Evaluation of sample preparation approaches and separation parameters in HPLC of plant bioactive compounds

Plant bioactive compounds can be defined as secondary plant metabolites produced within the plants besides the primary biosynthetic and metabolic routes. They are found to hold various types of important functions in the plants, such as protection, attraction or signalling, and most of them, which are present as "non-nutritive" compounds in plant food, may help to promote optimal health and to reduce the risk of chronic diseases. This communication discusses the results of our recent studies carried out to investigate a variety of factors that influence both the extraction from the plant matrix and the chromatographic behaviour of plant secondary metabolites and other biological molecules of agrochemical and plant physiology interest. These studies have been carried out with the purpose of developing novel analytical separation methods. The presentation evaluates the influence of different methods of sample preparation and of the mobile phase composition on the selective separation of representative compounds of plant interest in reversed-phase high performance liquid chromatography (RP-HPLC). Appropriate selection of the mobile phase in RP-HPLC involves the evaluation of the equilibrium in solution that might take place between the analytes and the components of the liquid phase. The ionogenic nature of most of the considered compounds requires the control of pH, which is performed using suitable buffering agents incorporated into the mobile phase, whereas their elution is performed by varying the content of an organic solvent in the aqueous starting eluent. The constituents of the buffer solutions do not limit their action at controlling the protonic equilibrium of the ionogenic groups of the analytes in solution. They also might interact with the analytes, for examples by an ion-pairing mechanism, with the result of altering their chromatographic retention, which is modulated by the chemical composition and concentration of the organic solvent, which progressively increases during the analysis in gradient elution mode. The application of the results of our studies to investigate the changes of secondary metabolites associated with the establishment and maintenance of diseases in infected plants and on the occurrence of bioactive compounds in edible plants is also discussed.