Chromatography Forum

I have a method for sunscreens that seems to work fine. However, when I tested the robustness of the method by varying the flow rates, I get very different peak areas.

The raw data:
4,574,402 uVs - 0.9 mL/min
4,131,267 uVs - 1.0 mL/min
3,783,385 uVs - 1.1 mL/min

What's weird is that the relationship seems to be linear as well (R2 = 0.9952). Any thoughts on why this would be?

This is absolutely normal behavior. Since the peak area is calculated from signal-intensity (peak height) over a time period.

If you run your analysis with e.g. 0.5 mL/min instead of 1.0 mL/min the signal-intensity is constant but the time your substance stays in the flow cell and absorb the light will be factor 2 higher.

Therefore using the half flow-rate will cause the double peak area while peak height is still constant.
Otherwise, using the double flow-rate will result in 50 % of the peak area since your compound stays only the half of the time in the flow cell.

I hope this makes at least some sense to you.

Agree: typical.

A hardy perennial of a question - try a search of the archives.

Peter

That does make a lot of sense. I think I was caught up on the fact that the amount of analyte doesn't change but didn't think about how much longer the sample was in the path of the detector. Thanks everyone!

I suppose the detector is UV so its signal depends on concentration, not bulk. So when the flowrate increases, the concentration is the same, but the integration time is reduced, so you have lower peak area.