Dynamics of Capillary Isoelectric Focusing in the Absence of Fluid Flow:  High-Resolution Computer Simulation and Experimental Validation with Whole Column Optical Imaging

Abstract

A 150-component, dynamic electrophoresis simulator was developed and applied to the description of capillary isoelectric focusing (CIEF) of amphoteric substances in quiescent solution. The simulator is shown to be capable of producing high-resolution pH 3-10 focusing data with 140 individual carrier ampholytes (20/pH unit) and at current densities that are used in CIEF, i.e., under conditions that were hitherto unaccessible by dynamic computer simulation. Having a focusing capillary of 5-cm length, the predicted focusing dynamics for amphoteric dyes obtained at a constant voltage of 1500 V (300 V/cm) are shown to qualitatively agree with data obtained by whole-column optical imaging. The simulation data provide detailed insight into the dynamics of the focusing process for the cases with the focusing column being sandwiched between 40 mM NaOH (catholyte) and 100 mM phosphoric acid (anolyte) or having the column ends only permeable for OH- and H+ at cathode and anode, respectively. Simulation data reveal that the number of sample boundaries migrating from the two ends of the column to the focusing positions is always equal to the number of sample components. The number of detectable migrating sample boundaries, however, can be lower. Whole-column optical imaging is demonstrated to be the method of choice for following the approach to equilibrium. With that detection format, transient sample peaks can be recognized and properly identified. This would also be possible with a scanning detector moving rapidly and repeatedly along the column but cannot be accomplished by a stationary detector placed at a specified location. The data presented demonstrate that the model together with imaging monitoring can be used to optimize the CIEF separation conditions.

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

“…14. Focusing of the three dyes follows a double-peak approach, is complete within about 5 min and was found to compare well to experimental data obtained by whole-column imaging [53]. The predicted effect of the stabilizing phase on the shift of components, pH gradient and conductivity distribution is shown with the 100, 1000 and 5000 min data (Fig.…”

“…Under these conditions, an almost stepless pH 3-10 gradient is formed (Fig. 14C) and focusing is predicted to proceed via a double peak approach [53]. This is well seen for components focusing in the center of the gradient (see pI 7.00 carrier depicted in Fig.…”

“…This is illustrated here with ZE, ITP, IEF and EKC separation examples. The data shown were produced with GENTRANS, the simulator based on the work of Bier, Mosher, Thormann, Breadmore and coworkers [1,37,38,44,[49][50][51][52][53][54]75].…”

“…Initially the sample was uniformly distributed inside the focusing column of 5 cm length (0rxr5 cm) and electrode compartments were filled with NaOH (catholyte; x45 cm) and phosphoric acid (anolyte; xo0 cm). The entire electrophoresis column length employed for simulation was 12.5 cm, the number of segments was 2500 and the constant voltage applied was 1500 V. In this configuration, most of the potential drop is across the 5 cm focusing space [53]. Separation proceeds as described for Fig.…”

“…New models with sophisticated integration schemes emerged and the number of applications increased. Models that are currently in use in various laboratories are the 1-D simulators GENTRANS of Mosher et al [44,49,[51][52][53][54][129][130][131][132] and SIMUL5 of Hruška et al [48] (available at http://www.natur.cuni.cz/ Gas), which succeeded SIMUL 4.0 [47]. Interesting new models are those of Bercovici et al [69] (available as the Stanford's Public Release Electrophoretic Separation Solver (SPRESSO), http://microfluidics.stanford.edu/spresso), the 2-D model of Shim et al [70,71,135,136] and the models of Yang and his collaborators [68,138].…”