One-Step Focusing of Ampholytes With Electrophoretic Mobilization: Concepts Assessed by Computer Simulation.

The dynamics of three one-step focusing protocols described in the literature for IEF-MS analyses of proteins are assessed by computer simulation. Focusing of 101 carrier ampholytes (pI range 3.0-11.

Mobilization in two-step capillary isoelectric focusing: Concepts assessed by computer simulation.

The mobilization step in a two-step capillary isoelectric focusing protocol is discussed by means of dynamic computer simulation data for systems without and with spacer compounds that establish their zones at the beginning and end of the focusing column. After focusing in an electroosmosis-free environment (first step), mobilization (second step) can be induced electrophoretically, by the application of a hydrodynamic flow, or by a combination of both means. Dynamic simulations provide insight into the complexity of the various modes of electrophoretic mobilization and dispersion associated with hydrodynamic mobilization.

Dynamic computer simulations of electrophoresis: 2010-2020.

The transport of components in liquid media under the influence of an applied electric field can be described with the continuity equation. It represents a nonlinear conservation law that is based upon the balance laws of continuous transport processes and can be solved in time and space numerically. This procedure is referred to as dynamic computer simulation.

Instabilities of the pH gradient in carrier ampholyte-based isoelectric focusing: Elucidation of the contributing electrokinetic processes by computer simulation.

Electrokinetic processes that lead to pH gradient instabilities in carrier ampholyte-based IEF are reviewed. In addition to electroosmosis, there are four of electrophoretic nature, namely (i) the stabilizing phase with the plateau phenomenon, (ii) the gradual isotachophoretic loss of carrier ampholytes at the two column ends in presence of electrode solutions, (iii) the inequality of the mobilities of positively and negatively charged species of ampholytes, and (iv) the continuous penetration of carbonate from the catholyte into the focusing column. The impact of these factors to cathodic and anodic drifts was analyzed by simulation of carrier ampholyte-based focusing in closed and open columns.

Computer simulation of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers with a free or immobilized neutral chiral selector.

A detailed computer simulation study of the isotachophoretic migration and separation of norpseudoephedrine stereoisomers for cases with the neutral selector added to the leader, immobilized to the capillary wall or support, or partially present in the separation column is presented. The electrophoretic transport of the analytes from the sampling compartment into the separation medium with the selector, the formation of a transient mixed zone, the separation dynamics of the stereoisomers with a free or immobilized selector, the dependence of the leader pH, the ionic mobility of norpseudoephedrine, the complexation constant and selector immobilization on steady-state plateau zone properties, and zone changes occurring during the transition from the chiral environment into a selector free leader are thereby visualized in a hitherto unexplored way. For the case with the selector dissolved in the leading electrolyte, simulation data are compared to those observed in experimental setups with coated fused-silica capillaries that feature minimized electroosmosis and zone detection with conductivity and absorbance detectors.

Simulation and experimental study of enzyme and reactant mixing in capillary electrophoresis based on-line methods.

The establishment of an efficient reaction mixture represents a crucial part of capillary electrophoresis based on-line enzymatic assays. For ketamine N-demethylation to norketamine mediated by the cytochrome P450 3A4 enzyme, mixing of enzyme and reactants in the incubation buffer at physiological pH was studied by computer simulation. A dynamic electrophoretic simulator that encompasses Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure driven flow was utilized.

Impact of Taylor-Aris diffusivity on analyte and system zone dispersion in CZE assessed by computer simulation and experimental validation.

Application of pressure-driven laminar flow has an impact on zone and boundary dispersion in open tubular CE. The GENTRANS dynamic simulator for electrophoresis was extended with Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure-driven flow. Effective diffusivity of analyte and system zones as functions of the capillary diameter and the amount of flow in comparison to molecular diffusion alone were studied for configurations with concomitant action of imposed hydrodynamic flow and electroosmosis.

Computer simulation of electrophoretic aspects of enantiomer migration and separation in capillary electrochromatography with a neutral selector.

A computer simulation study describing the electrophoretic separation and migration of methadone enantiomers in presence of free and immobilized (2-hydroxypropyl)-β-CD is presented. The 1:1 interaction of methadone with the neutral CD was simulated by using experimentally determined mobilities and complexation constants for the complexes in a low-pH BGE comprising phosphoric acid and KOH. The use of complex mobilities represents free solution conditions with the chiral selector being a buffer additive, whereas complex mobilities set to zero provide data that mimic migration and separation with the chiral selector being immobilized, that is CEC conditions in absence of unspecific interaction between analytes and the chiral stationary phase.

Sampling strategies for capillary isoelectric focusing with electroosmotic zone mobilization assessed by high-resolution dynamic computer simulation.

The impact of initial sample distribution on separation and focusing of analytes in a pH 3–11 gradient formed by 101 biprotic carrier ampholytes under concomitant electroosmotic displacement was studied by dynamic high-resolution computer simulation. Data obtained with application of the analytes mixed with the carrier ampholytes (as is customarily done), as a short zone within the initial carrier ampholyte zone, sandwiched between zones of carrier ampholytes, or introduced before or after the initial carrier ampholyte zone were compared. With sampling as a short zone within or adjacent to the carrier ampholytes, separation and focusing of analytes is shown to proceed as a cationic, anionic, or mixed process and separation of the analytes is predicted to be much faster than the separation of the carrier components.

High-resolution electrophoretic simulations: performance characteristics of one-dimensional simulators.

Three comprehensive one-dimensional simulators were used on the same PC to simulate the dynamics of different electrophoretic configurations, including two migrating hybrid boundaries, an isotachophoretic boundary and the zone electrophoretic separation of ten monovalent anions. Two simulators, SIMUL5 and GENTRANS, use a uniform grid, while SPRESSO uses a dynamic adaptive grid. The simulators differ in the way components are handled.

Dynamic computer simulations of electrophoresis: a versatile research and teaching tool.

Software is available, which simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. These dynamic models are based upon equations derived from the transport concepts such as electromigration, diffusion, electroosmosis and imposed hydrodynamic buffer flow that are applied to user-specified initial distributions of analytes and electrolytes. They are able to predict the evolution of electrolyte systems together with associated properties such as pH and conductivity profiles and are as such the most versatile tool to explore the fundamentals of electrokinetic separations and analyses.

Dynamic computer simulations of electrophoresis: three decades of active research.

Dynamic models for electrophoresis are based upon model equations derived from the transport concepts in solution together with user-inputted conditions. They are able to predict theoretically the movement of ions and are as such the most versatile tool to explore the fundamentals of electrokinetic separations. Since its inception three decades ago, the state of dynamic computer simulation software and its use has progressed significantly and Electrophoresis played a pivotal role in that endeavor as a large proportion of the fundamental and application papers were published in this periodical.

High-resolution computer simulation of electrophoretic mobilization in isoelectric focusing.

Cationic and anionic electrophoretic mobilization for focusing of hemoglobins (Hb's) in the presence of 100 carrier ampholytes covering a pI range of 6.00-7.98 was studied by computer simulation at a constant current density of 300 A/m(2).

High-resolution computer simulation of the dynamics of isoelectric focusing: in quest of more realistic input parameters for carrier ampholytes.

The impact of the systematic variation of either DeltapK(a) or mobility of 140 biprotic carrier ampholytes on the conductivity profile of a pH 3-10 gradient was studied by dynamic computer simulation. A configuration with the greatest DeltapK(a) in the pH 6-7 range and uniform mobilities produced a conductivity profile consistent with that which is experimentally observed. A similar result was observed when the neutral (pI = 7) ampholyte is assigned the lowest mobility and mobilities of the other carriers are systematically increased as their pI's recede from 7.

High-resolution computer simulation of the dynamics of isoelectric focusing using carrier ampholytes: focusing with concurrent electrophoretic mobilization is an isotachophoretic process.

Focusing of four hemoglobins with concurrent electrophoretic mobilization was studied by computer simulation. A dynamic electrophoresis simulator was first used to provide a detailed description of focusing in a 100-carrier component, pH 6-8 gradient using phosphoric acid as anolyte and NaOH as catholyte. These results are compared to an identical simulation except that the catholyte contained both NaOH and NaCl.

High-resolution computer simulations of stacking of weak bases using a transient pH boundary in capillary electrophoresis. 1. Concept and impact of sample ionic strength.

The dynamics of focusing weak bases using a transient pH boundary was examined via high-resolution computer simulation software. Emphasis was placed on the mechanism and impact that the presence of salt, namely, NaCl, has on the ability to focus weak bases. A series of weak bases with mobilities ranging from 5 x 10(-9) to 30 x 10(-9) m2/V x s and pKa values between 3.

High-resolution computer simulation of the dynamics of isoelectric focusing of proteins.

A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High-resolution focusing data of four hemoglobin variants in a convection-free medium are presented for pH 3-10 and pH 5-8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5-10 min, whereas at 600 V/cm separation is predicted to be complete between 2.

High-resolution computer simulation of the dynamics of isoelectric focusing using carrier ampholytes: the post-separation stabilizing phase revisited.

A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the stabilizing phase in isoelectric focusing under conditions that were hitherto inaccessible. High-resolution focusing data are presented for pH gradients spanning 7 units (pH 3-10 and pH 4-11 with 20 carrier ampholytes/pH unit) and 3.5 units (pH 7-10.