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What would cause low signal from higher concentrations?
Posted: Thu Oct 20, 2022 9:04 pm
by jpappas_PNB
Running a standard curve on HPLC and my linearity is coming out poor (0.9772) due to the highest concentration being lower than what it should be to make the line fit properly for my data points. I've made multiple new standard solutions to ensure it is not error in pipetting etc.
Is there a deeper issue at hand with my detector or something possibly that could cause this?
Re: What would cause low signal from higher concentrations?
Posted: Thu Oct 20, 2022 10:26 pm
by millerk8561
Hard to say, could have been a bad injection? Does the same exact error persist if you reinject the same samples?
Re: What would cause low signal from higher concentrations?
Posted: Fri Oct 21, 2022 12:47 pm
by jtmarten
How high is the concentration?
I've not run into this, but is there a saturation point for the column or detector?
Re: What would cause low signal from higher concentrations?
Posted: Fri Oct 21, 2022 1:50 pm
by James_Ball
Will depend on the column dimensions, detector type and many other things.
Can you give more information as to the HPLC setup and the analyte that is giving the problem.
Saturation is one problem that can happen. If the concentration is beyond the limit of the detector then it can give strange results.
Re: What would cause low signal from higher concentrations?
Posted: Sun Oct 23, 2022 10:31 pm
by wss
Running a standard curve on HPLC and my linearity is coming out poor (0.9772) due to the highest concentration being lower than what it should be to make the line fit properly for my data points. I've made multiple new standard solutions to ensure it is not error in pipetting etc.
Is there a deeper issue at hand with my detector or something possibly that could cause this?
To me it sounds like some concentration-dependent process is starting to occur somewhere in the system, which may just be inherent to your compound or may be exacerbated by your conditions. As James said, can you describe more details as to what conditions you're using and the analyte you're targeting? It could also just be a bad injection as miller mentioned.
Re: What would cause low signal from higher concentrations?
Posted: Tue Oct 25, 2022 12:57 pm
by Pear
Running a standard curve on HPLC and my linearity is coming out poor (0.9772) due to the highest concentration being lower than what it should be to make the line fit properly for my data points. I've made multiple new standard solutions to ensure it is not error in pipetting etc.
Is there a deeper issue at hand with my detector or something possibly that could cause this?
Can you post your calibration graph? Perhaps a linear fit is not best, maybe your concentration range is quadratic?
Re: What would cause low signal from higher concentrations?
Posted: Thu Oct 27, 2022 10:45 am
by lmh
I assume your peak hasn't gone flat-topped or saturated the detector? What is the detector anyway?
Re: What would cause low signal from higher concentrations?
Posted: Thu Oct 27, 2022 12:55 pm
by DR
How's the peak shape for this in general? Really broad peaks are tough to integrate consistently as they get smaller and this can also be a contributing factor to nonlinearity due to inaccuracy.
As previously mentioned, if you're getting "mesa" shaped peaks, that's an indication of having saturated the detector, so you're losing linearity at the high end.