Chromatography Forum

Hi everyone,

I have run the gradient HPLC on my samples which were dissolved in the solution (Acetonitrile: Water: Acetic acid) (3:6:1). I have got the desirable peaks from this type of samples. I have to say that my mobile phases are 20% and 55% acetonitrile.

I have collected the fractions during the specific time range and freeze dried them to reduce the volume. As it gets over-dried, I have dissolved it in 60% acetonitrile. After running this new sample with gradient HPLC, I have got negative peaks. I am not sure what is the reason of getting this negative results. Is it related to the high percentage of acetonitrile presented in the sample solution?

If you have any experience regarding to solve the negative peak problem, please give me your advice.

Thanks a lot,
Elham

Elham,

You don't state what detection system you are using, however, a negative peak just means that something in your sample has less absorbance than your mobile phase. It is not due to the different acetonitrile % since even if you were operating at a very low UV wavelength where ACN absorbs (ACN has a 190 nm UV cutoff), the difference would produce a positive peak not a negative one.

Another possibility arises if you are using a diode-array detector with a reference wavelength set. If anything in your sample absorbs more at the reference wavelength than at the measurement wavelength, that will cause a negative peak.

So, as vickig suggested, we need more details about your system: what kind of detector, what wavelength, etc.

That said, using a diluent that is stronger than your initial mobile phase is always risky, but usually more for peak shape problems than for negative peaks as such.

Let's see representative chromatograms