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.
Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpKa = pKamonoanion - pKamonocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pKa values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pKa values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pKa and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download).
Capillary zone electrophoresis (CZE) in classical buffer-based background electrolytes (BGEs) and carrier ampholytes-based capillary electrophoresis (CABCE) using narrow pH cuts of carrier ampholytes (CA) as constituents of quasi-isoelectric BGEs have been applied to separation and characterization of synthetic human and salmon gonadotropin-releasing hormones (GnRH) and their derivatives and fragments. The selectivity, separation efficiency, resolution and speed of CZE and CABCE analyses have been compared within a wide pH range of the BGEs (3.50-9.75) using two mixtures of structurally related GnRH peptides as model analytes. A baseline separation of mixture 1 (human GnRH and its three fragments) was achieved in CA-based BGEs at pH 3.5 and in both classical and CA-based BGEs within the pH range 7.00-9.75. Full separation of mixture 2 (salmon GnRH, its two fragments and human GnRH fragment) was obtained in both types of BGEs at acidic pH values 3.5 and 4.00 and at neutral pH 7.00. In addition to the separation of related GnRHs, their effective electrophoretic mobilities were determined and from the dependences of mobilities on pH, the isoelectric points (pI) of analyzed peptides were estimated. The pI values obtained by CABCE were in a good agreement with those determined by CZE in classical BGEs but in some cases rather different from those predicted by theoretical calculations.
