Free zone electrophoresis

Abstract

The free zone electrophoresis method described in this monograph can be used for the fractionation of small molecules, large molecules, and particles. Its versatility is illustrated in runs with inorganic ions, organic ions, nucleic acid bases, nucleosides, nucleotides, proteins, nucleic acids, subcellular particles, viruses, and erythrocytes. The principles of the construction of the electrophoresis equipment are described in detail. The separation chamber is a horizontal tube which slowly rotates round its long axis. The rotation eliminates the need for a supporting medium. A mathematical treatment of this rotational stabilization is given. The equipment is fitted with a unique U.V.-scanning system and is automated so that it requires no attention after the introduction of the sample. Because the “boundary anomaly” effects cause the concentration of any ion inside a migrating zone to be different from that outside, the scanning system can also be used to detect substances which have no U.V.-absorption if the buffer contains appropriate U.V.-absorbing ions. Free zone electrophoresis is intended primarily for analytical purposes but can also be used for preparative experiments on a small scale. The amount of material required is about the same as in paper electrophoresis. The mobility values obtained by free zone electrophoresis agree well with those determined by the classical Tiselius moving boundary method. Free zone electrophoresis also permits determinations of electroendosmotic mobilities, and thereby ζ-potentials of the surface of the revolving electrophoresis chamber. The empirical relationship between recorder deflection and zone concentration fits the theoretically derived curve closely. Using these calibration curves an accuracy of about 4% was obtained in quantitative determinations. Some sections also apply to electrophoresis methods other than free zone electrophoresis, e.g. the sections dealing with the electrophoretic migration velocity profile, the elimination of the electroendosmotic disturbances, and the detection of substances which have no U.V.-absorption.