Abstract: The present invention relates to a method for separating the components of a mixture M by electrophoresis in a single capillary, wherein: (A) the compounds of the mixture M are introduced and migrate inside the capillary according to the capillary electrophoresis technique; (B) a fraction F of the compounds separated in step (A) is isolated in the capillary and said isolated fraction F is forced to migrate towards the vicinity of one end of the capillary while maintaining this fraction inside the capillary; (C) upon the application of a pressure difference between the ends of the capillary, a second separation medium MS2 different from the first separation medium MS1 of step (A) is introduced into the capillary through the end of the capillary in the vicinity of which the fraction F is located, whereby the fraction F migrates to the other end of the capillary while filling the capillary with the separation medium MS2; and (D) an capillary electrophoresis is carried out in the capillary thus filled with the se