Chromatography Forum

Hi folks,

We recently injected a solution of Nicarbazin (in DMF) with a mobile phase of acetonitrile and phosphate buffer (pH 2. onto C18 column and saw two peaks...one small followed by one large. PDA scan of both suggested both are same or similar compounds.

Looking at our validation data, I noticed all was fine. We ran the same sample on two other machines with the same outcome. Is the compound sticking to the sampling needle? Even changing the rinse solvent to pure DMF (from 90/10 Water/MeOH) did not help. Bad injector seals perhaps? Or could it be something to do with the column, at the inlet end?

Any ideas? Thanks in advance.

You may be seeing the "strong diluent" effect. When the sample is dissolved in something stronger than the initial mobile phase, the sample tends to get "smeared" into the column instead of equilibrating nicely. The symptom can be tailing, fronting, splitting, shoulders, etc, or even a secondary peak.

If possible, try to dilute your sample with the mobile phase and see if the problem goes away.

If it doesn't, what's the possibility that you actually have two compounds with similar chromophores (UV spectra in solution are notoriously information-poor)?

Hi,

I guess the DILUENT is creating problems for you. The stronger the diluent as compared to the mobile phase the more is the possibility of it not getting equilibrated. Try using the same or rather similar DILUENT (in composition) to the mobile phase. May be the problem will minimise

Chreers,

Yogesh

Thanks Tom and Yogesh, appreciate your excellent suggestions.

I also noticed the two peaks in the blank sample (which is just DMF). Sequential injection of consecutive blank samples saw the intensity of these two peaks gradually diminished. Very strange indeed....perhaps Tom is correct in that there really are 2 closely related compounds/chromophores for Nicarbazin.

Nicarbazin IS a 1:1 of complex of two compounds. You should get two peaks.

Thanks Uwe.....I just found that out. Nicarbazin is actually a 1:1 mixture of 4,4'-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethyl pyrimidine; DNC being the main active drug. Hence, Nicarbazin is usually assayed in relation to DNC.

Still, I need to figure out what's going on in my blank injections.