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.

Primesep 100 and Primesep 200 columns can be used as a universal column for analysis of wide range of compounds. These mixed-mode reversed-phase ion-exchange HPLC columns can provide a valuable alternative to traditional reversed-phase column. Amines, amino acids, quaternary amines, and various zwitter-ions can be analyzed along with hydrophobic compounds and organic and inorganic counter-ions. In this application, 8 compounds with different hydrophobic, hydrophilic, basic and acidic properties are separated based on their properties. Primesep 100 column is a mixed-mode HPLC column with a C12 carbon chain and carboxylic acid on the surface with pKa of 1. Primesep 200 column is a mixed-mode HPLC column with a C12 carbon chain and carboxylic acid on the surface with pKa of 2. These columns can be used with 100% organic (ACN) and 100% aqueous mobile phases. This HPLC method can be adopted as a generic and robust approach for analysis of acidic, basic and neutral compounds within the same run.

The presence of three drugs, dextromethorphan, amitriptyline and trimipramine in saliva were analyzed by mixed-mode chromatography on a Primesep D reversed-phase anion-exchange column. No sample preparation is required as proteins from saliva are not retained at these conditions and elute in the void of the column due to cation-exclusion effect. Both column and proteins are positively charged at current conditions. Mobile phase consists of ACN-water-formic acid and is compatible with LC/MS detection technique. This general HPLC method can be used for analysis of hydrophobic basic drugs in biofluids.