I carried out an experiment aimed at confirming the presence of aspirin and to quantify the abundance of asprin using standard addition. And using HPLC instrumentation.
From the result, the chromatogram shows increase in the peak height as the concentration increases due to spiking and I thought this was a good result.
However the peak areas are not increasing, the increase then decrease, there is no steady increase .
Please what would be the reason. And how will this affect the error when peak area verse concentration graph is plotted.
Thank you
Hi Frances, it looks like you have a problem with your autosampler - especially your injector. I would do reproducibility tests with different concentrations in your case. That means you inject each concentration 10x or 20x and look at your peak areas and peak heights. With these values you can create an average and the standard deviation and then you can see relatively quickly where the problem is located. Do you have leaks somewhere (e.g. screw connection of the sample loop)?
No I do not have any leak
What about the autosampler could cause the problem
Thanks
Your sample will probably be drawn up via a syringe into the sample loop. If the syringe is not working reproducible you have a problem with the reproducibility of your analysis. Do you have the possibility (in the software) to set up a multiple of the sample loop? If yes, then your fluctuations may decrease or disappear completely (e.g. with a 5 µL loop you draw up 20 µL, i.e. the sample loop is overfilled by a factor of 4. The excess (in this case 15 µL) ends up in the waste).
Hello Frances,
It would be useful if you could provide a few whole chromatograms in pdf, png or jpg format.
Kind regards,
Ade Kujore
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Most probably the variations in the peak areas could be because of inconsistent injections. If the syringe picks up air bubbles during injections, the areas can vary widely. Make one standard sample and inject multiple times and see what is the RSD value of the area counts. You will come to know the precision of your injections.
Have you done the stability assay? Aspirin may under go for degradation in some of the mobile phase used. It depends upon the mobile phase used and of course some other conditions also. This can be confirmed from the chromatograms only.
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