Combining per-aqueous and chiral reversed phase separation modes towards an enhanced comprehensive 2-dimensional...

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.