By Anonymous on Tuesday, October 1, 2002 - 09:03 am:
I'm in the middle of validating a RP-HPLC method using a C8 column. The mobile phase is 0.05% TFA in water and 0.05% TFA in acetonitrile. The gradient is from 15% to 100%. The problem I have is when we changed the flow rate from 0.8 mL/min to 1.1 mL/min in the rubostness test, the peak heights and the area counts of the main peak dropped 20%. The %AUC results of the main peak were still the same. I repeated this experiment on different instruments and also changed the flow rate back and forward. There seems no degradation and the phenominon is reproducible. Even though this is a %AUC method, I'm concerned about this dramatic area counts change.
By Anonymous on Tuesday, October 1, 2002 - 12:11 pm:
The area counts will change with flowrate changes. The faster the flowrate, the less time the analyte spends in the detector and therefore the less response it will have. Since you have increased your flowrate by approx. 25%, you should have an area decrease approximately the same percent.
If you want to increase your response (area and sensitivity) you can reduce your flowrate. We have had to do this when developing methods for very low responding drug products. Yes the run is longer and the peak width broader, but it works.
Hope this helps,
DH
By Anonymous on Wednesday, October 2, 2002 - 07:14 am:
See at discussion in Archive Jan 01 - June 01: Should Flow Rate Effect Detector Response Like This.
It contain a good explanation on this effect.
By Anonymous on Wednesday, October 2, 2002 - 06:46 pm:
Easy illustration is to turn off your pump for a few seconds in the middle of the elution time of a peak to watch the area counts for that peak go way up. Works with a non-destructive detector, like a UV, of course.
By A. Buske on Monday, October 7, 2002 - 02:47 am:
The peak area depends - beside on the concentration - on other factor as wavelength, flow rate, detector make, sampling rate etc...
However that’s not what you are interested in when doing robustness testing. Peak area might change, but the measured analyte content should be stable. So inject standard solution, make a calibration under that conditions, analyse your sample(s), determine the content and don’t care for peak areas.
It is sometimes very interesting to see what parameters influence your result.
A. Buske