A methodology for estimation of the unknown physical parameters in a detailed model for chromatographic processes is proposed. The model describes the concentration of the solute in the mobile phase and considers external/internal mass transfer resistance. The external volume and its mixing effect are also considered. The unknown model parameters estimated are the bed void, the axial dispersion coefficient, the liquid film mass transfer coefficient, the effective diffusion coefficient and the apparent bead porosity. All the parameters in the model were subsequently determined in three classes of experiments, the external mixing behaviour, the mobile phase behaviour and the stationary phase behaviour. The estimates are based on the sum of the least squares of the residuals between the experimental breakthrough curves and the model response. The methodology is exemplified by frontal experiments with lysozyme, bovine serum albumin and immuno globulin gamma on a well-defined column set-up. When the mixing properties in the external volume had to be accounted for, to avoid adding broadening behaviour to the dispersion coefficient, the film mass transfer coefficient and the diffusion coefficient were also investigated. The results show that it is possible to estimate the unknown parameters using the proposed methodology.