Impact of reversed phase column pairs in comprehensive two-dimensional liquid chromatography

•We studied the selection of RP column pairs for LC × LC analysis of herbal samples.•We show that fcov depends on the choice of 2nd dimension phase.•The fcov from the carbon-clad phase (CCP) was twice the fcov from the SB-C18 phase.•More peaks were detected with a CCP in the second dimension than with a SB-C18.•Rs was greater with a CCP in the second dimension than with a SB-C18.

A major issue in optimizing the resolving power of two-dimensional chromatographic separations is the choice of the two phases so as to maximize the distribution of the analytes over the separation space. In this work, we studied the choice of appropriate reversed phases to use in on-line comprehensive two-dimensional liquid chromatography (LC × LC). A set of four chemically different conventional bonded reversed phases was used in the first dimension. The second dimension column was either a conventional bonded C18 phase or a carbon-clad phase (CCP). The LC × LC chromatograms and contour plots were all rather similar indicating that the selectivities of the two phases were also similar regardless of the reverse phase column used in the first dimension. Further, the spatial coverage seen with all four first dimension stationary phases when paired with a second dimension C18 phase were low and the retention times were strongly correlated. However, when the C18 column was replaced with the CCP column much improved separations were observed with higher spatial coverages, greater orthogonalities and significant increases in the number of observed peaks.