Abstract: An isotachophoresis method for preconcentrating and isolating a plurality n of charged analytes (Ai, with i=1 to n) contained in a sample is disclosed, wherein each one of the analytes Ai has an effective electrophoretic mobility ?Ai obeying the fully ordered relationship ?A1>?A2>etc.>?An, comprising the step of preparing a mixture of said sample and a number n?1 of spacer compounds (Sk, with k=1 to n?1) wherein each one of said spacer compounds (Sk) has an effective electrophoretic mobility ?Sk obeying the fully ordered relationship ?Ak>?Sk>?Ak+1. An axial electric field is applied along the longitudinal axis of a separation channel, thereby causing a preconcentration and separation of the analytes and spacers forming respective focused spacer zones and focused analyte zones that flow along the longitudinal axis.