Investigation of the Flavonoids in Croatian Propolis by Thin-Layer Chromatography

Summary Flavonoids and phenolic acids with a variety of biological activity are considered to be the main compounds in propolis–a natural product produced by the honey bee. TLC can be used for rapid screening of pharmacologically active components and to establish the difference between different propolis samples. Our goal was to optimize chromatographic conditions for separation of flavonoids and phenolic acids and to apply the optimized method for analysis of propolis samples from different geographic regions of Croatia. For chromatographic analysis we used 20 cm × 20 cm glass-backed TLC plates coated with 0.25 mm layers of silica gel 60 F254. Ethanolic standard solutions (80%) of the flavonoids and phenolic acids (10 μL) were applied to the plates. Chromatograms were developed at room temperature by ascending development in previously saturated vertical, flat-bottomed glass chambers with glass lids. Visualization was performed in short- and long-wavelength UV light and in long-wavelength UV light after spraying with different reagents. After calculation of R F values numerical taxonomy methods were used to test the efficiency of 11 mobile phases and to optimize chromatographic conditions for separation of 19 standard solutions. We established the most appropriate mobile phases (chloroform–methanol–(98–100%) formic acid, 44.1 + 3 + 2.35, and n-hexane–ethyl acetate–glacial acetic acid, 31 + 14 + 5) for separation of standards. The results obtained were used for analysis of propolis samples. TLC was shown to be a highly suitable method for rapid analysis of propolis samples. It can be used to establish differences between the amounts of pharmacologically active compounds in propolis from different geographic regions of Croatia.