This paper presents a practical flow-through method to separate anisole and ethyl phenylacetate, respectively, from a polystyrene mixture. The microfluidic separation uses different diffusive dynamics of the substances transverse to the lamination flow formed in a microchannel. The effect of inlet flow rates and ambient temperature on separation is examined. Additionally, the possibility of the separation of the light substance from the mixture with different molecular weight is shown numerically and experimentally. The separation efficiency is explained by the facts that the relaxation time depends on the inlet flow rate and that the diffusivity depends on the ambient temperature. This method can be applied to separate monomers from aggregates.