bio-oil by targeted analysis. The whole procedure was applied to the characterization of a bio-oil using helpful representations such as mass-plots, van Krevelen diagrams and heteroatom class distributions. Finally the results were compared with those obtained with a Fourier Transform ion-cyclotron resonance mass spectrometer (FT-ICR/MS).

Development of a supercritical fluid chromatography method with ultraviolet and mass spectrometry detection for the characterization of biomass fast pyrolysis bio oils.

The characterization of complex mixtures is a challenging issue for the development of innovative processes dedicated to biofuels and bio-products production. The huge number of compounds present in biomass fast pyrolysis oils combined with the large diversity of chemical functions represent a bottleneck as regards analytical technique development. For the extensive characterization of complex samples, supercritical fluid chromatography (SFC) can be alternative to usual separation techniques such as gas (GC) or liquid chromatography (LC). In this study, an approach is proposed to define the best conditions for the SFC separation of a fast pyrolysis bio-oil. This approach was based on SFC data obtained directly from the bio-oil itself instead of selecting model compounds as usually done. SFC conditions were optimized by using three specific, easy-to-use and quantitative criteria aiming at maximizing the separation power. Polar stationary phases (ethylpyridine bonded silica) associated to a mix of acetonitrile and water as polarity modifier provided the best results, with more than 120 peaks detected in SFC-UV.

Oxygen speciation in upgraded fast pyrolysis bio-oils by comprehensive two-dimensional gas chromatography.

Biomass fast pyrolysis is considered as a promising route to produce liquid for the transportation field from a renewable resource. However, the derived bio-oils are mainly oxygenated (45-50%w/w O on a wet basis) and contain almost no hydrocarbons. Therefore, upgrading is necessary to obtain a liquid with lower oxygen content and characterization of oxygenated compounds in these products is essential to assist conversion reactions. For this purpose, comprehensive two-dimensional gas chromatography (GC × GC) can be investigated. Oxygen speciation in such matrices is hampered by the large diversity of oxygenated families and the complexity of the hydrocarbon matrix. Moreover, response factors must be taken into account for oxygenate quantification as the Flame Ionisation Detector (FID) response varies when a molecule contains heteroatoms. To conclude, no distillation cuts were accessible and the analysis had to cover a large range of boiling points (30-630 °C). To take up this analytical challenge, a thorough optimization approach was developed. In fact, four GC × GC column sets were investigated to separate oxygenated compounds from the hydrocarbon matrix. Both model mixtures and the upgraded biomass flash pyrolysis oil were injected using GC × GC-FID to reach a suitable chromatographic separation. The advantages and drawbacks of each column combination for oxygen speciation in upgraded bio-oils are highlighted in this study. Among the four sets, an original polar × semi-polar column combination was selected and enabled the identification by GC × GC-ToF/MS of more than 40 compounds belonging to eight chemical families: ketones, furans, alcohols, phenols, carboxylic acids, guaiacols, anisols, and esters. For quantification purpose, the GC × GC-FID chromatogram was divided into more than 60 blobs corresponding to the previously identified analyte and hydrocarbon zones. A database associating each blob to a molecule and its specific response factor (determined by standards injection at different concentrations) was created. A detailed molecular quantification by GC × GC-FID was therefore accessible after integration of the corrected normalized areas. This paper aims to present a detail level in terms of characterization of oxygenated compounds in upgraded bio-oils which to our knowledge has never been reached so far. It is based on an original column set selection and an extremely accurate quantification procedure.

Reversal of elution order in a single second dimension by changing the first column nature in comprehensive two-dimensional gas chromatography.

This paper explains how one single stationary phase can involve two different elution orders for linear alkanes, cyclic alkanes, aromatics and phenols using comprehensive two-dimensional gas chromatography. For this purpose, a coal-derived middle distillate was injected in nonpolar×semipolar and polar×semipolar configurations implying the same second dimension stationary phase (trifluoropropyl). Results show that even if the same column is utilised as a second dimension, the group-type elution order is reversed from one combination to the other. This can be explained as follows:for the polar×semipolar combination, each fraction eluting from the first dimension contains species that differ so much in terms of boiling points, that volatility plays a key role in the second isothermal separation. This is exemplified by the separation of a phenol and demonstrated using the proportional relationship between retention times, vapour pressures and activity coefficients. Moreover, van't Hoff plots (plots of ln k vs. 1/T) demonstrated the influence of the elution temperature from the first dimension on the second dimension separation. Therefore, available choice of stationary phase's combinations is much higher considering that one single column leads to very different retentions for similar compounds. Finally, this can explain why a reverse orthogonality approach is usually proficient for the separation of polar compounds.

Using gas chromatography to characterize a direct coal liquefaction naphtha.

Speciation of oxygenated compounds in direct coal liquefaction naphthas is essential considering their important roles in coal conversion reactions. This study attempts to characterize them as fully as possible using gas chromatographic systems. Firstly, GC-MS was deployed allowing the identification of a few ketones, alcohols, and phenols. This conventional analysis was complemented by the application of GC-GC-FID aiming to overcome the coelutions highlighted when using one-dimensional gas chromatography. Heart-cutting and comprehensive two-dimensional gas chromatography were used and the comprehensive system led to better performances as expected considering the complexity of the matrix. In fact, it allowed the identification of more than a hundred of oxygenated compounds belonging to five chemical families: alcohols, ketones, furans, acids and phenols. Average response factors of each of these families were determined by GC×GC-FID using calibration curves and vary from 1 (hydrocarbons) to 2.50 (carboxylic acids). Thanks to a breakthrough columns set involving a trifluoropropyl stationary phase, alcohols and phenols which represent around 14% of the sample were fully identified. A detailed quantification of these species was carried out for the first time in such matrices using the determined response factors. It was concluded that 90% (w/w) of the alcohols are aromatic (phenols), 5% (w/w) are cyclic and 5% (w/w) are linear. A quantification of hydrocarbon families was also achieved and shows that the matrix is mostly naphthenic (56%, w/w), but also contains aromatics (22%, w/w) and paraffins (8%, w/w). This detailed characterization leads to a better understanding of coal conversion processes and is essential to convert them into synthetic fuels.

Considerations on orthogonality duality in comprehensive two-dimensional gas chromatography.

The term "orthogonal" in comprehensive two-dimensional gas chromatography (GC × GC) has a double sided meaning as it stands for a separation resulting from the combination of two independent retention mechanisms (Giddings, J. C. J. High Resolut. Chromatogr. 1987, 10, 319) but also for a 2D separation where the components are evenly distributed over the entire 2D space. It is shown in the present study that a nonorthogonal GC × GC system associating a polar stationary phase in the first dimension (poly(ethylene glycol)) to a nonpolar one in the second dimension (poly(dimethyl siloxane)) leads to a structured chromatogram, a high peak capacity, and a great 2D space occupation. This idea is demonstrated through the characterization of oxygenated compounds in a coal-derived middle distillate. Results show a clear separation between oxygenated species and hydrocarbons which are classified into linear alkanes, cyclic alkanes, and aromatics. A breakthrough configuration combining a polar poly(ethylene glycol) first dimension and a trifluoropropyl methyl stationary phase in the second dimension enabled a unique identification and quantification of linear, cyclic, and aromatic alcohols. This configuration which could be considered as nonorthogonal still involves two different retention mechanisms: polarity and boiling point in the first dimension and electronic interactions in the second dimension. It is selective toward electronegative poles of alcohols and phenols. The contributions of these two configurations compared to a conventional orthogonal system as well as their roles for oxygenated compounds speciation are highlighted. This contribution is measured through three 2D space occupation factors. It appears through these two examples that orthogonality is intimately linked to analyte properties, and a general concept of dimensionality must be considered.

Investigating comprehensive two-dimensional gas chromatography conditions to optimize the separation of oxygenated compounds in a direct coal liquefaction middle distillate.

Considering the global energetic context, diversifying fuels is of growing importance and many new alternatives are promising. Coal liquefaction products definitely appear among the new generation substitutes. These product's characteristics are very far from fuel specifications as they are mainly composed of naphthenes, aromatics, polycondensed naphthenic and aromatic structures and heteroatomic compounds (nitrogen and oxygen), with a very low paraffin content. Identification and quantification of oxygen-containing species in coal-derived liquids are of considerable importance to understand their behaviors in further processing. However, these species have not been characterized as fully as the predominant hydrocarbon components. Literature shows that these compounds consist mainly in alkylated phenolic and furanic structures. Therefore, comprehensive two-dimensional gas chromatography has been investigated to provide enhanced molecular characterization of these complex samples. Several different configurations involving innovative column configurations were tested. Each of them was optimized by testing different column lengths, modulation periods, and oven conditions. A comparison of the contribution of each column configuration was carried out regarding four main criteria: individual separation of oxygenates, group type separation, resolution, and space occupation. One of them enabled an outstanding separation of paraffins, naphthenes, monoaromatics, diaromatics and targeted O-compounds in a direct coal liquefaction product. It was therefore subjected to further experimentations using a time-of-flight mass spectrometer to validate the identification and unravel more than fifty oxygenated molecular structures. A group-type quantification was also established for four column arrangements and gives the distribution of paraffins, naphthenes and aromatics. It can be concluded from this study that a non-orthogonal arrangement involving a highly polar column in the first dimension was the most adapted one.

Using comprehensive two-dimensional gas chromatography for the analysis of oxygenates in middle distillates I. Determination of the nature of biodiesels blend in diesel fuel.

In the current energetic context (increasing consumption of vehicle fuels, greenhouse gas emission etc.) government policies lead to mandatory introduction in fossil fuels of fuels resulting from renewable sources of energy such as biomass. Blending of fatty acid alkyl esters from vegetable oils (also known as biodiesel) with conventional diesel fuel is one of the solutions technologically available; B5 blends (up to 5%w/w esters in fossil fuel) are marketed over Europe. Therefore, for quality control as well as for forensic reasons, it is of major importance to monitor the biodiesel origin (i.e. the fatty acid ester distribution) and its content when it is blend with petroleum diesel. This paper reports a comprehensive two-dimensional gas chromatography (GC x GC) method that was developed for the individual quantitation of fatty acid esters in middle distillates matrices. Several first and the second dimension columns have been investigated and their performances to achieve (i) a group type separation of hydrocarbons and (ii) individual identification and quantitation of fatty acid ester blend with diesel are reported and discussed. Finally, comparison of quantitative GC x GC results with reference methods demonstrates the benefits of GC x GC approach which enables fast and reliable individual quantitation of fatty acid esters in one single run. Results show that under developed chromatographic conditions, quantitative group type analysis of hydrocarbons is also possible, meaning that simultaneous quantification of hydrocarbons and fatty acid esters can be achieved in one single run.