PDA Peak Purity

[Uwe Neue]

What is your mobile phase?

[aceto_81]

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

[MestizoJoe]

Mobile phase is 85% [50mM phosphate buffer, 5mM octane sulfate at pH 2] 12% MeOH 3% ACN

[Uwe Neue]

At the very low wavelength, you may be seeing the solvation of your analyte with methanol.

[HW Mueller]

Uwe, do you mean "interference from MeOH"?
It looks clearly as if a "zero" of baseline was never done here, so the specs of mobile phase components interfere.

[Alex Buske]

It looks very much like unappropriate "baseline correction". The dip at 200 nm could simply be "calcultated" because to much solvent signal is subtracted.
Try to refine the baseline correction. Or don't use it and compared spectra at the beginning and end of the peak. Or use a spectra range starting from 210nm (below everything is highly overlaid bei solvent absortion and therefore doesn't contain much information anyway).

Alex

[ksharp]

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.

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.

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.

[aceto_81]

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.

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.

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.

But if you compare the spectra at one side:
16.051 with 16.092 with 16.235 with...

Then you will find variation, which made me think that this is no random variation...

Ace

[praveenpaliwal]

MestizoJoe

Sir how could u insert images of PDA spectra

[zheyin]

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

How could u have a small impurity that is much wider than the main compound?
(You can see the same absorbance trace at 16 min and 16.9 min for that impurity?)

I think it cannot be one impurity.

[zheyin]

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.

You said the only possible false positive is when the s/n is low.
But my recent experiment of pure ref std shows that at higher conc, the purity fails (comparing purity factor and threshold) while at lower conc, it passes.
Both numbers are high 9999.xx something.
I am very confused.

[tom jupille]

I clarified it a bit in the third paragraph by including "saturation of the detector".