DAD : spectral homogeneity

[lotussss]

Hello,

My question is about spectral homogeneity. What mean study of spectral homogeneity in hplc?

Thanks.

[Uwe Neue]

Spectral homogeneity refers to the uniformity of the UV-vis spectrum over the peak. If the spectrum is the same, then the likelyhood that there is a coelution is very small.

[lotussss]

But, if there is a small quantity of impurety, you can't see the difference. Yesterday my next employer ask to me if i know the study of spectral homogeneity? I said OK. But he ask to me about this study with agilent chemstation software. what propose this software?

[Ary]

Its been a very long time since I used the Agilent software but if I remember correctly it looks at the UV spectra from various points across a chromatographic peak and compares them. It then assigns a number from 0-1000 to indicate how alike these spectra are. 1000 indicates the spectra are identical and therefore the peak is "pure". If you have coeluting peaks the idea is that the UV of the two components will be different and so the UV spectra at say the beginning middle and the end of a chromatographic peak will be different and so the peak purity figure will be less than 1000. In practice if the compounds have very similar UV profiles this peak purity figure is far less reliable. As far as I'm aware this is still the case but I would be happy to stand corrected if things have moved on.

[Uwe Neue]

I have not worked for a while with this technique either, but from what I remember, the technique (we used the Waters software) was VERY sensitive to impurities in the peak. It was often rather the other way round: we had to lower the threshold to declare a peak to be spectrally homogeneous or "pure".

[lotussss]

In spite of I haven't use this technique (...), I work with empower of waters.
Could you tell me where is this option in the software and how use it? Initialy need you any standard to calibrate this option or it is just a calculation of the software such as variation's area according to choice of width's, height's(...) peak.

[Uwe Neue]

It is in the PDA software. Look for "Match Angle", as the key word, and in the manual.