Chromatography Forum

Hello,

while developing a LC method for some sulphonamides, we find some strange behaviour of our API.
After dilution with water, we get a degradation peak.
Maybe this sound really strange, but if we don't dilute further, only using the first dilution, we don't get the degradation.

We tested our API in plastic bins: an amount API + an amount water, inject this => no degradation. Same solution further diluted => degradation peak.

We also took an aliquote from the plastic bin, put in in a volumetric flask made of glass, leave it for 5 minutes and inject this, without diluting => no degradation

We were thinking about glass interaction, but that doesn't stroke with the last experiment.

Any idea about what is going on?

Ace

Have you tested the pH of your water source?

We tested the pH, and also tested different water sources: MilliQ, destillated, and even bottle water.
All with no difference

Ace

I assume your sample, regardless of concentration is always dissolved in the same injection solvent composition?.

Does the peak area keep increasing with time or temperature in the dilute solution? If it stay constant, I'd also be looking for contamination
of the sample, water, column, or instrument.

I would dilute with pH buffers and see if it still appears consistently.

Please keep having fun,

Bruce Hamilton

I assume your sample, regardless of concentration is always dissolved in the same injection solvent composition?.

Does the peak area keep increasing with time or temperature in the dilute solution? If it stay constant, I'd also be looking for contamination
of the sample, water, column, or instrument.

I would dilute with pH buffers and see if it still appears consistently.

Please keep having fun,

Bruce Hamilton

The injection solvent composition was always water.
The peak area is constant after dilution, but not always the same.
We tried with diluted HCl, but still the same story: sometimes we've got a peak, sometimes not.

We even tried to rinse our volumetric glasses just before dilution with water, with no effect.

Thanks for you suggestions

Ace

Is your API in a solution before you add water? What is the pH after you add water? What is the pH when you see degradation?
Could it be that your degradation peak is just dirt you are adding inadvertently?

could please list every thing you used for 2nd dilution but not the 1st dilution, I mean everything.

I guess one of the things should be the "killer"

could please list every thing you used for 2nd dilution but not the 1st dilution, I mean everything.

I guess one of the things should be the "killer"

The dilutions where degradation is occuring is only with "pure" water:
demi water and MilliQ water (we tested both).


Aceto