Supplementary Appendix 1 to A Practical Approach to Modelling of Reversed-Phase Liquid Chromatographic Separations: Advantages, Principles, and Possible Pitfalls

This information is supplementary to the article “A Practical Approach to Modelling of Reversed-Phase Liquid Chromatographic Separations: Advantages, Principles, and Possible Pitfalls” that was published in the March 2018 LCGC Europe issue.

Determination of dwell volume

• Replace the column with a 100 cm x 0.1 mm capillary to generate enough pressure to ensure proper function of check valves.

• Use water as solvent A and 10 mg/L of uracil in water as solvent B.

• Set the flow rate to the flow that should be modelled.

• Programme a 1 µL injection of water to ensure that the auto-sampler is in the flow path and contributes to the dwell volume.

• Run a gradient with a 5-min isocratic hold followed by a 10-min linear gradient with the same slope as the average gradient used for collection of the retention modelling data, for example 10%B at 0 min, from 10 to 20%B between 5 and 15 min, from 20 to 10%B between 25 and 25.1 min.

• UV detection at 259 nm at 20 Hz to ensure a well-defined curve.

• The dwell volume is defined using the gradient start and stop time and time point for 50% of maximal signal as described in Figure 2(a).

• In order to evaluate the linearity (Figure 2) an extra run is made where the range is extended to 0–100%B.  An extra gradient is necessary since flow rate and gradient range influence the determination of dwell volume.