Chromatography Forum

We have taken a method from our supplier.The compound is synthetic antibiotic
Its Pka is 1.8.
m.P-A: WATER: NETHANOL (95:5)
M.P-B: METHANOL
COLUMN:Zorbax-XDB-C18-150X4.6mm,5mc
FLow ;1 mL/min
UV:254
Gradient
T/%B : 0/20, 10/30, 25/40,30/60,40/60,45/20
The method was successfully tranferred from supplier lab to ours.The peak rt is 15 min.When one inject sample ,no problem for the first injection.However as injections progress a peakat rrt 0.9 starts elluting.But no problems of stability either.The method runs good in originating lab.What could be the problem?

You state that a peak appears at 0.9 min ? The T zero for your method is around 1.7 minutes so you must be looking at an artifact (injector or pressure pulse). Does it change in abs, area, height, retention time for each injection ?

He said RRT of 0.9 (assuming it wasn't a typo) so around 13.5 minutes based on a 15 min RT for the peak of interest.

Yet another example of "more information needed" to help with the original question.

Hi, In fact, no problem with first imjection.From second injection onwards,a small gradient-elution type hump will form and this hump slowly takes tha shape of a peak after some injections.This will hump or peak elutes at RRT 0.9

The peak is likely a mobile phase contaminant. You can confirm this by extending your re-equilbration time by 2x and 3x. If the peak intensity increases with equilibration time then the contaminant is in one of your mobile phase reserviors.

Remove and clean the aqueous reservior with methanol and replace the solvent filter.

Let me know if this helps.