Recent trends in two-dimensional liquid chromatography

Multi-dimensional liquid chromatography (MD-LC) continues to gain in popularity for applications where conventional one-dimensional liquid chromatography is insufficient to solve the analytical problem at hand. In this review we have focused on articles published in the years 2019 to early 2023 and look for trends using our previous review published in 2018 as a baseline. We have also explored usage patterns related to involvement of industrial laboratories in the published research. The two major areas of technical development have been continued work on modulation strategies that help mitigate problems associated with mobile phase mismatch when coupling complementary separation mechanisms, and development of computer-aided method development strategies. Progress in these areas is making 2D-LC easier to use, and it appears that this is translating to a shift toward more involvement by industrial laboratories. Indeed, over 34% of the more than 200 publications on 2D-LC in the last four years have had at least one-industry affiliated author. A recent inter-laboratory comparison study focused on the performance of a sophisticated multi-stage, multi-dimensional separation for therapeutic protein characterization is an exemplary indication of the increasing investment of industrial laboratories to MD-LC, and we expect this trend to continue for the foreseeable future

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