Effects of Mobile Phase Composition on The Separation of Catecholamines by Liquid Chromatography with Electrochemical Detection

In this study, the effects of ion-pairing agents and other ingredients of mobile phase on the separation of nor adrenaline (NA), adrenaline (A) and dopamine (DA) were studied in reversed-phase high performance liquid chromatography with electrochemical detection. The effects of two ion-pairing agents, hexane-sulphonic acid sodium salt (HSA) and pentane-sulphonic acid sodium salt (PSA), have been compared with widely used octane-sulphonic acid sodium salt (OSA). In phosphate based mobile phase, increasing the concentration of OSA and HSA increased the capacity factors (K) of all catecholamines (CAs) in the standard mixture and higher concentration of HSA was required to reach the same values. PSA had minimal effect on the retention times at the same molar concentration range. In ammonium acetate based mobile phase, increasing the concentration of the ammonium acetate significantly reduced the K values of all CAs when HSA was used as an ionpairing agent. Alteration of the ammonium acetate concentration in PSA containing mobile phase had minimal effect on K values. In contrast to the results obtained with phosphate buffer, increasing the pH of the ammonium acetate based mobile phase resulted in an increase in the K values of all compounds investigated. HSA was found to be a suitable ion-pairing agent for liquid chromatographic separation of CAs in either phosphate or ammonium acetate based mobile phases and the applicability of the method was demonstrated by analysis of CAs in different rat tissues after alumina extraction.