Study of overload for basic compounds in reversed-phase high performance liquid chromatography as a function of mobile phase pH

The retention and overloading properties for eight basic solutes and two quaternary ammonium compounds were studied over the pH range 2.7–10.0 using phosphate and carbonate buffers. At low pH, a hybrid inorganic–organic silica-ODS phase (XTerra RP-18, 15 cm × 0.46 cm) showed substantial loss in efficiency when sample masses exceeded about 0.5 μg; these results were similar to those obtained previously on pure silica ODS and wholly polymeric phases, suggesting a common overloading mechanism. At pH 7–8.5, substantial improvements in loading capacity were obtained on XTerra due apparently to the unexpectedly strong influence of small decreases in solute ionisation. Data from the quaternary compounds suggested that silanol ionisation on this phase was still small even at intermediate pH. For many bases, loading capacity continued to improve as the pH was raised to 10, in line with the decrease in the proportion of ionised solute. However, for the highest pKa solutes, peak shape worsened at high pH, possibly due to the negative influence of increasing column silanol ionisation.