Mixed-mode HPLC columns allow to analyze compounds with drastically different properties in one run. Acidic, basic, and neutral compounds can be separated in one run using either isocratic or gradient conditions. In this application, neutral hydrophilic (uracil, phenol and hydroquinone), neutral hydrophobic (toluene), hydrophilic acidic (benzoic acid), hydrophilic basic (lutidine) and hydrophobic basic (amitriptyline) are separated using gradient of ACN. Neutral compounds are retained by reversed-phase mechanism, hydrophilic acidic compound become more hydrophobic at lower pH and retain by reversed-phase mechanism too. Basic compounds are retained by cation exchange mechanism, and hydrophobic basic compounds are retained by reversed-phase and cation-exchange mechanisms. All compounds are resolved within 17 minutes on a short column. Method can be applied to various polar and hydrophobic compounds, which can be separated on one column and in one run. Mixed-mode columns can operate in single or combination of several modes: reversed-phase, ion-exchange, ion-exclusion and HILIC. This mixed-mode HPLC column can be used as a general column for separation of wide range of compounds.