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Selection of Separation Conditions for HPLC and HPLC/MS of Aromatic Sulphonic Acids and Acid Azo Dyes

Abstract Liquid chromatography of organic anionic compounds usually cannot be performed without ionic additives to the mobile phase. Buffered mobile phases usually do not provide sufficient suppression of the ionization of strong acids, such as anionic dyes and aromatic sulfonic acids used in the dye production, or as whitening agents or detergents in various industrial and household applications. Hence, other modes are usually necessary for successful separation of these compounds, including ion‐exchange chromatography, ion‐interaction reversed‐phase chromatography, or ion‐pair chromatography. The retention mechanisms in these modes are still a controversial issue. In the present work, various theoretical models describing the retention of strong organic acids with bulky hydrophobic structural elements are reviewed. Conventional ionic additives employed in these separation modes usually are not volatile enough for HPLC/MS applications, such as quaternary ammonium salts commonly used as ion‐pairing reagents in ion‐pair chromatography. This inconvenience can be overcome either by removing the nonvolatile additives from the mobile phase before introducing the column effluent to the ion source of the mass spectrometer, or by using more volatile compounds as their substitution. Suitable volatile reagents should be selected taking into account their effects on the retention, separation selectivity, efficiency, and band symmetry of various compounds differing in structures. Another important point that should be considered in method development is possible suppression of ionization by the additives, which should be avoided.