In this study, preparative medium pressure liquid chromatography (MPLC) was used for the purification of some phenolic compounds including hydroxytyrosol, chlorogenic acid, and punicalagin from olive mill waste water (OMWW), European cranberrybush (Viburnum opulus L.) juice, and pomegranate peel, respectively. While the polyphenols of OMWW and European cranberrybush juice were extracted with ethyl acetate (EtOAc), methanol was used for pomegranate peel extraction. At the end of the extraction process, the solvents were removed by rotary evaporator and the residue passed into the water phase. After the centrifugation and microfiltration procedure, the injection of samples was carried out by MPLC. The outflow of the colon was collected at specific periods and the predetermined polyphenols for purification were identified with High-Pressure Liquid Chromatography with UV Detector (HPLC-UV). After the collection of defined fractions, chlorogenic acid, and punicalagin were concentrated with a rotary evaporator and transformed into particle form by freeze drying. Hydroxytyrosol was produced as concentrated liquid by using a rotary evaporator. The purities of the obtained compounds were determined as 90.2%for hydroxytyrosol, 92.5%for chlorogenic acid, and 97.1%for punicalagin. Moreover, the inhibition percentages of these compounds on 2,2-diphenyl-1-picrylhydrazyl (DPPH) were established, respectively, as 76.01, 84.90, and 94.64.