Study of the Behavior of Water-Soluble Vitamins in HILIC on a Diol Column

An effort has been made to investigate the chromatographic behavior and to understand the basic mechanisms in HILIC-based separation of water-soluble vitamins with highly varied properties on a diol column. The water content of the mobile phase is of utmost importance because it directly affects the type and extent of interactions of the solutes with the stationary phase and with the buffered mobile phase. A mixed-mode partitioning-surface adsorption mechanism enables most precise description of their chromatographic retention and separation. The point at which surface adsorption becomes apparent, however, depends on the properties of the solutes on the given stationary phase, and on the presence of buffer salt ions. Adjustment of mobile phase pH and use of different buffer salts can be used to modify electrostatic interactions among the solutes, active silanols, and counter-ions. The role of hydrogen bonding was clarified by substitution of ACN by solvents with moderate to strong hydrogen bonding potential. Analytes which are neutral at the working pH start to interact with the stationary phase when the ACN content is increased to 80%. Negatively charged analytes are adsorbed on the stationary phase when the ACN content is approximately 86%, because augmentation of the counter-ions weakens electrostatic repulsion by the active silanol groups. On the other hand, the electrostatic attraction of thiamine contributes significantly to its retention even when using mobile phases with high water content.