A system was developed that generates two gradients simultaneously, 180 degrees out of phase. Essential elements of the system were a pump delivering solvent A at a constant velocity, a pump delivering solvent B at a constant velocity, two mixing chambers, and a ten-port valve. By use of this gradient device and two reversed-phase columns packed with 1000-A pore diameter, 8-microns particle size macroporous poly-(styrene-divinylbenzene) resin, six proteins were separated in 40 s in a single gradient cycle.