Hey everyone,
im struggling to explain whats the cause of the following phenomena:
We are using HPLC to validate the purity of our compounds. So in the best case we obtain chromatograms with a single peak an maybe some minor impurities. However, i started noticing, that only in the case of fluorescein compounds multiple peaks are appearing in the chromatogram. We are using a DAD detector and the spectra of these "duplica" is all the same, indicating is should be the same compound. Also NMR of the sample shows a single, clean product. All other measured substances are showing just a single peak. Does anyone has an explanation for this behavior?
The column is a reversed phase C18 column
Im running a gradient from 0 to 100% B
A= 5% ACN and 0.1 % MSA in Water
B= 5% Water and 0.1% MSA in ACN
Detection in the chromatorgam shown is 210 nm
thanks,
Jan
The multiple peaks for fluorescein compounds in your chromatogram may result from pH-dependent structural variations and isomeric forms. Despite these multiple peaks, the compound is chemically identical, as confirmed by consistent UV spectra and NMR results.
Many years ago I had a similar problem encapsulating Acyclovir in liposomes.
In my case it was the addition of acetic acid to the sample.
It turned out that the molecule had two pKa's and one peak was acyclovir and the other was an ionic pair of acyclovir with acetic acid. Obviously the absorption spectrum was the same.
Some questions:
Sample preparation is the same?
However, 1H NMR is much less sensitive to impurities than HPLC. It is also possible to detect an impurity with the same chromophore. Have you considered MS?
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