Free-flow electrophoresis method for separating analytes

A free-flow electrophoresis method for separating at least one analyte of interest from a mixture of analytes, wherein the method comprises

• flowing a separation medium through a separation chamber in a flow direction;
• applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, and applying the mixture of analytes to the separation medium, whereupon the at least one analyte of interest is separated from the mixture of analytes,
• or
• applying the mixture of analytes to the separation medium, and applying an electric field in the separation medium by an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode, whereupon the at least one analyte of interest is separated from the mixture of analytes;
• collecting fractions of the separation medium with at least one fraction comprising the at least one analyte of interest separated from the mixture of analytes;
• characterized in that
• the separation medium comprises two or more individual separation media, wherein the two or more individual separation media differ in their pH value, and wherein each of the two or more individual separation media comprise at least one anion of at least one acid and at least one cation of at least one base, wherein the at least one acid is the same in each of the two or more individual separation media and the at least one base is the same in each of the two or more individual separation media.