High Voltage Electrophoresis

Filter paper is still employed and found very satisfactory for use in high voltage electrophoresis. The technique is particularly useful for the separation of low molecular weight substances such as amino acids, peptides and polypeptides. One problem that must be overcome when using high voltage electrophoresis is the dissipation of the heat generated by applying appropriate cooling.  There are a number of ways of doing this.  One method, when employing flat-plate electrophoresis, is to make the lower plate of insulated metal fitted with cooling coils and the top plate is made of glass.

In tank-electrophoresis, during the separation process, the filter paper is immersed in a cooled liquid medium such as a chlorinated hydrocarbon. This form of high voltage electrophoresis apparatus is depicted in figure 10. The applied voltage can be extremely high, for example, several thousand volts and the paper sheets can be as large as 18 inches by 46 inches and even larger.  In the apparatus shown in figure 10, carbon tetrachloride was used for cooling purposes. The technique of high voltage paper electrophoresis has a very wide field of application, too many to mention individually. The technique is particularly useful for ‘fingerprinting proteins.

In practice the protein is hydrolysed to amino acids and low molecular weight polypeptides and then subjected to a two dimensional procedure that involves a separation in one direction by the usual technique of paper chromatography and then subjected to electrophoresis, the direction of electrophoretic migration being in a direction normal to that of the chromatographic development. The amino acids are distributed over the paper surface in a unique  and characteristic pattern.

Figure 10. High Voltage Electrophoresis Apparatus (Tank Type)

Figure 11. A Two Dimensional Separation of Amino Acids(Paper Chromatography and Paper Electrophoresis)

This procedure is particularly useful in allowing differences between similar proteins to be accurately and precisely identified. An example of this analytical procedure is shown in figure 11.