Resolution isoelectric focussing

As the sample constituent will always move towards their respective isoelectric points, it is not crucial where the samples are applied. Isoelectric focussing gives very high resolutions of samples, and it is particularly suitable for separating isoenzymes, as differences between isoelectric points of only 0.01 pH units are sufficient for separation by this technique. 

Where the mobilities of the sample ions are very similar, their resolution may be enhanced by including, with the sample, synthetic ampholytes called spacer ions. These have mobilities intermediate to those of the sample ions and hence help to separate them by taking up positions between the sample ions. The spacer ions are similar to the ampholytes used in isoelectric focussing. 

The resolution of isoelectric focussing can be expressed as A p/, the minimum pH difference to resolve two compounds. If compounds have similar pis, resolution can be improved by using pH gradients with a narrower range and smaller increments. Other factors that influence Apl include the applied electric field strength E, the diffusion coefficient D of the protein and the change of its mobility at different pH. 

This technique was discovered by H. Svensson in Sweden and has a high-resolution powra. A simple comparison would help establish the method s supremacy over other methods while paper electrophoresis resolves plasma proteins into six bands, isoelectric focussing resolves It into atleast 40 bands. 

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