Isomers of organic compounds usually have very similar properties. Separation of such isomers by HPLC requires intensive screening of various columns and mobile phase conditions. In case of hydrophilic compounds, this task becomes even more challenging due to the lack of or limited retention on traditional reversed-phase (C18 or C8) HPLC columns. Three isomers of aminobenzoic acid (2-aminobenzoic acid, 3-aminobenzoic acid and 4-aminobenzoic acid) were successfully separated on a Primesep 100 reversed-phase/cation-exchange column. Baseline separation within 10 minutes was achieved and produced a perfect peak shapes for all three analytes. The method can be used a generic approach for separation of ionic hydrophobic and ionic basic isomers by mixed-mode chromatography.

Application Notes: Procainamide is a common antiarrhythmic agent used to treat cardiac arrhythmias. According to USP methods procainamide hydrochloride contains not less than 98% and no more than 102% procainamide based on the dried basis. The USP HPLC method for the separation of procainamide and 4-aminobenzoic was developed on Legacy L1 column according to the US Pharmacopeia methodology. L1 classification is assigned to reversed-phase HPLC column containing C18 ligand. Support for the material is spherical silica gel with particles size 3-10 um and pore size of 100-120A. Resolution between critical pairs corresponds to rules and specifications of UPS.

Reference: USP 35- NF30