transient bidirectional isotachophoresis
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In modern isoelectric focusing (IEF) systems, where (i) convective mixing is prevented by gels or small cross-sectional area separation channels, (ii) current densities vary spatially due to the presence of electrode vessels with much larger cross-sectional areas than those of the gels or separation channels, and (iii) electrophoretic and diffusive fluxes do not balance each other, stationary, steady-state pH gradients cannot form (open-system IEF). Open-system IEF is currently described as a two-stage process: A rapid IEF process forms the pH gradient from the carrier ampholytes (CAs) in the first stage, then isotachophoretic processes degrade the pH gradient in the second stage as the extreme pI CAs are moved into the electrode vessels where they become diluted. Based on the ratios of the local effective mobilities and the local conductivities ( μLeff(x)$\mu _{\rm{L}}^{{\rm{eff}}}( x )$ / κ(x)$\kappa ( x )$ values) of the anolyte, catholyte, and the CAs, we pointed out in the preceding paper (Vigh G, Gas B, Electrophoresis 2023, 44, 675-88) that in open-system IEF, a single process, transient, bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on. In this paper, we demonstrate some of the operational features of the tbdITP model using the new ITP/IEF version of Simul 6.
The carrier ampholytes-based (CA-based) isoelectric focusing (IEF) experiment evolved from Svensson's closed system IEF (constant spatial current density, absence of convective mixing, counter-balancing electrophoretic and diffusive fluxes yielding a steady state pH gradient) to the contemporary open system IEF (absence of convective mixing, large cross-sectional area electrode vessels, lack of counter-balancing electrophoretic- and diffusive fluxes leading to transient pH gradients). Open system IEF currently is described by a two-stage model: In the first stage, a rapid IEF process forms the pH gradient which, in the second stage, is slowly degraded by isotachophoretic processes that move the most acidic and most basic CAs into the electrode vessels. An analysis of the effective mobilities and the effective mobility to conductivity ratios of the anolyte, catholyte, and the CAs indicates that in open system IEF experiments a single process, transient bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on until it is turned off. In tbdITP, the anolyte and catholyte provide the leading ions and the pI 7 CA or the reactive boundary of the counter-migrating H3 O+ and OH- ions serves as the shared terminator. The outcome of the tbdITP process is determined by the ionic mobilities, pKa values, and loaded amounts of all ionic and ionizable components: It is constrained by both the transmitted amount of charge and the migration space available for the leading ions. tbdITP and the resulting pH gradient can never reach steady state with respect to the spatial coordinate of the separation channel.
In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment-where the carrier ampholyte (CA) mixture is placed between the anolyte- and catholyte-containing large-volume electrode vessels (open-system IEF)-a two-stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two-stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open-system IEF experiment.
