A method for separating an analyte to be separated from a composition of analytes by free flow isoelectric focusing comprising: forming within a free flow electrophoresis chamber a pH function profile between an anode and a cathode, comprising a pH separation plateau which average pH corresponds essentially to the isoelectric point (pi) of an analyte to be separated and which has a pH range delimited by an upper pH limit and a lower pH limit; a pH function between the anode and the pH separation plateau having an average pH lower than the pH of the pH separation plateau or a higher electrical conductivity than the pH separation plateau, or both a lower average pH and a higher electrical conductivity than the pH separation plateau; and a pH function between the cathode and the pH separation plateau having an average pH greater than the pH of the pH separation plateau or a higher electrical conductivity than the pH separation plateau, or both a greater average pH and a higher electrical conductivity than the pH separation plateau; wherein the pH separation plateau and the pH function profiles between the cathode and anode are formed by respective buffer systems; introducing a sample comprising an analyte having a defined pi to be separated from a mixture of analytes into the FFE chamber wherein the sample can be introduced in the pH separation plateau, in a zone at the anodic side or in a zone at the cathodic side of said pH separation plateau; and eluting the analytes from the FFE chamber, and optionally recovering all or a portion of the analytes in one or a plurality of fractions; optionally wherein the method further comprises identifying the pi of an analyte to be separated from a composition of analytes prior to the electrophoretic separation in case the pi of said analyte is not known. forming within a free flow electrophoresis chamber a pH function profile between an anode and a cathode, comprising a pH separation plateau which average pH corresponds essentially to the isoelectric point (pi) of an analyte to be separated and which has a pH range delimited by an upper pH limit and a lower pH limit; a pH function between the anode and the pH separation plateau having an average pH lower than the pH of the pH separation plateau or a higher electrical conductivity than the pH separation plateau, or both a lower average pH and a higher electrical conductivity than the pH separation plateau; and a pH function between the cathode and the pH separation plateau having an average pH greater than the pH of the pH separation plateau or a higher electrical conductivity than the pH separation plateau, or both a greater average pH and a higher electrical conductivity than the pH separation plateau; wherein the pH separation plateau and the pH function profiles between the cathode and anode are formed by respective buffer systems; a pH separation plateau which average pH corresponds essentially to the isoelectric point (pi) of an analyte to be separated and which has a pH range delimited by an upper pH limit and a lower pH limit; a pH function between the anode and the pH separation plateau having an average pH lower than the pH of the pH separation plateau or a higher electrical conductivity than the pH separation plateau, or both a lower average pH and a higher electrical conductivity than the pH separation plateau; and a pH function between the cathode and the pH separation plateau having an average pH greater than the pH of the pH separation plateau or a higher electrical conductivity than the pH separation plateau, or both a greater average pH and a higher electrical conductivity than the pH separation plateau; wherein the pH separation plateau and the pH function profiles between the cathode and anode are formed by respective buffer systems; introducing a sample comprising an analyte having a defined pi to be separated from a mixture of analytes into the FFE chamber wherein the sample can be introduced in the pH separation plateau, in a zone at the anodic side or in a zone at the cathodic side of said pH separation plateau; and eluting the analytes from the FFE chamber, and optionally recovering all or a portion of the analytes in one or a plurality of fractions; optionally wherein the method further comprises identifying the pi of an analyte to be separated from a composition of analytes prior to the electrophoretic separation in case the pi of said analyte is not known.