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When samples are dissolved for HPLC analysis, organic solvents are often used to enhance the solubility of the sample components. However, when the diluent becomes significantly stronger than the mobile phase, peak shape distortions may develop during injection. This is a serious problem for weakly retained analytes under 100% aqueous mobile phase conditions. Under these extreme conditions, even small amounts of solvent in the diluent can cause serious broadening or distortion effects. However, if the organic solvent used in the diluent elutes significantly after the analyte, the peak shape of the analyte will be relatively unaffected, even when a relatively strong solvent such as THF is used. This phenomenon is counterintuitive considering the usual practice of keeping the sample diluent as weak as possible. This report describes the potential analytical utility and limitations of this approach for the analysis of weakly retained analytes.

When mobile-phase salt content is increased, cationic analytes often show increased retention. This effect is generally attributed to chaotropic or ion pairing effects. However, a cation exclusion mechanism could explain the same effects. In this study, experimental conditions were manipulated to enhance cation exclusion effects and reduce chaotropic/ion pairing effects by using (1) low ionic strength mobile phases to reduce electrostatic screening, (2) a buffer anion (dihydrogen phosphate) that exhibits minimal chaotropic/ion pairing effects, and (3) columns that show evidence of a weak positive charge. Urea was used as neutral void marker and glycinamide (in protonated form) as cationic void marker. It was assumed the difference in retention volumes between void markers would reflect an "excluded volume", inaccessible to cationic analytes. As ionic strength was lowered, it appeared as much as 80% of the pore volume became inaccessible to the glycinamide cation at the lowest ionic strength tested (1.4 mM). Three model cationic analytes showed retention loss approximately proportional to the excluded volume as ionic strength was decreased. This suggests that, under certain conditions, cation exclusion may become the dominant mechanism in mediating the retention of cationic analytes as the mobile-phase salt content is varied.