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Application of Hydrophilic Interaction TLC Systems for Separation of Highly Polar Glycosidic Compounds from the Flowers of Selected Verbascum Species

Separation of highly polar compounds, for example, iridoids and triterpene saponins, present in natural samples is a challenging task. Because of their nonvolatility and the lack of chromophores, their analysis by means of gas chromatography or high-performance liquid chromatography (HPLC) is difficult. The use of normal as well as reversed-phase systems fails to resolve highly polar compounds that are strongly adsorbed on polar stationary phases and poorly retained on alkyl-bonded stationary phases usually used in HPLC. Because of these facts, separation has been performed in hydrophilic interaction systems by means of thin-layer chromatography (TLC). Planar chromatography offers also a possibility to use different derivatizing agents to visualize the resolved compounds. Another problem encountered in the analysis of such compounds present in plant extracts is that they occur as multicomponent mixtures. In this article, two-dimensional (2D) TLC systems were used for the resolution of compounds present in the polar fractions of the different Verbascum spp. flower extracts. TLC separations were performed on silica gel plates, with two different mobile phases used in the perpendicular directions. First, the analyzed samples were developed with AcOEt-MeOH-H2 O-25% aqueous NH3 (55:35:9:1, v/v/v/v) and redeveloped with MeOH-AcOEt-H2 O-HCOOH (10:90:26:22, v/v/v/v) in a perpendicular direction. The resolved compounds were visualized using the vanillin-sulfuric acid reagent. The obtained videoscans were further used for preliminary comparative studies of the investigated species. It is for the first time that 2D hydrophilic interaction systems, characterized with different pH values, are being applied for the analysis of highly polar compounds present in selected Verbascum spp. flower extracts. Anew approach of using an image-processing program for the comparative studies and for method validation is also presented.