The Role of Ion Pairing Agents in Liquid Chromatography (LC) Separations

Abstract

Ion pairing agents are frequently used in liquid chromatography (LC), especially in reversed-phase liquid chromatography (RPLC), to increase the separation and retention of charged analytes. However, their use in other modes of HPLC, such as sizeexclusion HPLC (SE–HPLC) and ion exchange chromatography (IEC) has not been extensively explored. In RPLC, the target analytes may have charged functional groups, which makes it difficult to maintain them on the hydrophobic stationary phase. This difficulty is often overcome by the inclusion of an ion pairing agent, which introduces ionic contacts and strengthens the hydrophobic connections between the analytes and stationary phase. The separation mechanisms in both SE–HPLC and IEC rely on size and charge differences, respectively, between analytes. Thus, they are naturally constructed to separate analytes without the requiring extra ion pairing agents. In this study, we introduced an ion pairing agent (<0.1% formic acid) to the protein sample, and as a result, an alteration in the elution profile (separation efficiency, selectivity, retention of analytes, and resolution) has been achieved. When samples were introduced with an ion-pairing agent in SE–HPLC, there was a considerable improvement in low molecular weight species (LMWs) separation with four extra peaks with higher resolution (up to 2%), without significant alteration in total area percentage. Similarly, a minor new acidic variant peak was resolved in the weak cation-exchange LC (WCX–LC) analysis of the formic acid-enhanced sample, with the resolution being increased by 3%. The results show that using ion pairing agents should be explored in other modes of HPLC as well as for applications where they may offer enhanced chromatographic separations.