Optimizing transfer and dilution processes when using active solvent modulation in on-line two-dimensional liquid chromatography

Two-dimensional liquid chromatography (2D-LC) is becoming increasingly popular for the analysis of complex samples, which is partly due to the recent introduction of commercial 2D-LC systems. To deal with the mobile phase incompatibility between highly orthogonal retention mechanisms, such as hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC), several strategies have been introduced over the years. One of these strategies is active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension before transfer to the second dimension. This strategy has gained a lot of attention and holds great potential, however, no clear guidelines are currently in place for its use. Therefore, this study aims to investigate how the ASM process can be optimized when using highly incompatible LC combinations, such as HILIC and RPLC, in a simplified selective comprehensive 2D-LC set-up (sHILIC x RPLC) to suggest guidelines for future users. Using a representative sample, the dilution factor (DF), the duration of the ASM phase, the filling percentage of the sample loops, and their unloading configuration are investigated and optimized. It is observed that a DF of 10 with an optimal ASM phase duration, a sample loop filling of maximum 25%, and an unloading configuration in backflush mode, result in the best peak shapes, intensities, and re-coveries for early eluting compounds, while keeping the total analysis time minimal. Based on these results, some general recommendations are made that could also be applied in other 2D-LC modes, such as comprehensive 2D -LC (LC x LC), heart-cutting 2D-LC (LC-LC), and other chromatographic combinations with mobile phase in-compatibility issues.