PURPOSE:To make it possible to separate polypeptides with MW of 10,000 or less in MW order within a short time, by adapting a gel permeation chromatograph method using a mobile phase obtained by adjusting ethanol containing 1-5mol urea to acidic state. CONSTITUTION:One side of an aquous gel permeation column 1 using silica as a packing agent is connected to a mobile phase tank 3 receiving a mobile phase obtained by holding ethanol containing 1-5mol urea to an acidic state through a specimen injection port 2 and the other side thereof is connected to a detector 4. When polypeptides being a specimen are injected in the column 1 from the specimen injection port 2, the hydrophilic property of the column 1 is increased by ethanol in the liquid mobile phase and polypeptides receive the action of the liquid mobile phase held to the acidic state to suppress the Coulombic action with the column packing agent as low as possible and polypeptides are dominated only by a molecular size to be adsorbed by the column packing agent. By this method, polypeptides are eluted from the column 1 in MW order. As a result, polypeptides with MW of 10,000 or less can be eluted in MW order within a short time.
