Protocatechuic acid (PCA) possess antioxidant, anticancer, and antidiabetic activity. It is a novel active substance against infectious disease bursal virus infection and avian influenza virus H9N2 infection. The separation of PCA was experimentally investigated using tri-n-butyl phosphate (TBP) in natural non-toxic diluents (sunflower oil, soybean oil, sesame oil). Dimerisation constant, partition coefficient, distribution coefficient, extraction efficiency, and loading ratio were determined. A single cycle achieved a maximum 90% separation. The trend observed for separation in diluent was sunflower oil > soybean oil > sesame oil. Mass action law, relative basicity and Langmuir model were used to represent the reactive extraction equilibrium of protocatechuic acid using TBP. The toxic effects can be averted by evasion of direct contact of organisms with organic phase and by the utilization of natural non-toxic diluents. The diffusion coefficients of protocatechuic acid were estimated using Wilke-Chang equation and Reddy-Doraiswamy equation. The number of theoretical stages (NTS) for counter current extraction was found to be 5 using modified Kremser equation. The results can be employed to design reactive extraction column. (C) 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.