In the work the chromatographic separation of a mixture of two large molecule compounds (analytes) - methyl esters of. deoxycholic (D) and cholic (C) acids was performed. Ternary mixture of solvents containing: hexane, ethyl acetate and methanol as eluent modifier was used as a mobile phase. The influence of the eluent composition on the separation process was analysed, the dependency of isotherm parameters and mass transport kinetics on the eluent composition was determined. For the analysis the transport - dispersive model consisting of the mass balances equations of the analytes and the modifier lumped with isotherm equations was used.