Patent ID: 7169275
Filing Date: 2007-01-30
Classification: G01N

Abstract:
1. A method for separating particles selected from the group consisting of cells, cell constituents, organelles, biomolecules, and parts or complexes thereof, in a free-flow electrophoresis (FFE) apparatus with a separation chamber defined by a floor, a cover and spacers to maintain these at a distance from each other, a dosage pump for conveying a separation medium that enters the separation chamber by way of medium feed lines and that leaves said chamber by way of outlets, an anode and a cathode located at a distance from each other for applying an electric field in the separation medium that flows between these two electrodes, sample injection points for adding a mixture of particles to be separated in the separation medium, and fractionation points for removing the particles in the separation medium that have been separated by FFE; which method comprises the following steps: (a) providing a separation medium; (b) conveying the separation medium with the dosage pump via the medium feed lines into the separation chamber; (c) flowing through the separation chamber with the separation medium; (d) fractionating the separation medium via discrete outlets; (a) applying an electrical field in the separation medium; (b) coupling particles to be separated with selectively chosen charge labels for generating charge-modified particles, which due to their selectively modified net surface charge display a different migratory behavior in FEE than non-charge-modified particles; (c) adding a mixture of charge-modified particles to be separated to the separation medium in the separation chamber by way of the sample injection points; (d) removing the particles that have been separated by FFE in the separation medium by way of the fractionation points; (e) collecting the fractions with the separated particles; wherein two focusing areas are created in the vicinity of the anode or cathode by introducing a focusing buffer between the separation medium and these two electrodes, whereby this focusing buffer is fed to the focusing areas via separate feed lines so that it flows in the same direction like the separation medium, the focusing buffer being much more electrically conductive than the separation medium such that a fluidic boundary is created between the separation medium and the focusing buffer in an area between the anode and cathode, and whereby charge-modified particles are focused within the separation medium and close to these fluidic boundaries at the border of the focusing areas.