Chromatography Forum

I was wondering if anyone has had a similar problem as mine;
The samples (not diluted with MF) and the resolution solution (diluted with MF) have 2 peaks (one from API and the other from impurity). The API peak has RT=15min and the Imp peak has RT=12min. But, when I run the individual evaluation standards 0,2% API and 1% Imp (diluted with MF) their RT's are 4 minutes late?!
MF or any other of whatsoever change was not been made while analysis was running. Same MF was used for the system and for diluting the solution. The method is isocratic,, flow 1ml/min! Column T is ambient, and the samples are not being cooled. Inj.volume 20mcl. Column Superspher Select B 250x4.
The analysis was also conducted on a different system and the results were the same: RT of peaks in the individual evaluation standards are 4 minutes late than those in the samples...
Any ideas?

Tnx

What are the compositions of the mobile phase and sample diluent? Is there a pH effect going on, ion pairing, something like that? Have you tried dissolving your samples in MP?

The composition is:
MF A: Octan-1-sulphonic acid Sodium salt and glacial acetic acid in water (70%)
MF B: ACN (30%)

By the way this is a method that it is being used for a couple of years now (it is pharmacopoeal method), and till now there were no problems of whatsoever...

But I have noticed that the ion-pairing agent that we used for this analysis it's a brand new container and a different lot.no than the ones used before...

But I have noticed that the ion-pairing agent that we used for this analysis it's a brand new container and a different lot.no than the ones used before...

That reagent, and preparing both in matching solvents, would be where I'd start my investigation.

Yep, also classic problem solving techniques - remake the MPs and samples

I suggest that you mix two solutions (spike) to make sure that you compare apples to apples.