Evolution of the theoretical description of the isoelectric focusing experiment: II. An open system isoelectric focusing experiment is a transient,bidirectional isotachophoretic experiment

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 H₃O⁺ and OH⁻ ions serves as the shared terminator. The outcome of the tbdITP process is determined by the ionic mobilities, pK_a 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.