A new operating method, namely three-port operation in a three-zone simulated moving bed (TT-SMB), was developed to improve the separation performance of simulated moving bed (SMB) chromatography. In the TT-SMB, the switching period consists of two steps; extract outlet port is closed at the first step for raffinate production and raffinate outlet port is closed at the second step for extract production. As a result, three ports were used in each step of the three-zone SMB operation. The separation performance of TT-SMB was compared with conventional three- and four-zone SMBs in terms of purity, recovery, productivity, and eluent consumption. A conventional three-zone SMB operation usually produced the contaminated portion at the product outlet node during the switching period. However, such phenomenon could be prevented by using the TT-SMB operation because one product outlet port was closed at each step. Compared to the conventional three-zone SMB, all performance parameters improved with the use of TT-SMB, and the improvement in purity and recovery was significant (up to 7%). Adjusting the step ratio played a key role in the trade-off between the performance parameters. The purity, recovery, and productivity of TT-SMB at well-designed step ratio conditions are up to 3% higher than those of the conventional four-zone methods (1-1-1-1 configuration). (C) 2014 Elsevier B.V. All rights reserved.