Abstract: As an excellent moderator and reflector material, graphite are widely used in nuclear reactors. Over the past few decades, accompanied by the decommission of nuclear reactors, the world has accumulated over 250000 tons of irradiated graphite, which are temporarily retained in interim storage facilities and reactors stores. The safe disposal of radioactive waste is related to the safety of the environment and staff, as well as the public perception of the nuclear power industry. The processing and disposal of irradiated graphite have attracted worldwide attention. Various methods have been developed for irradiated graphite treatment, including incineration, thermal decontamination, electrochemical and chemical treatment, molten salt oxidation, and so on. Due to its great reduction in the initial volume of the irradiated graphite, the gasification method is considered as a promising approach. However, after gasification, the existence of ¹⁴CO₂ and ¹⁴CO in the off-gas limited the application of gasification treatment. To remove the ¹⁴C in the off-gas after irradiated graphite treatment, the carbon isotope separation with chromatography at low temperature is studied in the present work. A chromatography device is constructed, and the ¹⁴CO separation performance is estimated with standard gas(the content of ¹³CO is about 1.1%). The influence of carrier gas flow rate, retention amount of liquid nitrogen, and injection quantity of CO on the separation ability are investigated. The results show that an increase in carrier gas flow rate leads to a worse ¹³CO separation performance. The enhancement of the liquid nitrogen retention is in favor of the ¹³CO separation, but with the same injection amount of CO, the improvement of ¹³CO separation will reach a platform. Less CO injection is beneficial for ¹³CO separation, and in the present work, the best ¹³CO separation is obtained with an CO injection amount of 5 L. Meantime, it is found that when the CO injection is increased, to obtain an obvious ¹³CO separation, the retention of liquid nitrogen is critical.