Separation and quantification of plant secondary metabolites and other biological molecules by advance analytical separation methods

This communication reports the results of our recent studies carried out to developing methods for the separation and quantification of plant secondary metabolites and other biological molecules of agrochemical interest, either by capillary electrophoresis (CE) with UV absorbance detection or by high performance liquid chromatography (HPLC) coupled to mass spectrometry. Most of the secondary metabolites investigated are phenolic compounds, which are ubiquitous in the plant kingdom and can be found as free molecules (aglycones), in the form of conjugates (usually as glycosides) and esters, or as oligomers or polymers. Most chromatographic and electrophoretic separations of these analytes are performed in aqueous solutions whose composition is one of the main factors influencing their separation performance. This aspect is particularly relevant for a significant number of phenolic compounds bearing different concomitant functionalities, consisting of ionisable and/or hydrogen-bonding groups, hydrophobic regions, and hydrophilic moieties. Such multifunctional molecules may interact to different extents with the various components of the surrounding aqueous solution and with either the stationary phase or the capillary wall, in chromatography and in capillary electrophoresis, respectively. We illustrate and discuss a variety of factors that influence both electrophoretic and chromatographic behaviour of phenolic compounds of particular interest in plant biology and phytochemistry. The presentation evaluates the influence of the composition of either the electrolyte solution (BGE) or the mobile phase on the selective separation of representative phenolic compounds in capillary zone electrophoresis (CZE) and in reversed phase liquid chromatography (RP-HPLC), respectively. Appropriate selection of the composition of either the BGE or the mobile phase involves the evaluation of the equilibrium in solution that might take place between the analytes and the components of such solutions. The result is the possibility of tailoring selectivity and efficiency of the considered separation systems by incorporating suitable buffering agents and additives into the BGE or the mobile phase, respectively. Practical applications of these approaches to separation and quantification of phenolic compounds in different plant matrices are then discussed.