The Formation of the pH Gradient

 

Unfortunately, the pH gradient necessary for isoelectric focussing cannot be formed by simply mixing suitable electrolytes. This is because, under a potential gradient, the electrolytes will migrate as well as the substances being separated, and a stable pH gradient will not be formed. It is therefore necessary to generate the pH gradient by, for example, the prolonged hydrolysis of a suitable mixture of amphoteric substances (called ampholytes) having a wide range of isoelectric points. As a result of the electrolytic process, the environment round the anode will become more acidic and that around the cathode will become more alkaline with a continuous pH gradient between. Each type of ampholyte in the mixture will accumulate at its respective isoelectric point. The function of pH with distance will depend on the types of ampholytes selected. Special mixtures of aminocarboxylic acids have been developed and made commercially available called Ampholine that cover the molecular range from 300 to 600 and a pH range from 3 to 10. If this material is employed with sucrose then a system with a density gradient can also be formed.

 

A wide range of substances has been used as suitable ampholytes but certain requirements must be met for optimum results.  The amplolytes must be chosen to give a linear pH gradient if possible (under some circumstances slight deviation from true linearity may possibly be tolerated) and, despite the electrolytic process, tthe pH gradient must remain stable for the total period of electrophoresis. Another important electrical property that should be sought is a constant electrical conductivity along the whole length of the pH gradient. As the field strength will be proportional to the conductivity any change in conductivity will result in impaired focussing and reduced resolution. In addition, it is obvious that there should be no conductivity gaps (positions of high impedance) which would also result in poor focussing.

 

An example of the separation of three substances having pI values of 4, 6 and 8 respectively is depicted theoretically in figure 13.

 

 

 

Figure 13. The Theoretical Separation of Three Substances (pI 4,6 and 8) Using Isoelectric Focussing.

I should be noted that the electrophoretic action tends to compress the peaks and, thus, improve resolution; however, at the same time, as the concentration of the solute in the band increases the concentration gradient also increases resulting in greater diffusion which tends to disperse the band and oppose the compression of the band from electrophoretic migration. It follows, that substances of high molecular weight and, consequently, low diffusivities are likely to exhibit the more narrow bands and, thus, higher resolution.