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Online two-dimensional liquid chromatography can be used under various separation modes but is sometimes limited in terms of orthogonality, especially for the analysis of neutral compounds. The use of SFC in the second dimension offers a wide choice of mobile and stationary phases, suggesting retention properties complementary to those of the first dimension. Initial works on online LCxSFC coupling published in the literature highlighted the first difficulties of solvent compatibility or potential problems with bubbles created by CO 2 in loops. The present work highlights the impact of the interface between the LC and SFC dimensions on the performance of the 2D separation. Six different configurations have been evaluated, differing by the addition of a makeup flow in the first dimension, a division, or a separation of flow paths in the second dimension. Their performances with empty loops eliminated the need for complex trapping columns, regardless of the LC mobile phase nature. Injection in partial fill mode was possible without any problems of CO 2 bubble formation, solvent miscibility, pressure, or modulation repeatability in each of the studied configurations. Selected configurations even allowed for the use of a mobile phase split upstream of the valve, so that the online LCxSFC configuration could be as flexible as LCxLC instrumentation in terms of the first dimension flow rate or second dimension injected volume. The obtained results allowed for building guidelines presenting the best interfaces to set up depending on the nature of the mobile phases in both dimensions. Two applications on monomers of lignin and industrial polymers confirmed preliminary results on configuration testing and illustrated that online LCxSFC could now be easily implemented. Precision tests on retention time were conclusive and are promising for the future use of this technique in industrial routines.

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.

Pseudomonas aeruginosa lung infections are a major cause of death in cystic fibrosis and hospitalized patients. Treating these infections is becoming difficult due to the emergence of conventional antimicrobial multiresistance. While monosaccharides have proved beneficial against such bacterial lung infection, the design of several multivalent glycosylated macromolecules has been shown to be also beneficial on biofilm dispersion. In this study, calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized. The characterization of their inhibitory properties on Pseudomonas aeruginosa aggregation, biofilm formation, adhesion on epithelial cells, and destruction of alveolar tissues were performed. The antiadhesive properties of the designed glycoclusters were demonstrated through several in vitro bioassays. An in vivo mouse model of lung infection provided an almost complete protection against Pseudomonas aeruginosa with the designed glycoclusters.

Counter-current chromatography (CCC) is a preparative separation technique working with the two liquid phases of a biphasic liquid system. One phase is used as the mobile phase when the other, the stationary phase, is held in place by centrifugal fields. Limonene is a biorenewable cycloterpene solvent coming from orange peel waste. It was evaluated as a possible substitute for heptane in CCC separations. The limonene/methanol/water and heptane/methanol/water phase diagrams are very similar at room temperature. The double bonds of the limonene molecule allows for possible π-π interactions with solutes rendering limonene slightly more polar than heptane giving small differences in solute partition coefficients in the two systems. The 24% higher limonene density is a difference with heptane that has major consequences in CCC. The polar and apolar phases of the limonene/methanol/water 10/9/1 v/v have -0.025 g/cm(3) density difference (lower limonene phase) compared to +0.132 g/cm(3) with heptane (upper heptane phase). This precludes the use of this limonene system with hydrodynamic CCC columns that need significant density difference to retain a liquid stationary phase. It is an advantage with hydrostatic CCC columns because density difference is related to the working pressure drop: limonene allows one to work with high centrifugal fields and moderate pressure drop. Limonene has the capability to be a "green" alternative to petroleum-based solvents in CCC applications.