Scouting of different separation strategies for phenolic compounds in comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC × LC) offers unique opportunities to resolve structurally diverse analytes that cannot be fully characterized by conventional one-dimensional methods. In this work, different LC × LC configurations combining reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) were systematically investigated for the separation of a 48-compound phenolic standard mixture. Special attention was devoted to the discussion of key method parameters affecting retention behaviour and selectivity in the different LC × LC configurations. Three setups, namely, HILIC × RP-LC, RP-LC × RP-LC, and RP-LC × HILIC were optimized using focusing modulation with active trapping and evaluated in terms of orthogonality, peak capacity, resolution, dilution factor, analysis time, and solvent consumption. Among the tested configurations, HILIC × RP-LC provided the best results in terms of both separation efficiency and operational sustainability with higher corrected peak capacity, reduced dilution, and enhanced throughput. On the one hand, RP-LC × RP-LC offered robust performance with broad applicability due to stationary-phase availability, whereas, RP-LC × HILIC showed complementary selectivity but lower eco-sustainability. The Red, Green, Blue (RGB) additive colour model was applied to integrate analytical, environmental, and productivity metrics, identifying HILIC × RP-LC as the most effective configuration. Finally, the optimized method was applied to the characterization of phenolic profiles in herbal liqueurs, enabling detailed fingerprinting of complex natural matrices. These findings highlight the importance of rationally selecting LC × LC configurations and demonstrate the potential of HILIC × RP-LC with focusing modulation for efficient, sustainable, and information-rich profiling of phenolic compounds.