Combining per-aqueous and chiral reversed phase separation modes towards an enhanced comprehensive 2-dimensional liquid chromatographic based chiral screening platform

The differentiation of enantiomers in complex mixtures is crucial in various fields such as food science, pharmaceuticals, and environmental studies. While this is achievable in principle through comprehensive two-dimensional liquid chromatography (LC × LC), practical challenges emerge when the mobile phase used in the first dimension (¹D) is too strong or incompatible with the one in the second dimension (²D), leading to peak broadening and reduced resolution. These drawbacks become particularly evident when analyzing a diverse range of chiral compounds. In order to obtain timely elution of such compounds, typically spanning a range of hydrophobicity, the use of gradients (in both dimensions) is inevitable. This in turn leads to changing mobile phase compositions transferred from ¹D to ²D, and hence to variations in the effectiveness of the modulation process. Lowering of the eluotropic strength of the mobile phase used in ¹D allows to mitigate such problems. In this study, a novel achiral × chiral platform for fully automated screening of chiral compounds was developed. In the ¹D of this platform, reversed HILIC (or per-aqueous liquid chromatography (PALC)) was employed, utilizing a commercially available HILIC column, which ensures robust and reproducible results with a water-rich mobile phase. In ²D, chiral chromatography with a broad range of gradients was utilized. Moreover, the impact of varying concentrations of organic solvent transferred to the ²D on enantioseparation was investigated. The water-rich mobile phase in the ¹D facilitates the complete refocusing of organic solutes before entering the second dimension, preventing the loss of resolution in the ²D. This also allows for longer sampling times, consequently longer ²D running times, reducing the need for ultra-fast columns in the ²D. Furthermore, it enables the application of various mobile phase compositions in the ²D. Finally, this tool was successfully used to analyze compounds in urine and plasma matrices.