Investigation of the effects of solvent-mismatch and immiscibility in normal-phase × aqueous reversed-phase liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive separation technique that allows achieving high peak capacities and information on chemical correlations. Unfortunately, its application in industrial practice is still not widespread due to limiting factors such as complex method development, tedious method optimization and solvent-incompatibility (such as solvent-strength mismatch or immiscibility experienced during fraction transfer). A severe case of solvent-incompatibility is encountered in the comprehensive coupling of normal-phase LC and reversed-phase LC (NPLC × RPLC). NPLC × RPLC is considered a desirable LC × LC system, especially for the characterization of synthetic polymers, due to the high orthogonality of the two retention mechanisms. However, its experimental realization often suffers from solvent-injection effects in the RPLC dimension, such as peak-deformation, peak-splitting, or even unretained elution (“breakthrough”) of sample components. Such a decrease in performance or loss of retention is highly dependent on the types of solvents used. To explore the boundaries of solvent compatibility, we applied large-volume injections (LVI) of reference analytes (e.g. alkyl benzenes; ethoxylate and propoxylate polymers) dissolved in water-immiscible sample solvents, such as dichloromethane, n-hexane, and isooctane in fast water-based gradient RPLC separations (using methanol or acetonitrile as eluent). It was found that, when using highly aqueous initial gradient conditions, hydrophobic sample diluents were retained and eluted during the applied gradient. Depending on the relative retention of the retained diluent and the sample analytes, good chromatograms for LVI of immiscible solvents were obtained, comparable with injections under ideal conditions. The conclusions from injection experiments in aqueous RPLC were verified by coupling an NPLC system with a gradient from isooctane to tetrahydrofuran and an RPLC system with a gradient from water to acetonitrile in an online comprehensive NPLC × RPLC separation of a mixture of propoxylate polymers. The separation provided separation of the polymers based on their number of hydroxyl end-groups (NPLC) and oligomer chain-length (RPLC), without suffering from significant band-broadening effects due to solvent-mismatch upon injection in the second-dimension RPLC system.