Ampholyte-free Isoelectric Focusing Research Articles

Continuous micropreparative trapping in carrier ampholyte-free isoelectric focusing

The micro-isolation of a minor ampholyte from a mixture of four ampholytes using carrier ampholyte-free isoelectric focusing (IEF) is described. The separation is achieved in two consecutive steps. In the first step, a concentrated mixed zone of minor ampholyte-containing major ampholytes is formed when the original mixture is loaded into the column by electromigration. During this loading, a flux of solvolytic ions from both ends of the column is set up such a way that one end of the zone formed is immobilized in the column. It is achieved by selection of the pH range, which includes the p I value of the minor ampholyte but not the p I values of the major ampholytes. The minor ampholyte is captured in this zone completely, whereas the major ampholytes migrate through this zone to the outlet reservoir. After the zone has acquired a reasonable volume, the loading step is interrupted. In the second step, either the complete contents of the column are separated by conventional carrier ampholyte-free IEF in a pH range that includes the p I values of all ampholytes, or using a smaller pH range the minor ampholyte can be separated and isolated completely. Zone formation was modelled by computer simulation for a two-ampholyte mixture and verified experimentally for a four-ampholyte mixture in an instrument for electrically controlled IEF by using coloured low-molecular-mass ampholytes.