Analysis of Highly Polar Compounds by Ion-Pair Liquid Chromatography

Many polar compounds of biochemical and pharmaceutical relevance exhibit low retention in reversed-phase liquid chromatography (RPLC), making their separation challenging. While hydrophilic interaction liquid chromatography (HILIC) columns are commonly used for such analyses, they require mobile phases with high organic solvent content. This work explores an alternative approach using RPLC with conventional C18 columns and mobile phases containing low percentages of acetonitrile, along with small amounts of the surfactant sodium dodecyl sulfate (SDS). This combination significantly enhances the retention of highly polar compounds. When the SDS concentration is sufficiently low, below the critical micellar concentration in water (8 mM), the retention increase follows a linear pattern. The retention behavior of polar compounds with different properties (nucleosides, methylxanthines, sulfonamides, and the diuretic hydrochlorothiazide) is examined using mobile phases in the submicellar region, with SDS concentrations ranging from 0 to 0.3 mM, acetonitrile contents between 10 and 20% (v/v), and temperatures varying from 25 to 55 °C. Changes in peak half-widths are also analyzed. Since SDS adsorbs onto the stationary phase, modifying its surface, the equilibration time has been investigated as a critical factor affecting retention reproducibility, influenced by the SDS concentration, acetonitrile content, and temperature. The results emphasize the need for complete equilibration to ensure reliable and consistent results.