Help

[shadow]

Hi, I am using a waters alliance system to do RP and I am getting different areas for all peaks when I change the flow rate in the gradient. Areas are consistent if flow is the same, but they differ greatly when changed. Chromatographic parameters are the same, inj vol is the same, the vial is the same, nothing changed except flow rate, higher flow rate = lower area, can anyone explain this? I have seen this in different systems with different columns and I do not understand it, any help is appreciated thank you.

[skunked_once]

Higher flow rate results in shorter duration of the peaks in the flow cell. Shorter duration = less absorbance = lower peak areas.

[Gerhard Kratz]

To change Flow rate during a run is not the best way to get a robust method and reliable results. In some cases used, but I would recommend to restart method development. A flow gradient will change equilibrium on the stationary phase, baseline drift. Best is to work with constant flow and constant pressure. When you have peaks who are not basline separated and you want to use flow rate changes to get a better resolution, maybe another chromatographic mode is a better way. You use RP, try HILIC, for example.

[shadow]

Thanks for the replies.
Skunked, if that is the case then if I adjust my sampling rate I should offset the effect and get similar areas?

Gerhard, i meant in different runs, but now that you mention it, how do you achieve constant pressure and volume on a normal HPLC run? I read an article on a separation done using constant pressure at the expense of the flow rate being increased and it worked well, peaks were sharp at the end of the run.

Thanks

[HPLCaddict]

Thanks for the replies.
Skunked, if that is the case then if I adjust my sampling rate I should offset the effect and get similar areas?

No. The sampling rate just tells the detector the interval between the collected data points. It does not modify the residence time of the analyte in the detector cell.

Gerhard, i meant in different runs, but now that you mention it, how do you achieve constant pressure and volume on a normal HPLC run? I read an article on a separation done using constant pressure at the expense of the flow rate being increased and it worked well, peaks were sharp at the end of the run.

Well, this constant-pressure HPLC can be interesting, but right now this is purely academic work. It's not possible with standard HPLC equipment. You might use different flow-rates during a run, though. I like to do this at the end of a run to speed up cleaning/reequillibration steps. I'd keep my fingers away from flow-rate changes during the separation step itself, as this will decrease the robustness of the method.

[shadow]

Thank you for the advice!