Structural elucidation of complex polyesters polyols from bio-lubricant using off-line liquid chromatography x supercritical fluid chromatography coupled with Orbitrap mass spectrometry.

Synthetic complex esters and polyol esters are incorporated as partially bio-based and biodegradable alternatives to petroleum base oils in lubricant formulations, to provide specific properties or performance and to help reducing their carbon footprint in certain cases. A sample can contain over 400 molecules of high chemical similarity including numerous isomers. To resolve such complexity, a separation technique with large peak capacity coupled to high-resolution mass spectrometry (HRMS) is essential. In this study, comprehensive off-line LCxSFC hyphenated with an Orbitrap analyzer was used for the structural elucidation of a synthetic bio-lubricant composed of a polyol reacted with fatty acids of varying length or with repetitive units of polyesters of ricinoleic acid. Retention in the LC first dimension was mostly due to the degree of oligomerization of ricinoleic acid within the polyester and to the chain length of the fatty acid. The SFC second dimension highlighted the esterification degree of the polyalcohol and the number and positions of fatty acids double bonds. The combination of both dimensions permitted the separation of isomers. The coupling of SFC with Orbitrap analyzer allowed an accurate assignment of molecular formulas. Finally, the fragmentation in the ionization source confirmed the attributed structures. By introducing a clear distribution of the chemical structures in the retention space, LCxSFC-HRMS provided a powerful analytical method for the comprehensive molecular characterization of the complex polyester polyols sample.

Characterization of positional isomers of drug intermediates by off-line RPLC x SFC hyphenated to high resolution MS.

Many steps are needed in the synthesis of a new active pharmaceutical ingredient (API). In a practical case proposed by a French pharmaceutical company, an intermediate synthesis step, needed to protect 8 hydroxyl groups before oxidation, could produce a mixture of neutral compounds containing up to 652 structures being positional isomers of 18 molecular formulas. Some mixtures allowed obtaining the desired API, others did not. An efficient analytical method was needed to characterize these neutral positional isomers and identify the mixtures to reject. Two samples were provided by the pharmaceutical company: Sample A was conform, Sample B was not. 8 RPLC columns were used with different gradients to screen Sample A. Next, the best RPLC separation was used as the second dimension fast analysis in a comprehensive 2D-RPLC systems. Two columns were used as first dimension: a fluorinated one and a zirconium based one. An order of magnitude was gained in peak capacity, but a better sample characterization was still needed. An off-line RPLC x SFC x Q-TOF/MS analysis was performed collecting 96 RPLC fractions and analyzing them by SFC with Q-TOF/MS detection. A home-made software associated the 96 SFC MS chromatograms to produce either base peak (BPC) or extract ion (EIC) contour plots that allowed for a satisfying characterization of the samples. Subtracting the EIC of expected m/z compounds from the Sample B BPC contour plot produced a unique new contour plot clearly pointing out unexpected compounds explaining the failure of the synthesis and possibly allowing improving the synthesis process.

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.