Chromatography Forum

Hi everyone,

In the scope of one of my previous posts, for the earliest eluted impurity (RRT ~ 0.45) we are observing peak splitting as the organic content at first line of the gradient increases (e.g. from A90:B10 to A60:B40).

Although the diluent is a very complex mixture, wouldn't this trend be occurring in the reversed order?

I kindly ask any thoughts/ideas as support.
Thanks in advance.

Mobile Phase A: 10mM K2HPO4 Buffer, pH 7
Mobile Phase B: MeOH : ACN (50:50)
Injection volume: 5 µL
Diluent: [DCM/EtOH 80:20] (10%) / [0.1% FA in MeOH/H2O 70:30] (90%)
Nominal concentration: 0.1 mg/mL API

Peak deformation because of solvent/mobile phase mismatch is a very complex function of a whole lot of parameters - initial mobile phase composition being only one of them. Another parameter is retention time of the peak - the earlier a peak in the chromatogram, the more it is prone to peak deformation. If you increase the organic content in the initial mobile phase, you decrease the mismatch between solvent and initial mobile phase - but you also shift the peaks to earlier retention times, meaning they get more susceptible to peak deformation. In your case, obviously, this outweighs the increase of organic content in the mobile phase, so your problem gets worse.

Peak deformation because of solvent/mobile phase mismatch is a very complex function of a whole lot of parameters - initial mobile phase composition being only one of them. Another parameter is retention time of the peak - the earlier a peak in the chromatogram, the more it is prone to peak deformation. If you increase the organic content in the initial mobile phase, you decrease the mismatch between solvent and initial mobile phase - but you also shift the peaks to earlier retention times, meaning they get more susceptible to peak deformation. In your case, obviously, this outweighs the increase of organic content in the mobile phase, so your problem gets worse.

Thanks HPLCaddict for your thoughts.

Changing diluent strenght is challenging due to the complex API/polymer solubility. In that sense, is there any any change to do in mobile phase strenght?
I was wondering if initiating with 90:10 to avoid splitting and very fast down to 45:55 (isocratic step to separate the critical pair: impurity RRT 0.99/main peak) could eventually bring any improvement in sensitivity.
Based on what we has been observing, initiate at 45:55 could split the peak in two but in a way that allows satisfactory integration at baseline?!
Just some "wandering" thoughts.

Any ideas would be very helpful.

Thanks in advance.