37 Isolation and Analysis Methods of Volatile Compounds from Flowers and Leaves Valeria Radulescu 1* • Eliza Oprea 2 • Silvia Chiliment 1 1 University of Medicine and Pharmacy “Carol Davila”, Faculty of Pharmacy, Department of Organic Chemistry, 6 Traian Vuia, 020956, Bucharest, Romania 2 University of Bucharest, Faculty of Chemistry, Department of Organic Chemistry, 4-12 Regina Elisabeta, 030018, Bucharest, Romania Corresponding author: *valeriaradulescu@netscape.net Keywords: essential oil, GC-MS, headspace analysis, infusion, Salvia officinalis, solid phase extraction, tincture ABSTRACT The paper has an introductory part, which presents the basic principles for different methods of isolation and determination of volatile compounds from plants. In the experimental part, a comparison is made between the chemical composition of different plant products (essential oil, infusion and tincture), using as research material flowers and leaves of Salvia officinalis. The essential oil from leaves and flowers of S. officinalis was obtained through hydro-distillation in a Neo-Clevenger apparatus. The volatile compounds from infusion and tincture were extracted using solid phase extraction (C18-SPE) and liquid-liquid extraction with hexane and dichloromethane. The volatile oil diluted with dichloromethane and the infusion and tincture extracts were analysed by gas chromatography coupled with mass spectrometry. The results were also compared with those obtained through headspace at 50°C and 80°C. The chemical composition of volatile oils extracted from leaves and flowers are very similar. The only significant difference is a higher contribution of monoterpenic hydrocarbons in flowers (8.59%) compared to leaves (4.54%). The analysis of volatile compounds from S. officinalis leaves shows that through infusion and maceration most of the monoterpenic hydrocarbons are lost. In headspace analysis, the amount of volatile compounds at an equilibrium temperature of 80°C is more than five times higher than at 50°C (the average ratio is 5.24). 1. INTRODUCTION The volatile compounds from ornamental as well as medicinal plants are mainly responsible for their pleasant smell but also for the plants’ therapeutical effects. Studies concerning the chemical composition of these volatile compounds have a long history, their dynamics following a path parallel to that of analytical techniques. The chemical composition of volatile compounds is very complex, due to the large variety of chemical structures – which in turn gives rise to a large variety of physical and chemical properties – and also due to the quantitative differences between compounds, the ratio between the largest and the smallest reaching values as high as 1 : 100,000. In many cases, the compounds responsible for the pleasant/ unpleasant smell of the plant or for its therapeutic effects are not the major ones; these effects may be caused by compounds existing only in very small quantities. Therefore, in order to study the complex chemical composition of volatile compounds from plants, advanced analytical techniques must be used, these allowing the identification of compounds even in minute quantities. Such a technique, used on a very large scale, is the GC-MS (gas chromatography – mass spectrometry) coupling. The results obtained by this kind of analysis vary, depending on the methods used for separating and isolating the volatile compounds from the plants, and also on the manner of preparing the sample for chromatographic analysis. Different parts of medicinal plants are used in phytotherapy, under the form of essential oils, but also as infusions or tinctures. The latter ones are easier to prepare, but fewer studies are made concerning their chemical composition. 1.1. Isolation of volatile oils Isolation of essential oil, contained in various parts of the plant, is carried out using a standardized system of hydrodistillation in a Neo-Clevenger apparatus (Fig. 1), described by various Pharmacopoeias (British Pharmacopoeia 2003). The distillate is collected in a graduated tube and the aqueous phase is automatically recirculated into the distillation flask. This method also allows a quantitative determination of the essential oil. After being isolated, the volatile oil can be diluted with a volatile solvent (dichloromethane) and then analysed by GC-MS. In the case of very low levels of volatile oil in the plant, this separation method cannot be used. Instead, the volatile compounds may be extracted from the hydro-distillate obtained through extraction with an adequate solvent, or through solid phase extraction (SPE).