I have done HPLC analysis of my nitramine samples. After doing calibration (R square 0.99) i am getting negative concentrations during analysis. Please suggest me the reason behind it
You can receive the negative concentration if your calibration curve not have 0 as point or not extrapolated by 0. If you continue your curve and it meet Y above 0 in this case your analyte less than your lower calibration point can be negative.
You can receive the negative concentration if your calibration curve not have 0 as point or not extrapolated by 0. If you continue your curve and it meet Y above 0 in this case your analyte less than your lower calibration point can be negative.
Please clarify , are you getting negative peaks of your main component after performing the right validation.
Your calibration table should not have '0' listed as a point. Instead, the data should be allowed to show the true intercept for greater accuracy.
If, based on your own calibration table, your integrated area determinations are computed to be lower than "0", then your calibration curve is probably in error and you need to go back and measure %RSD of each point (and make sure you have enough points to cover the whole range AND a good enough curve fit to the points) to determine the accuracy and standard deviation.
Note: A common cause of this is matrix effect. When comparing pure, clean standards to actual samples, sometime the results will be very different than expected. This is due to contributions from the matrix the actual samples are in. Proper sample prep may be needed to overcome this and/or the use of internal standards to compensate for losses.
A poor quality method usually results in poor quality data. A properly developed method addresses any sample prep issues during development.
This is strange! Can you upload your standard curve? I would like to know about this as I have never seen negative concentration while calculating according to standard curve.
"below the baseline". That implies "Negative peaks", not concentrations (which may produce an erroneous calculation of a negative value, but in fact simply indicate failure of the method used). *Even if you included a point for zero (0) in your calibration table, you would still have the same problem if the peak really did drop below the baseline and was integrated. A zero has nothing to do with your question.
If your baseline is flat (this is a question) and your peaks dip down below it, then back up to meet the baseline again (as Jasim asks because this is a completely different question and problem), then we request that you please share with us the precise details of your HPLC method plus a chromatogram (with scales) showing this. Such basic information is needed to determine if this observation is a result of an interpretation error, wrong integration settings, inappropriate detection settings, poor equilibration, co-elution, contamination or a poor quality method. Any of those items could be responsible for the change you observed and depending on which one caused it, the solution will be different.
Have you done to check limit of detection (LOD) and limit of Quantification (LOQ) calibration curve?
maybe concentration lower than LOD or LOQ
or you need check the other parameter from calibration curve like anova, Vx0, mendel test etc
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