A three-fraction simulated moving bed (3F-SMB) unit for separating feed mixtures into three fractions is presented. Criteria for the selection of the flow rate ratios in the four sections of the unit under linear chromatographic conditions are developed. These criteria translate into a region of complete three-fraction separation in the (m(2), m(3)) plane. The performance of the 3F-SMB unit is investigated through detailed simulations using as a model system the separation of the four nucleosides: 2'-deoxyadenosine, 2'-deoxythymidine, 2'-deoxyguanosine, and 2'-deoxycytidine. For nonlinear chromatographic conditions, simulations on the same model system allowed qualitative conclusions on the performance of the 3F-SMB to be drawn. Further, the possibility of using the 3F-SMB unit to implement a cleaning-in-place (CIP) step within a standard SMB is shown and validated through simulations, using as a model system a mixture of two nucleosides and one highly retained impurity.