The analysis of essential oils contained in the different sage (Salvia L.) species by means of TLC/densitometry M. Sajewicz 1, Ł. Wojtal 1, D. Staszek 1, Ł. Krzystanek 1, M. Hajnos 2, T. Kowalska 1, M. Waksmundzka-Hajnos 3 1 Institute of Chemistry, Silesian University, 9 Szkolna Street, Katowice, Poland 2 Department of Pharmacognosy, Medical University of Lublin, 1 Chodźki Str., Lublin, Poland 3 Department of Inorganic Chemistry, Medical University of Lublin, 6 Staszica Street, Lublin, Poland Introduction Essential oils have been recognized for ages now as substances of plant origin that possess highly appreciated curative, cosmetic, and nutritional properties. In purely chemical terms, essential oils are the multi-component mixtures of mono-, di-, tri-, and sesquiterpenes. Structurally, these compounds can belong to the groups of hydrocarbons, alcohols, aldehydes, ketones, esters, and ethers. At ambient temperature, many of them appear as liquids and display an oily consistency. Thin Layer Chromatography (TLC) / Densitometry Preparative TLC Preparative TLC separation of the essential oils was performed at the temperature of -10 O C and the investigated samples were separated into the fractions. Stationary phase: silica gel (the PLC plate 20 cm  20 cm, silica gel 60, 2 mm, cat. # , Merck, Darmstadt Germany); mobile phase: toluene + ethyl acetate, (95:5; v/v). Each fraction was carefully scraped out from the glass plate, then ca. 5 mL methanol was added to each of them, and the entity was ultrasonicated for 30 min. Finally, silica gel was filtered out and the solutions were analyzed with use of GC-MS. The results are shown in Figs 2-4. Fig.1. A comparison of the densitograms obtained from the analytical thin layer chromatograms developed for the five different sage species (i.e., Salvia lavandulifolia L., Salvia triloba L., Salvia nemorosa L., Salvia staminea L., and Salvia hians L.), depending on the temperature of running the TLC experiment. (a) 22 O C and (b) -10 O C. The employed chromatographic plates were covered with silica gel (layer thickness 0.25 mm, Si60 F cm x 20 cm; Merck, Darmstadt, Germany; cat. # ). Mobile phase: toluene + ethyl acetate (95:5; v/v).. (a) (b) Acknowledgement The work of one author (Łukasz Wojtal) was partially supported by the PhD scholarship granted to him in 2008 within the framework of the ‘University as a Partner of the Economy Based on Science’ (UPGOW) project, subsidized by the European Social Fund (EFS) of the European Union. Fig.2. Densitogram of the preparative layer chromatogram for the essential oil derived from Salvia lavandulifolia with use of the Deryng apparatus and spotted on to the plate in the 40-µL aliquot. Development temperature:-10 o C. Below, gas chromatograms are presented of the two preparatively separated fractions. In fractions 2, the following compounds were identified: eucalyptol, thujone, and camphor (based on the NIST library of the spectra). Fig.3. Densitogram of the preparative layer chromatogram for the non- diluted essential oil derived from Salvia triloba with use of the Deryng apparatus and spotted on to the plate in the 40-µL aliquot. Development temperature: -10 o C. Below, gas chromatograms are presented of the two preparatively separated fractions. In fractions 2, the following compounds were identified: eucalyptol, thujone, and camphor (based on the NIST library of the spectra). Experimental GC-MS: The TRACE 2000 model gas chromatograph with the MS TRACE Finnigan model mass detector and the CTC Combi PAL model autosampler. Working conditions of the gas chromatograph: Column, DB-5; 30 m  0.25mm (i.d.)  0.25µm (the film thickness) Carrier gas, helium, p = 100kPa Temperature program, 40°C (3 min); 40 to 150°C ( 8°/min); isothermal conditions, 150°C (15 min) Temperature of injector, 150°C Ionization energy, 70 eV Sample volume: 3 µL Vapour distillation in the Deryng apparatus The dried plant material (50 g) was placed in the round-bottomed flask and 400 mL water was added. Vapour distillation was carried out for 3 h with use of the Deryng apparatus. The procedure is described in Polish Pharmacopoeia VI [1]. Fig.4. Densitogram of the preparative layer chromatogram for the essential oil derived from Salvia hians with use of the Deryng apparatus, diluted in methanol (conc. 2%, v/v) and spotted on to the plate in the 100-µL aliquot. Development temperature: -10 o C. Below, gas chromatograms are presented of the six preparatively separated fractions. Conclusions  Generally, TLC of the volatile organic compounds contained in the essential oils of the sage species is a difficult analytical task.  Due to volatility of the essential oil components, these samples are relatively unstable. Analytical task becomes even more difficult, when TLC is applied as a separation tool, due to the open bed of stationary phase.  Temperature influences the analysis of the volatile compounds contained in the essential oils by means of TLC. Lowering of temperature considerably improves both, separation performance and the yields of the separated fractions.  This study is an introduction to a larger research project devoted to the analysis of the volatile organic compounds contained in the essential oils derived from the different sage species by means of TLC. Reference 1.Polish Pharmacopoeia VI, Polish Pharmaceutical Society, Warsaw, 2002