Movement of a charged species tends toward the electrode of opposite charge, but mixing and diffusion randomize the motion. However, operating in a gel or in a matrix such as paper can reduce the random motion so that electrical migration predominates. The pH at which the net charge of a particle or species approaches zero is called its isoelectric point. The net charge difference between a particle with its tightly associated ions and the bulk of the solution is called the zeta potential. Two particles with charges of the same sign will repel each other.
Slabs of gels are suitable for purification of small amounts of some materials by electrophoresis. However, larger amounts can be obtained by a continuous technique. A sheet of paper is hung vertically with a ceramic plate of a cooling unit touching it. See the next figure:
The flexible hoses indicated by 1 and 2 on this photo are the intake and return lines respectively for cold air. Sample entry points are shown by 10. Rather heavy filter paper is used. Its bottom edge is serrated to funnel the dripping liquid to collection tubes. Usually there are tubes for all the drips, but only 3 are shown. The colors represent materials with different isoelectric points. The application point need not be at the top center. All of the top edge gets buffer solution. As the applied solution with the buffer flows down the sheet, charged materials separate. Tabs or ears on the paper sheet dip into electrode chambers to complete the electrical circuit.
The voltages for continuous electrophoresis are rather high (several hundred volts), and simple heating due to current flow would accentuate mixing and diffusion were there no cooling. The small side view insert on the figure shows how the paper rests on a non-conducting box through which cold water flows.
One of our BASIC exercises demonstrates movement of charged particles or ions in solution in response to an electrical field, but you should try the new Java exercise instead. You see the species moving as if they were applied separately at different locations along the center line. In a real system, they would be mixed at one single starting location and would separate under the influence of the electric field.
The next portion about continuous electrophoresis shows what would happen in a continuous-flow electrophoresis as the solvent carries the species downward through an electric field applied from the sides. Note that an isoelectric point near the specified pH results in little motion. The size and shape of a particle or molecule are also important because these determine the drag force.
Large scale electrophoretic separations are unknown because the technique does not translate well to large sizes, mainly because it is difficult to remove the heat generated. Prior to dramatic improvements in chromatographic techniques, continuous electrophoresis was popular for purifying proteins such as enzymes. After several years of neglect, advances in electrophoresis seem to be leading to a resurrection. On an analytical scale, electrophoresis is invaluable. When calibrated with known compounds, identity and concentrations of the compounds come from inspection of the locations and sizes of spots after standardized electrophoresis.
For analytical electrophoresis, samples are placed on moist paper or in a well of gelatinous material. Usually the pH is uniform throughout the matrix, but there are compounds termed ampholytes that can establish a pH gradient. With uniform pH, application of an electromagnetic force (EMF) with electrodes at the extremities of the paper or gel will start migration. Charged species will move at rates determined by their net charges and their sizes because drag forces slow migration. For analytical measurements, the sample size is measured precisely. After a time, the EMF is disconnected and spots are identified by staining or by some type of spectrophotometry. When the pH is not uniform because of the ampholytes, a species will migrate until it encounters a pH equal to its isoelectric point. There is no longer a driving force, so migration stops. Very sharp separations are possible, and the technique is called isoelectric focussing.
