AbstractWe describe a novel approach to generate dynamic pH gradients suited to fractionate or purify samples of biomolecules or particles such as proteins and viruses in tiny volumes. The method combines diffusion and electromigration between micro‐scaled channels embedded in hydrogel. For the used geometry and in accordance with numerical calculations the gel‐channel system reaches a tuneable, steady‐state pH gradient after a few minutes. For quantification of experimentally generated pH‐profiles, the concentration independent extinction ratio of phenol red at two wavelengths is used. The proposed electrophoretic flow‐cell is simple and flexible since no Immobilines are required to establish the pH gradient.