The semicontinuous separation/concentration of two solutes with different molecular size by the size exclusion cyclic separation method is based on the opposite swelling response of two gals to a temperature change. Experimental results of separation and concentration of solutes are verified qualitatively by the theoretical models based on the local equilibrium assumption. Separation of two solute is shown by the breakthrough curves in coupled gel columns. In closed coupled columns, the volumetric space for the large molecules which are totally excluded from the gels be-comes smaller, creating a concentrating effect as the gel swell when temperature changes. A mechanistic model is suggested to predict the large molecule concentration to increase to its solubility limit as cycle repeats.