Indirect UV detection of hydrophilic ionized compounds in reversed-phase liquid chromatography by use of a UV-absorbing ion of the same charge

Abstract

Ion-pair reversed-phase liquid chromatographic systems have been developed that allow the detection and quantitation of low amounts of hydrophilic ionized substances, which otherwise display little or no capability for optical detection. The mobile phase contains a UV-absorbing ion of the same charge and hydrophobic character as the samples, most often together with a hydrophobic counter-ion to give the samples suitable retention; the stationary phase consists of octadecylsilica. Chromatograms featuring two system peaks are usually obtained, and these give rise to some deviations from the usual response pattern. In the present work the influence on retention and detection sensitivity of the non-absorbing counter-ion, the UV-absorbing ion and the uncharged organic modifier have been studied systematically. Principles for optimizing the detector response by changing the mobile phase composition have been deduced. Examples of applications of the indirect detection technique to the analysis of hydrophilic compounds of pharmaceutical and biological interest, such as amino acids, water-soluble vitamins and piperazine, are presented.