Performance of chromatographic columns is of major importance in the development of more efficient separation methods. So far, a common practice is to study the column behavior in isocratic elution by modifying the flow rate and fitting the theoretical plate height values versus the mobile phase linear velocity, according to the Van Deemter equation. In this work, an approach is presented to extend the measurement of efficiency to linear gradient elution, where the mean retention factor is kept constant at each assayed flow. This avoids a possible source of uncertainty due to the change in the distribution equilibria profile, and makes the mean interactions with the stationary phase in gradient elution similar to those in isocratic elution. The approach was applied to Zorbax Eclipse XDB C18 and Chromolith SpeedROD C18 columns, using four sulphonamides as probe compounds. The results are compared with those obtained in isocratic elution, and gradient elution where the gradient program is kept constant while the flow is varied, giving rise to changes in the mean retention factor at each flow rate. The mean compression factor obtained experimentally was higher than expected, with mean values of 0.98 and 1.02 for the Zorbax and Chromolith columns, whereas the predicted values were 0.87 and 0.92, respectively. Better efficiencies were obtained at lower flow and higher gradient slope. The resolution shows similar values in isocratic and gradient elution when the mean retention factor is kept constant. When the gradient slope is constant, the resolution is usually smaller, although it improves at higher flows.
Keywords: Column characterization; Data treatment; Dispersion parameters; Liquid chromatography; Peak profile; Van Deemter plots.
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