Evaluation of the phase ratio for three C18 high performance liquid chromatographic columns.

For a chromatographic column, phase ratio Φ is defined as the ratio between the volume of the stationary phase Vst and the void volume of the column V0, and it is an important parameter characterizing the HPLC process. Although apparently simple, the evaluation of Φ presents difficulties because there is no sharp boundary between the mobile phase and the stationary phase. In addition, the boundary depends not only on the nature of the stationary phase, but also on the composition of the mobile phase. In spite of its importance, phase ratio is seldom reported for commercially available HPLC columns and the data typically provided by the vendors about the columns do not provide key information that would allow the calculation of Φ based on Vst and V0 values. A different procedure for the evaluation of Φ is based on the following formula: log k'j=a log Kow,j+log Φ, where k'j is the retention factor for a compound j that must be a hydrocarbon, Kow,j is the octanol/water partition coefficient, and a is a proportionality constant. Present study describes the experimental evaluation of Φ based on the measurement of k'j for the compounds in the homologous series between benzene and butylbenzene for three C18 columns: Gemini C18, Luna C18 both with 5 µm particles, and a Chromolith Performance RP-18. The evaluation was performed for two mobile phase systems at different proportions of methanol/water and acetonitrile/water. The octanol/water partition coefficients were obtained from the literature. The results obtained in the study provide further support for the new procedure for the evaluation of phase ratio.

Competitional hydrophobicity driven separations under RP-LC mechanism: application to sulfonylurea congeners.

Retention of a model set of sulfonylurea compounds has been studied under RP-LC conditions, considering competitional effects brought by different alcohols (ethanol, 1-propanol, 2-propanol, 1-butanol, 1-pentanol, and 1-octanol) used as additives in the organic component of the mobile phase (methanol). The capacity factors determined for the model compounds decreased with the increase of the hydrophobic character of the organic additive in the mobile phase. The amount of the additive within the organic component of the mobile phase was kept constant (1% as volumetric ratio). Retention was studied at different mobile phase compositions (aqueous to organic component ratios). Different functional fitting models were used to correlate retention to the content of the organic component in the mobile phase. Extrapolation of retention expressed as capacity factor to a mobile phase composition free of organic component is well correlated to the hydrophobic characteristics of the organic additives. The adsorption model was used for tuning the experimental find-outs. The possibility of controlling retention through the competitive effects induced by hydrophobic additives in the mobile phase is highlighted.