CIEF of components following sequential injection of ampholytes and the sample zone offers unique advantages for analysis. The most important one of these is the efficient separation of amphoteric compounds having pIs outside the pH range of the ampholytes applied, but the resolution of the components can be increased by an adequate setup in the injection protocol. In this study, the effect of the pH of the anolyte and catholyte on the selectivity and speed of the isoelectric focusing was investigated. Changes in the pH values significantly influenced the resolution and the length of the pH gradient, while changes in the charge state of components were also observed. Three ampholyte solutions (from different suppliers) covering only two pH units were used for the analyses of substituted nitrophenol dyes in uncoated capillary. With appropriate setup, the components, with pIs not covered by the ampholyte pH range, migrated in charged state outside the pH gradient. This phenomenon is preferable for coupling isoelectric focusing to MS detection, by evading the undesirable ion suppression effect of ampholytes.