Influence of secondary equilibria in solution on separation performance of biomolecules in liquid chromatography and capillary electrophoresis

Influence of secondary equilibria in solution on separation performance of biomolecules in liquid chromatography and capillary electrophoresis Danilo Corradini, National Research Council (CNR), Institute for Biological Systems, Area della Ricerca di Roma 1 - 00015 Monterotondo Stazione, Rome, Italy. E-mail: danilo.corradini@cnr.it. Both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) can be operated by a variety of separation modes involving either specific or general physical chemical properties of the analytes and of the liquid phase, as well as the proper design of the separation system. The liquid phase employed in a multiplicity of separations carried out by either HPLC or CE consists of a buffered aqueous solution, which may contain one or more no-buffering additives, either neutral or charged, and can be mixed with an organic solvent. Liquid phases of such composition are largely employed in both reversed phase liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE), which are the prevailing chromatographic and electrophoretic separation techniques for a variety of biomolecules. This communication examines and discusses the influence of composition, pH and physical-chemical properties of the liquid phase on the separation of peptides, proteins and plant secondary metabolites by RP-HPLC and CZE. RP-HPLC uses non-polar chromatographic columns and polar hydro-organic solutions as the mobile phase. The separation process depends on the hydrophobic binding of the analytes from the mobile phase to the hydrophobic moieties bound to the stationary phase, which is regulated by the composition of the mobile phase that is either maintained constant (isocratic elution) or varied during the separation run (gradient elution). The main contribution to the eluotropic strength of the mobile phase is given by the surface tension and dielectric constant of the organic solvent employed in the hydro-organic solution. Therefore, the chromatographic retention decreases with increasing the concentration of the organic solvent or by using an organic solvent with lower surface tension coefficient or higher dielectric constant. CZE is performed in capillary columns using a background electrolyte solution (BGE) of uniform composition along the separation path and applying a constant electric field (E field) across the capillary length. The separation mechanism is based on differences in the electrophoretic mobilities of the charged analytes and, therefore, on their charge-to-hydrodynamic radius ratio, which depends on pH and composition of BGE. Characteristic feature of all separation modes in CE is the possible occurrence of an electrically driven flow of BGE across the separation pathway, the electroosmotic flow (EOF), which is generated by the action of the E field on the electric double layer formed at the interface between the conducting electrolyte solution and the charged surface of the capillary column in contact with BGE. Direction and velocity of EOF are also governed by pH and composition of BGE. Our studies to investigate the separation behavior of biological molecules in RP-HPLC and CZE have been focused on compounds bearing different concomitant functionalities, consisting of ionizable and/or hydrogen-bonding groups, hydrophobic regions, and hydrophilic moieties. Such multifunctional biomolecules include peptides, proteins and variety of plant secondary metabolites [1-3]. The ionogenic nature of these compounds requires the control of pH, which is performed using suitable buffering agents incorporated into the liquid phase employed for their separation by either CZE or RP-HPLC. The constituents of the buffer solutions do not limit their action at controlling the protonic equilibrium. Th