Resolving phytosterols in microalgae using offline two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Sterols are unsaponifiable lipids resulting from plant metabolism that exhibit interesting bioactive properties. Microalgae are a major source of specific phytosterols, most of which are still not fully characterized. The similarity in sterol structures and the existence of positional isomers make the separation of phytosterols challenging. A method was developed based on an offline two-dimensional (2D) system, reversed-phase liquid chromatography (RPLC)-supercritical fluid chromatography (SFC)/quadrupole time-of-flight (Q-ToF) mass spectrometry, for the identification of sterols in microalgae. Subsequent positive-mode MS/MS was used to confirm the identified phytosterols. The 2D chromatogram exhibited a pattern related to the positions of the double bonds, which were confirmed by standard injection, enabling structural elucidation. The analysis of the unsaponifiable fraction of two algae, namely Scenedesmus obliquus, a freshwater microalgae, and Padina pavonica, a marine macroalgae, highlighted the ability of the method to distinguish a large number of sterol isomers.

A powerful two-dimensional chromatography method for the non-target analysis of depolymerised lignin.

BACKGROUND: Lignin is an abundant natural polymer obtained as a by-product from the fractionation of lignocellulosic biomass. In the name of a circular economy, lignin should be valorised into valuable chemicals or biomaterials and utilised instead of petrochemicals. For the development of efficient valorisation processes, the structural characterisation of lignin can be highly beneficial. However, this is an arduous task, as the isolated (and sometimes processed) lignin mainly consists of various neutral monomers but also oligomers. In addition, the material contains isomers, which can be especially problematic to separate and identify. RESULTS: We present a powerful off-line comprehensive two-dimensional (2D) chromatography method combining liquid chromatography (LC), supercritical fluid chromatography (SFC), and high-resolution mass spectrometry for the non-target analysis of depolymerised lignin. The implementation of a 1-aminoanthracene column in the second dimension enabled a class separation of potential lignin monomers, dimers, trimers, and tetramers with an additional separation based on the number of hydroxyl groups and steric effects. The pentafluorophenyl column in the first dimension additionally improved the separation based on hydrophobicity. The comparison of off-line 2D LC × SFC to 1D SFC showed that besides the overall improved performance, the first method is also superior for the separation of isomers. Advanced data analysis methods (MS-DIAL, SIRIUS, and Feature-Based Molecular Network) were integrated into the non-target workflow to rapidly visualise and study the detected compounds, which proved to be especially beneficial for the characterisation of the separated isomers. SIGNIFICANCE: The method yielded the first 2D LC plot demonstrating a classification of lignin compounds, which can be applied to compare various lignin sources and processing methods. In addition, the technique demonstrated improved separation of compounds, including isomers, which was especially beneficial as 77 % of the detected compounds had at least one isomer in the same lignin sample.

Off-line two-dimensional separation involving supercritical fluid chromatography for the characterization of the wastewater from algae hydrothermal liquefaction.

An off-line multidimensional method involving liquid chromatography combined with supercritical fluid chromatography was developed for the characterization of the wastewater of hydrothermal liquefaction of microalgae Chlorella sorokiniana. The first dimension consisted of a phenyl hexyl column operated in reversed-phase mode, whereas the second dimension was performed on a diol stationary phase. Optimization of the kinetic parameters of the first and second dimensions were performed, taking into account the fraction collection system. The beneficial effect of working at high flow rate in both dimensions, as well as the need to work with short columns (50 mm) in the second dimension was evidenced. Injection volume was also optimized in both dimensions. The first dimension benefited from on-column focusing, while in the second dimension, untreated water-rich fractions could be injected without peak deformation. The performances of offline LCxSFC were compared to LC-HRMS, SFC-HRMS and LCxLC-HRMS for the analysis of the wastewater. Despite a long analysis time of 3.3h, the off-line separation coupled to high-resolution mass spectrometry exhibited a very large orthogonality with 75 % occupation rate of the separation space, reaching an effective peak capacity of 1050. While other evaluated techniques were faster, one-dimensional techniques failed to separate the numerous isomers while LCxLC exhibited lower orthogonality (45% occupation rate).

Effect of the injection of water-containing diluents on band broadening in analytical supercritical fluid chromatography.

In this study, the impact of introducing water in the sample solvent upon the injection in SFC is investigated. Adsorption of water on the stationary phase was indicated. Using a set of ten neutral test compounds and four ionizable test compounds, spread all along the co-solvent gradient, several parameters were scrutinized (i.e. water content in the sample diluent, nature of the sample diluent, nature of the co-solvent) in regards to peak broadening. From this systematic investigation, the competition for adsorption on the stationary phase between the analytes and the water molecules contained in the diluent was highlighted. The chromatographic peaks of neutral molecules eluting before water molecules were compressed and the ones eluting after were broadened. While the extent of this phenomenon was related to the peak position for neutral molecules, it was not observed on acidic molecules.

Classification of biphasic solvent systems according to Abraham descriptors for countercurrent chromatography.

The method development of liquid-liquid chromatography, either countercurrent chromatography or centrifugal partition chromatography, is slowed down by the selection of the biphasic solvent system that constitutes its column. This paper introduces a classification of 19 solvent systems, including the most popular systems based on heptane/ethyl acetate/methanol/water, some non-aqueous systems and some greener systems. This classification is based on Abraham descriptors determined through the partition coefficients of 43 probes. Among 21 determined models, nine of them allow an accurate prediction of partition coefficients from solute descriptors and another ten provide a description of the chromatographic interactions at the 5% significance level. A graphical tool (spider diagram) is built for the comparison of the chromatographic columns previously characterized with the solvation parameter model. The position of a solvent system in this spider diagram relates to the interactions at stake, thus the selection of columns offering similar or orthogonal interactions is facilitated, with no previous knowledge of the solute required. This semi-empirical strategy cannot fully predict the retention behavior but can judiciously orientate the user towards a limited number of solvent systems to be experimentally tested.

In Silico Screening of Comprehensive Two-Dimensional Centrifugal Partition Chromatography × Liquid Chromatography for Multiple Compound Isolation.

Isolation of unknown compounds for structural identification and the collection of target molecules to generate unavailable standards remain a challenge when dealing with complex samples. While tedious multistep purification is commonly used, it is not appropriate for a limited amount of sample or when a full recovery of expensive molecules is required. Two-dimensional preparative chromatography in a comprehensive mode provides an effective means to collect a large number of molecules in such a case. However, there is currently a lack of metrics to estimate preparative performances with a minimal number of experiments. An in silico comparative study of various pairs of chromatographic systems is proposed, focusing on the occupation rate and the homogeneity of peak spreading in the 2D separation space. Off-line combination of centrifugal partition chromatography (CPC) with liquid chromatography (LC) exhibits numerous advantages for 2D preparative separation. Our in silico approach was illustrated through the isolation of eight bioactive compounds with very similar structures from Cyclopia genistoides plant by CPC×LC. The column screening was performed considering predictive 2D plots in light of the preparative performance descriptors and compared to real 2D preparative separations.

Comparison between centrifugal partition chromatography and preparative liquid chromatography as first dimensions in off-line two-dimensional separation: Application to the isolation of multi-targeted compounds from Edelweiss plant.

Preparative two-dimensional chromatography is gaining interest in the elucidation of complex samples as it allows the recovery of a large number of molecules without the risks inherent to tedious multi-step sample preparation. While the second dimension is often selected to be liquid chromatography, it may be of interest to compare the specificities of two different techniques, namely liquid chromatography and centrifugal partition chromatography, to be used as first dimension. A fair comparison between off-line CPCxLC and prepLCxLC in selective comprehensive mode for preparative purposes is carried out in this study, illustrated by the isolation of five compounds from high-value Edelweiss plant. The method development of each configuration is achieved on laboratory scale instruments. The quality of separation is compared using 2D-contour plots. The prepLCxLC exhibits a large separation space that leads to an overall large peak capacity, which is of great interest for complex samples. But its limited loading capacity involves a large number of 2 D runs increasing the running costs for preparative purposes. On the other hand, CPCxLC provides a low peak capacity due to the poor efficiency provided by CPC. However, this liquid-liquid technique can be finely tuned to generate a high selectivity, decreasing the number of runs necessary to produce a limited number of target solutes.

Two-dimensional multi-heart cutting centrifugal partition chromatography-liquid chromatography for the preparative isolation of antioxidants from Edelweiss plant.

The Edelweiss plant has been recognized as a very valuable source of anti-aging principles due to its composition of antioxidants compounds: leontopodic acid A and 3,5-dicaffeoylquinic acid. In this work, off-line multi-heart cutting CPC-LC separation was set up at industrial scale in order to isolate and produce new high quality reference material of these two antioxidants from Edelweiss. For this purpose, CPC and HPLC methods were developed and optimized at laboratory scale and a comprehensive CPCxHPLC analysis of the crude extract was established. Thereby, the CPC method led to a first separation of the target compounds according to their partition coefficient in the solvent system and the HPLC method was performed on the recovered fractions to lead to a second separation. A 2D CPCxHPLC plot was established in order to know the fractions to select at the industrial scale. Then, the CPC and HPLC methods were transferred at industrial scale and the multi-heart cutting CPC-LC was performed in off-line mode. Using CPC with methyl ter-butyl ether-water 1:1 (v/v) solvent system and LC with Denali C18 column, 2g of crude extract sample were injected and leontopodic acid A and 3,5-dicaffeoylquinic acid were recovered with purity over 97%. The compounds were identified by MS and NMR.

Determination of surfactant bio-sourced origin by isotope-ratio mass spectrometry.

RATIONALE: To develop more eco-friendly laundry detergents, renewable surfactants synthesized from vegetal sources are increasingly being used. In a more stringent regulation context, the determination of bio-sourced surfactant origin thus appears essential to assess the claims of detergent manufacturers. Radiocarbon determination, the standard method for the analysis of bio-sourced materials, is an expensive technique, so there is a need for a cheaper method. METHODS: Here, the use of an elemental analyzer linked to isotope-ratio mass spectrometry (EA/IRMS) is evaluated as an alternative approach to the official method. The δ(18) O, δ(13) C and δ(2) H isotope-ratio values were determined to investigate the bio-sourced origin of surfactant raw materials and mixtures. RESULTS: A sample library of 26 commercial surfactants representative of detergent raw materials was first analyzed by EA/IRMS. The δ(18) O, δ(13) C and δ(2) H values allowed discrimination of synthetic and bio-sourced surfactants. Moreover, in this latter group, C4 plant-derived surfactants were distinguished by their δ(13) C values. Binary and ternary mixtures made of synthetic and bio-sourced surfactants were also analyzed and indicated a linear relationship between mixture isotope-ratio values and surfactant proportions. CONCLUSIONS: IRMS represents a viable alternative to radiocarbon determination for the evaluation of surfactant bio-sourced origin. It is a faster and cheaper technique, allowing discrimination of petroleum- and biomass-derived surfactants and identification of their carbon sources (C4 or C3 plants).

Stable isotope ratio measurements of royal jelly samples for controlling production procedures: impact of sugar feeding.

The carbon and nitrogen stable ratios of royal jelly (RJ) samples from various origins are determined using an elemental analyser linked online to an isotope ratio mass spectrometer to evaluate authenticity and adulteration. The (13)C/(12)C and (15)N/(14)N stable isotope ratios are measured in more than 500 RJs (domestic, imported and derived from feeding experiments) in order to obtain isotopic measurements that take into account seasonal, botanical and geographical effects. Authenticity intervals are established for traditional beekeeping practices, without feeding, in the range -22.48 to -27.90‰ for δ(13)C. For these samples, the δ(15)N values range from -1.58 to 7.98‰, depending on the plant sources of pollen and nectar. The δ(13)C values of the commercial samples vary from -18.54 to -26.58‰. High δ(13)C values are typical of sugar cane or corn syrups which have distinctive isotopic (13)C signatures because both plants use the C4 photosynthetic cycle, in contrast to most RJs which are derived from C3 plants. These differences in the (13)C-isotopic composition allow the detection of the addition of such sugars. RJs from traditional sources and from industrial production by sugar feeding are thus successfully distinguished.