Hello,
Today, when I analyzed my nectar sample (containing plant secondary metabolites, amino acids and proteins) using a HPLC with a UV detector (220nm) and a SEC column, NaOAC buffer as mobile phase, I got two big negative peaks which are repeatable. Wired to me. I am sure the instrument is all right and no air injected.
Is it possible that any components are fluorescent in the sample which could be excited on UV 220nm?
I think that you have chosen the wrong wavelenght for your UV detector or you have to use a mobile phase that is completely transparant at 220 nm.
Na acetate solutions have a significant absorbance at 220 nm, see fig 2 of
Article High throughput pyrogenic carbon (biochar) characterisation ...
This means that any sample component, or better any substance injected, having a lower absorbance than Na acetate at 220 nm, will cause a negative peak.
Hi,
I think Ladan Ajdanian and Jos P M Wielders have the best clue. Sometimes you can have a mobile phase that is less transparent to the wavelength than the analytes you are dealing with. You may take a spectrum of your mobile phase, if you will, to check whether this is true. Try to choose a mobile phase that is adequate for your analytes and wavelengths. Check the spectrum and the literature available about the new mobile phase before using it, if this is the case.
Jos Wielders have the right answer, the absorbance of the analytes are lower, then the NaAc. However, the negative peaks can be treated similarly to the positives, they have the same dependence from the concentration. It means you need not change your mobile phase. Even this negative peaks could more sensitive, because the analytes has very low molar absorbance coefficient, which would result very little positive peaks in "more transparent" mobile phase!
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