A semi-automated purification module for the cyclic separation of 99mTc was designed for production of [99mTc]TcO4– from γ irradiated 100Mo target. The separation process was carried out by using a 3-column purification system and the final product, [99mTc]TcO4–, was obtained in a total volume of 7 mL. To confirm proper separation achieved for 99mTc, a radio-labeling procedure using DTPA chelator was performed. The radiochemical purity was higher than 95%, which meets the strict radiopharmaceutical requirements. The yielded 99mTc can be separated with high efficiency from Mo in a quick and repeated way. Loss of 99mTc radioactivity during such a three-column separation process was not larger than 10%.