Evolution of the theoretical description of the isoelectric focusing experiment: I. The path from Svensson's steady state model to the current two-stage model of isoelectric focusing.

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 whose concentration decreased with their distance from the electrodes and whose diffusive flux 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 isoelectric focusing experiment - where the carrier ampholyte 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 carrier ampholytes 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. This article is protected by copyright. All rights reserved.