Influence of the composition of the liquid phase on the performance of separations in reversed phase liquid chromatography and in capillary electrophoresis of biomolecules

This communication discusses the influence of the composition of the liquid phase on the performance of separation of biomolecules by either capillary electrophoresis (CE) or reversed phase high performance liquidd chromatography (RP-HPLC). Appropriate selection of the background electrolyte solution (BGE) in CZE or 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 several biomolecules, such as phenolic compounds and other plant secondary metabolites, requires the control of the protonic equilibrium in solution, which is performed using suitable buffering agents incorporated into the BGE or the mobile, respectively. The constituentsof the buffer solutions do not limit their action at controlling the protonic equilibrium. They also might interact with the analytes, with the result of altering either their electrophoretic mobility or their chromatographic retention, respectively. In addition, the components of the BGE influence the generation of the electric double layer at the interface between the inner surface of fused silica capillaries and the electrolyte solution, which originates the electroosmotic flow in CZE. The discussion include the results of a study performed to identify the N-acylhomoserine lactone signalling molecules released by several nitrogen-fixing bacteria such as Azospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria, and Gluconacetobacter diazotrophicus, which are of interest in plant microbiology to study pathogenic or symbiotic interactions of bacteria with plant hosts. The AHLs, extracted from cell-free spent culture supernatants of the selected bacteria, are separated ad identified by HPLC-ESI-MS, using a narrow bore reversed phase column.