Experimental and theoretical dynamics of isoelectric focusing: IV. Cathodic, anodic and symmetrical drifts of the pH gradient

Abstract

The production of anodic, cathodic and symmetrical drifts of a pH 3.5-10 gradient formed by isoelectric focusing in polyacrylamide gels is demonstrated experimentally by manipulation of the electrolyte concentrations. Experimental behavior is reproduced by computer simulation of a model mixture of 15 hypothetical carrier ampholytes whose pIs span the pH range 3-10. The mechanism which produces the drifts is elucidated and approaches to minimize such drifts are discussed. The data suggest why most experimentally observed drifts are cathodic.

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

“…The nature of catholyte and anolyte in CIEF is of importance in order to control anodic and/or cathodic drift, which was described for slab gel IEF as a pH instability that causes a progressive loss of the acid and basic end of the pH gradient, respectively [16]. This drift could be a difficulty in AGP analysis, because this protein being very acidic and focused in the far end of the capillary, ampholytes loss caused by anodic drift can drag AGP sample down the reservoir.…”

Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

“…35 Dynamic computer simulation of electrophoresis has already demonstrated considerable value as a research tool. Since the 1980s, numerical simulations have been performed to better understand and describe IEF [36][37][38][39][40] and have shown a qualitative agreement between predictions and experimental results. Recent advances in computer simulation have led to the development of a simulator that can handle up to 150 components and voltages typically used in experiments.…”

Dynamic computer simulations of electrophoresis: A versatile research and teaching tool

“…In work exploring the impacts and uses of moving boundaries in IEF, simulations performed with GENTRANS revealed that the gradual decomposition of the pH gradient at the buffer gradient interfaces [53,158,159] (for an example see Fig. 15), electrophoretic mobilization after focusing [132] and IEF with concurrent electrophoretic mobilization [160] are ITP based processes (see Section 4.3).…”

“…Early work was performed with a current density typically much lower than that employed in the laboratory and with the number of components limited to 15, which provided qualitative information about the system, but could not be used in any quantitative capacity. Nevertheless, these simulations provided new insights into the separation, stabilizing and destabilizing processes of IEF of simple ampholytes and proteins in closed columns without electrolytes and in configurations with acid and base as anolyte and catholyte, respectively [158,163,[168][169][170]. Simulation was also employed to study the formation of stable pH gradients with weak monovalent buffers for IEF in free solution [146], for the investigation of the stability of stepwise pH gradients used in continuous flow electrophoresis [171], to visualize pH gradient formation together with protein focusing for IEF field flow fractionation [172,173] and to visualize continuous trapping of an ampholyte [174] and separation of proteins [175] in carrier free IEF.…”

“…Simulation was also employed to study the formation of stable pH gradients with weak monovalent buffers for IEF in free solution [146], for the investigation of the stability of stepwise pH gradients used in continuous flow electrophoresis [171], to visualize pH gradient formation together with protein focusing for IEF field flow fractionation [172,173] and to visualize continuous trapping of an ampholyte [174] and separation of proteins [175] in carrier free IEF. The most important concepts that emerged from this dynamic simulation work included the recognition (i) that separation in focusing is based upon the formation of transient moving boundaries [163], (ii) that focusing is based upon two phases, a relatively fast separation phase followed by a much slower stabilizing phase [163], (iii) that pH plateaus formed around neutrality during the stabilizing phase explain the plateau phenomenon often observed in IEF [168], (iv) that protein focusing in simple buffers proceeds via a transient multi-peak approach in which more than two transient peaks can occur [170], (v) that electrode assemblies have an impact on the IEF pattern formed [169] and (vi) that cathodic, anodic and symmetrical drifts of the pH gradient are produced whose magnitude and directions are dependent on the electrolytes used [158].…”