Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis

Abstract

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.

Continuous free‐flow electrophoresis

“…For example, application of FFE for the determination of the mobility curve (the dependence of mobility on pH of the buffer solution) of alcohol dehydrogenase was reported [72]. Correlation between electrophoretic mobility and the elution behavior in ion-exchange chromatography of more than a dozen proteins was followed by Nath et a].…”

“…Each of the 96 fractions was examined using gas chromatography/ mass spectrometry and 31 acids were identified, six of them for the first time in coffee [68]. Optimization of separation parameters, namely pH and conductivity of the background electrolyte, residence time, flow rate, applied voltage and chamber dimensions resulted in throughputs of up to 3 g of alcohol dehydrogenase/h with three simultaneous sample positions by FFZE and FFFSE, the purification factor being higher with ZE [73]. Preparative scale separation of proteins and enzymes, differing in apparent electrophoretic mobility by only 0.7 X lo-' cm2V-'s-' was reported under optimized conditions [74].…”

Recent developments in electrophoretic separations on microfluidic devices

“…Many of these focus on unique properties of the analytes that have not been fully examined for separations. Several of the new methods are related to electrophoresis and some of the standard bearers include isoelectric focusing (IEF) [1][2][3] and free-flow electrophoresis (FFE) [4,5]. Various processes, such as FFE, are well known on the preparative-scale, whereas others, such as IEF, have been well characterized for analytical-scale separations.…”

Prediction of the location of stationary steady‐state zone positions in counterflow isotachophoresis performed under constant voltage in a vortex‐stabilized annular column

“…The current in each discrete zone is the same, thus (5) The following equations for the total voltage drop across the column and total length of the column, respectively, for a system of n components in our sample are…”

“…ITP is a suitable fractionation and concentration technique for a variety of different sample types including food additives [1], drugs in biological samples [2], and recombinant proteins [3], and it is well suited for multiple scales ranging from microchip [4] to preparative applications [5]. Currently, there is a great deal of interest in using ITP in microchips due to their small sample size and rapid separation [4].…”