Identification of similar and orthogonal chromatographic thin-layer systems for two-dimensional separations of flavonoids and their analogues

In this paper a broad overview of the retention behaviour of twenty flavonoids and their analogues in 74 TLC systems is reported. The compounds were chromatographed on different stationary phases (non-polar and polar-bonded stationary phases — wettable RP18W, silica, and aminopropyl-modified and diol-modified plates) developed with a variety of binary mobile phases (aqueous and non-aqueous). The chemometric techniques principal component analysis and hierarchical clustering were used to evaluate similarities and differences among the chromatographic systems. For selection of the most suitable systems either to perform two-dimensional separation or to enhance overall resolution by merging two stationary phases, indices scoring the separating power of a given system or pair of systems were applied. When separation of all pairs of chromatographic systems was evaluated with the index considered, the theoretical separation achieved in these systems could be visualized as a colour map. This colour map enabled rapid examination of resolution and selection of orthogonal systems enabling acceptable separation of the compounds. On the basis of the colour map it was found that relatively efficient two-dimensional separation of the compounds could be achieved on amino-modified plates developed using 9:1 tetrahydrofuran-water in the first dimension then 9:1, 4:1, 3:1, or 7:3 acetonitrile-water in the second dimension. Theoretically, the best separation could be achieved by combining diol-modified and amino-modified stationary phases and using 3:2 methanol-water and 9:1 acetonitrile-water, respectively, as mobile phases.