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. They might interact with the analytes, for examples by an ion-pairing mechanism, with the result of altering either their electrophoretic mobility or their chromatographic retention, respectively. Multifunctional biomolecules with hydrophilic and hydrophobic moieties may also interact to different extents with nonionic components of the surrounding solution and with either the stationary phase or the capillary wall, in chromatography and in capillary electrophoresis, respectively. Both bare fused-silica and chemically or dynamically coated capillary columns have been employed in our studies carried out by CZE, using either plain aqueous or hydro-organic electrolyte solutions. RP-HPLC has been performed using alkyl-silica columns and hydro-organic mobile phases, under either isocratic or gradient elution mode. Comparison of selectivity, efficiency and resolution obtained with RP-HPLC and CZE using liquid phases of various composition are reported. Also compared is the complementary separation power of RP-HPLC and CZE for given classes of biomolecules, including peptides, proteins and polyphenols. It is evidenced that the appropriate selection of the composition of either the BGE or the mobile phase involves the evaluation of the physical-chemical properties of the liquid phase and the equilibrium in solution that might take place between the analytes and the components of such solution. The result is the well-known possibility of tailoring selectivity and efficiency of the considered separation systems by incorporating suitable organic solvents, buffering agents, and additives into the BGE or the mobile phase, respectively. The possibility of using either technique to elucidate the separation mechanism operating in the other one is also discussed. Practical applications of these studies to the development of analytical methods for the separation of biomolecules in complex matrices of plant origin are reported and pros and cons aspects of HPLC and CZE for the selected analytical separation problems are discussed too. References [1] L. De Gara, F. Orsini, I. Nicoletti, D. Corradini, Fundamental and practical aspects of liquid chromatography and capillary electromigration techniques for the analysis of phenolic compounds in plants and plant-derived food, Part 1: liquid chromatography, LCGC Europe 2018, 31, 480-490. [2] L. De Gara, F. Orsini, I. Nicoletti, D. Corradini, Fundamental and practical aspects of liquid chromatography and capillary electromigration techniques for the analysis of phenolic compounds in plants and plant-derived food, Part 2: capillary electromigration techniques, LCGC Europe 2019, 32, 8-14. [3] D. Corradini, Buffering agents and additives for the background electrolyte solutions used for peptide and protein capillary zone electrophoresis, TrAC, Trends in Analytical Chemistry, 2023, 117080, https://doi.org/10.1016/j.trac.2023.117080.
Influence of secondary equilibria in solution on separation performance of biomolecules in liquid chromatography and capillary electrophoresis
Corradini Danilo
2023
Abstract
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. They might interact with the analytes, for examples by an ion-pairing mechanism, with the result of altering either their electrophoretic mobility or their chromatographic retention, respectively. Multifunctional biomolecules with hydrophilic and hydrophobic moieties may also interact to different extents with nonionic components of the surrounding solution and with either the stationary phase or the capillary wall, in chromatography and in capillary electrophoresis, respectively. Both bare fused-silica and chemically or dynamically coated capillary columns have been employed in our studies carried out by CZE, using either plain aqueous or hydro-organic electrolyte solutions. RP-HPLC has been performed using alkyl-silica columns and hydro-organic mobile phases, under either isocratic or gradient elution mode. Comparison of selectivity, efficiency and resolution obtained with RP-HPLC and CZE using liquid phases of various composition are reported. Also compared is the complementary separation power of RP-HPLC and CZE for given classes of biomolecules, including peptides, proteins and polyphenols. It is evidenced that the appropriate selection of the composition of either the BGE or the mobile phase involves the evaluation of the physical-chemical properties of the liquid phase and the equilibrium in solution that might take place between the analytes and the components of such solution. The result is the well-known possibility of tailoring selectivity and efficiency of the considered separation systems by incorporating suitable organic solvents, buffering agents, and additives into the BGE or the mobile phase, respectively. The possibility of using either technique to elucidate the separation mechanism operating in the other one is also discussed. Practical applications of these studies to the development of analytical methods for the separation of biomolecules in complex matrices of plant origin are reported and pros and cons aspects of HPLC and CZE for the selected analytical separation problems are discussed too. References [1] L. De Gara, F. Orsini, I. Nicoletti, D. Corradini, Fundamental and practical aspects of liquid chromatography and capillary electromigration techniques for the analysis of phenolic compounds in plants and plant-derived food, Part 1: liquid chromatography, LCGC Europe 2018, 31, 480-490. [2] L. De Gara, F. Orsini, I. Nicoletti, D. Corradini, Fundamental and practical aspects of liquid chromatography and capillary electromigration techniques for the analysis of phenolic compounds in plants and plant-derived food, Part 2: capillary electromigration techniques, LCGC Europe 2019, 32, 8-14. [3] D. Corradini, Buffering agents and additives for the background electrolyte solutions used for peptide and protein capillary zone electrophoresis, TrAC, Trends in Analytical Chemistry, 2023, 117080, https://doi.org/10.1016/j.trac.2023.117080.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
