The resolution of a zone electrophoretic system or a chromatographic column is customarily defined in terms of the separation of two pure (homogeneous) species. For such separation the resolution increases indefinitely with time for analytic fractionation, or indefinitely with column length for elution separation. It is shown that microheterogeneity of either or both species imposes an upper limit on resolution. This may have a marked effect on conditions for optimal resolution, and on measurements of the apparent diffusion coefficient.