Analysis of Flavoniods and Phenolic Acids in Croatian Propolis by Two-Dimensional Thin-Layer Chromatography

Flavonoids constitute one of the largest groups of naturally occuring phenols and they are ubiquitous in green plants. Phenolic acids and flavonoids, with various biological activities, are considered to be main compounds in propolis – a natural product produced by the honeybee whose composition depends on its geographic origin. Our previous work showed that TLC is suitable method for rapid screening of pharmacologically active compounds. In our present study we wanted to develop a method more suitable for quantitative analysis. We used 2D TLC together with multiple develpoment of chromatograms to obtain better separation of the complex mixture. For chromatographic analysis we used 20 x 20 cm glass-backed TLC plates coated with 0.25 mm layers of silica gel 60 F254 (Merck, Darmstadt). First, we developed the chromatogram in mobile phase composed of chloroform : methanol : acetic acid (44.1:3:2.4) and after evaporation and rotation for 90°, we used n-hexane : ethyl acetate : acetic acid (31:14:5) for the second development [1]. Chromatograms were developed at the room temperature by ascendenting multiple development in previously saturated vertical flat-bottom glass chambers with glass lid (CAMAG, Switzerland). We investigated the presence of 17 flavonoids and 4 phenolic acids in our ethanolic propolis extract [2]. First, we established the RF values of all standards in the given mobile phases and divided them into 5 groups. Each group of standards (10 μ l of 80 % ethanolic solution in three different concentrations) together with the propolis sample was chromatographed on the same plate. Visualization was performed under short (λ = 254 nm) and long wave (λ = 366 nm) UV light, in long wave UV light after spraying with 5% ethanolic solution of AlCl3 and exposing to NH3 vapour and after spraying with different reagents (diazotized sulphanilic acid [3], Folin-Ciocalteu reagent, mixture of 1% ferric chloride and 1% potassium ferricyanide [4]). After establishing the presence of certain standard, composition of the mobile phases will be modified if it will be necessary. All present standards will be chromatographed again and densitometric evaluation of their concentration will be performed. [1] Jasprica I., Smolcic-Bubalo A., Mornar A. and Medic-Saric M.: J. Planar Chromatogr. 2004 (accepted) [2] Park Y. K. and Ikegaki M.: Biosci. Biotechnol. Biochem. 1998, 62, 2230-2232. [3] Markham K. R. :Techniques of Flavonoid Identification. Academic Press, London 1982. [4] Harborne J. B.: Phytochemical Methods. John Wiley & Sons, Inc., New York 1973.