Computer simulation and experimental validation of isoelectric focusing in ampholine-free systems

Bier, M.; Mosher, Richard A.; Palusinski, O.A.

doi:10.1016/s0021-9673(00)83062-9

Cited by 65 publications

(30 citation statements)

References 14 publications

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“…This is not completely reflecting reality as the anionic form of an ampholyte is more hydrated than the cationic and is thus assumed to possess a slightly smaller mobility compared to that of the cation [28]. For modeling of steady-state IEF, this has been shown to lead to profiles that are no longer completely symmetrical and this even for compounds with a pI of 7 [3,29]. Mobility inequalities of the species of ampholytes are also found in tables which list mobility values of compounds [30][31][32].…”

Section: The Use Of Unequal Mobilities Of Cationic and Anionic Speciementioning

confidence: 99%

High‐resolution computer simulation of the dynamics of isoelectric focusing: In quest of more realistic input parameters for carrier ampholytes

Mosher¹,

Thormann

2008

Electrophoresis

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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. When equal DeltapK(a) values and mobilities are assigned to all ampholytes a conductivity plateau in the pH 5-9 region is produced which does not reflect what is seen experimentally. The variation in DeltapK(a) values is considered to most accurately reflect the electrochemical parameters of commercially available mixtures of carrier ampholytes. Simulations with unequal mobilities of the cationic and anionic species of the carrier ampholytes show either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species) drifts of the pH gradient. The simulated cationic drifts compare well to those observed experimentally in a capillary in which the focusing of three dyes was followed by whole column optical imaging. The cathodic drift flattens the acidic portion of the gradient and steepens the basic part. This phenomenon is an additional argument against the notion that focused zones of carrier ampholytes have no electrophoretic flux.

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Section: The Use Of Unequal Mobilities Of Cationic and Anionic Speciementioning

confidence: 99%

High‐resolution computer simulation of the dynamics of isoelectric focusing: In quest of more realistic input parameters for carrier ampholytes

Mosher¹,

Thormann

2008

Electrophoresis

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“…The anionic form of an ampholyte is more hydrated than the cationic and can thus possess a slightly smaller mobility compared to that of the cationic species [32,33]. Such mobility inequalities were recently shown to predict a cationic movement of the pH gradient [24,34].…”

Section: Electrophoretic Ph Gradient Drift and Destabilization At Colmentioning

confidence: 99%

High‐resolution computer simulation of electrophoretic mobilization in isoelectric focusing

Thormann

Mosher²

2008

Electrophoresis

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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). Electropherograms that would be produced by whole column imaging and by single detectors placed at different locations along the focusing column are presented. Upon mobilization, peak heights of the Hb zones decrease, but the zones retain a relatively sharp constant profile and are migrating at a constant velocity. A further peak decrease occurs during readjustment at the locations of the original buffer/column interfaces, indicating that detection sensitivity is the lowest at these locations. An anionic carrier ampholyte mobility smaller than that of its cationic species produces a cathodic drift which is smaller than the transport rate used for electrophoretic mobilization. Compared to the case with equal mobilities of carrier ampholyte species, a small increase (decrease) is predicted for the cationic (anionic) mobilization rate within the focusing column. Simulation data suggest that electrophoretic mobilization after focusing and focusing with concurrent electrophoretic mobilization are comparable isotachophoretic processes that occur when there is an uninterrupted flux of an ion through the focusing column. Cathodic drift caused by unequal mobilities of the species of carrier ampholytes, electrophoretic mobilization, and decomposition occurring at the pH gradient edges are related electrophoretic processes.

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“…These restrictions were removed in the model developed for IEF by Palusinski et al (1981b) wherein assumptions regarding the shapes of the pH or the conductivity profiles are avoided. (See also Palusinski et al, 1981a;Bier et al, 1981;and Bier et al (1983). The current model is based on this work, expanded so as to cover a wide variety of electrophoretic modes.…”

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confidence: 99%

Theory of electrophoretic separations. Part I: Formulation of a mathematical model

1986

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A general model is developed for the electrophoresis of soluble materials. The model describes the evolution of concentration fields for a set of compounds which undergo transport by flow, diffusion, and migration in an electric field and simultaneously participate in rapid dissociation-association reactions. Modes of electrophoresis requiring special treatment can now be studied in a unified context. As an example of its utility, the model is used analytically to study a process known as isotachophoresis. In Part I1 two electrophoretic separation processes are simulated numerically, demonstrating the model's versatility. D. A. SAVILLE Department of Chemical EngineeringPrinceton University Princeton, NJ 08544 A. PALUSINSKI Biophysics Technology Laboratory and Department of Electrical and Computer Engineering University of ArizonaTucson, AZ 85721 SCOPEWhen a solution containing amphoteric compounds is exposed to an electric field, the migration of ions and uncharged species occurs in the presence of rapid dissociation-recombination reactions. If a zone containingseveral species is inserted in a column containinga homogeneous buffer, the various species will migrate at different rates according to their relative electrophoretic mobilities. Conversely, if the ends of the column are made impermeable to the amphoteric species, a stationary pH gradient will eventually be formed and sample constituents will migrate to their equilibrium isoelectric points under the influence ofthe electric field. These phenomena form the basis of several separation methodologies that have been difficult to model mathematically. The purpose of this paper is to present a generally applicable model of electrophoretic processes and apply it to a specific situation. The application is designed to illustrate the interplay between reaction, electromigration, and diffusion in simple configurations where complications due to lateral boundaries, bulk flow, and nonuniform temperature are supressed. The situation studied in detail is isotachophoresis in a one-dimensional column; more diverse applications that require numerical treatment of the equations are described in Part 11. CONCLUSIONS AND SIGNIFICANCEAlthough electrophoretic processes can be described by familiar conservation relations, the structure of the mathematical model differs from that used to describe systems with strong electrolytes due to the presence of rapid dissociation-recombination reactions that tie the concentrations of ionic species to those of the undissociated solutes. It is shown that since the reactions are fast relative to transport by diffusion and electromigration, it is possible to treat the reactions as being in local equilibrium. Similarly, the ratio of an electrical length (the Debye scale) to the physical scale of the process is small, and this leads to the electroneutrality approximation. The model that is developed consists of a set of conservation equations for the total concentration of each amphoteric compound and the current. Appended to this set of partial...

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Computer simulation and experimental validation of isoelectric focusing in ampholine-free systems

Cited by 65 publications

References 14 publications

High‐resolution computer simulation of the dynamics of isoelectric focusing: In quest of more realistic input parameters for carrier ampholytes

High‐resolution computer simulation of the dynamics of isoelectric focusing: In quest of more realistic input parameters for carrier ampholytes

High‐resolution computer simulation of electrophoretic mobilization in isoelectric focusing

Theory of electrophoretic separations. Part I: Formulation of a mathematical model

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