© 2022 John Wiley & Sons Ltd.Introduction: Urtica dioica (nettle) is a plant species of the Urticaceae family that grows in various parts of the world and exerts antioxidant, antibacterial, antiulcer, antiviral, and anti-inflammatory effects. Their leaves, roots, and seeds are used in various fields such as food, medicine, and cosmetics. Objectives: Inverse gas chromatography (IGC) was used to evaluate the surface characteristics and separation ability of U. dioica leaves, roots, and seeds. Characterization of these biomasses was performed by Fourier transform infrared spectroscopy (FTIR) analyses. Methodology: The surface properties of the biomasses including dispersive surface energy, adsorption enthalpy, Gibbs free energy, and acidity-basicity constants were determined at infinite dilution using various organic solvents. These properties were compared with each other. Dispersive surface energies were calculated using the Dorris–Gray, Donnet–Park, and Schultz methods. The accuracy of these methods and their applicability were evaluated. In the last stage of this study, the separation of xylene isomers was investigated by using U. dioica biomasses as stationary phases. Results: The surface functional groups were determined by FTIR analysis. As a result of the IGC studies, it was found that the adsorption of polar solvents on biomasses occurred exothermically and spontaneously. Besides, it was found that the surfaces of biomasses were basic. From the retention diagrams and selectivity coefficients, it was determined that xylene isomers were effectively separated. Conclusion: IGC is a promising, low-cost, easy-to-apply, and high-accuracy technique for the investigation of the surface properties of biomasses and their ability to separate isomers.