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I don't have a lot of experience in PDA peak purity measurements...actually, I have none...but with respect to the variation to which you refer: the same spectra are obtained on each "side" of the peak maximum at 16.408.So, I took the UV spectrum from the PDA at several time points within the peak. What I found was variation in the spectrum. I guess this can be considered noise. Correct me if I am wrong.
But if you compare the spectra at one side:I don't have a lot of experience in PDA peak purity measurements...actually, I have none...but with respect to the variation to which you refer: the same spectra are obtained on each "side" of the peak maximum at 16.408.So, I took the UV spectrum from the PDA at several time points within the peak. What I found was variation in the spectrum. I guess this can be considered noise. Correct me if I am wrong.
Compare:
16.051 with 16.840
16.092 with 16.811
16.235 with 16.723
16.323 with 16.639
They look the same. Just an observation. Good luck.
How could u have a small impurity that is much wider than the main compound?Maybe it is coincidence, but if you take a look at about 200 nm, you see a dip in the spectrum at the beginning of the peak, which slowly disappears, and back show up at the end of the peak.
This could be a small impurity with a spectrum that only has absorbance in the region < 205nm.
If you inject less volume, the underlaying peak can be too small to be detected.
Just some thoughts....
Ace
You said the only possible false positive is when the s/n is low.First of all, we have to realize the limitations of *any* PDA-based peak purity measurements. They will fail to detect an underlying peak (false negative) under four conditions:
1. The underlying peak has a very similar UV spectrum.
2. The underlying peak is very small.
3. The underlying peak has no UV absorbance over the wavelength range involved.
4. The underlying peak exactly coelutes with the peak of interest.
They will detect a non-existant underlying peak (false positive) only when the signal/noise ratio is low (very noisy baseline or very small peak of interest).
In this case (false positive?)Low-wavelength noise and/or saturation of the detector (as has been suggesting) is certainly a possibility. The other possibility is that you do, indeed have an underlying peak, which does not have a significant chromophore above 210 nm but *does* have an "end absorbance" below 210 nm (an aliphatic alcohol, for example).
What to do depends on your purpose. If its an assay (where you want to quantitate the main peak), then sitting at the absorbance maximum (presumably above 210 nm) would be fine. If you are looking for impurities, then there *is* a chance that you're missing something.
