Abstract
Continuous separation of cells, cell membranes, and proteins by electrophoretic techniques such as free-flow zone electrophoresis (FFZE) has a rich history since its introduction sixty years ago by Hannig. However, the results of FFZE, capillary zone electrophoresis, and other similar electrophoretic techniques are limited to analytical scale separations that are not readily extendable to the preparative scale. Moreover, a five- to ten-fold dilution of samples by buffer is common in separations by these techniques. Free-flow field step electrophoresis (FFFSE) is an electrophoretic technique that appears to be suitable for continuous simultaneous preparative separation and concentration and therefore has the power to overcome the above limitations. Here we apply FFFSE to a simple system of model proteins to show proof-of-concept of the technique. A continuous, preparative-scale separation of myoglobin from BSA with a throughput of 20 mg/h and a yield of >98% is shown to be successfully obtained using single-step FFFSE. Most important, it is shown that the preparative FFFSE experiment can be rationally designed, and the results predicted theoretically by use of electrokinetic data recorded in a simple analytical-scale FFZE experiment. This is the first paper to present a theory of separation in FFFSE. The separation is continuous , single-step , and environmental-friendly as no adjuvants are used, with no need for regeneration of components. The electrokinetic basis of the separation appears to be of a general nature. In further research we are testing the limits of the technique, by exploring its extension to more complex systems, and to higher preparative-scale throughputs.
Continuous Separation of Model Proteins by Free-Flow Field Step Electrophoresis and its Electrokinetic Basis