Abstract: 6,7-~3H estrone and dehydroestrone is separated and analysed by HPLC witha column of YWG--NH_2 (0.5×20cm). The mobile phase is ethyl alcohol/hexane(5/96, V/V) with a flow of 2 ml/min. The detection is carried out at UV 280mm.The liquid scintillator PPO is used at a flow ratio of scintillator and eluent 4:1.The limit of detection is in the order 10~(-7)g and of 10~-(10)Ci. When the ratio ofsolvents is 15/85, the mixture of estradiol, estriol, estrone and acetylestrone canbe completely separated. When the mobile phase was changed into ispropyl alcohol/dichloromethane (10/90, V/V), the isomers of dehydroestrone can be separated. What is more, using the same column and mobile phase, the sample6,7-~3Hestrone can be separated and purified. The amount of each sample is 7--8 mCi.The time of purification is less than 35 min. The percentage recovery of sampleamounts to 93.9%. The radiochemical purity of product is about 99% with aprecision of 0.1%.