4-Aminosalicylic acid (PAS) is an antibiotic used in treatment of tuberculosis. It is a polar amino acid with limited retention on traditional C18 (reversed-phase) columns. Other isomers of aminosalicylic acid exist, but the main impurity in PAS is 5-Aminosalicylic acid, which also serves as anti-inflammatory drug. Both compounds are isomers with similar empirical structure and properties. These two isomers were separated on a Primesep 100 column with UV, ELSD and LC/MS compatible mobile phase. Method can be used a generic approach for separation of isomers of basic and zwitter ionic compounds. Isomers are retained and separated based on reversed-phase and cation-exchange properties. Retention time is controlled by the amount of acetonitrile, buffer concentration and buffer pH. Buffer pH is affecting ionization of these two compounds and thus serves as a powerful tool to adjust selectivity of separation.