Chromatography Forum

I have a HPLC-DAD method to determine 5 pharmaceuticals (sulfamethoxazole, carbamazepine, clofibric acid, ibuprofen and diclofenac) with a gradient (CAN and 25 mM phosphate buffer pH 3). Flow rate 1.2 mL/min. Column: Phenomenex Kinetex 100 cm. Last May After I changed the column I realize that peak areas were smaller than before (about 1/3 of the initial area for all peaks).
So I checked the voltage of the lamps. The voltage was a bit low, but not so much. In any case I changed the lamps for new ones but the problem was not solved.
I have also checked the autosampler and the pressure when injecting seems OK & I have checked the volume that is taking from the autosampler which also seems OK as well as reproducibility. I also checked the noise of the baseline before and after of the lamp exchange being both of them very similar.
Pumps seem to work OK, no leaking is observed.
I have run the method without with the precolumn+column, only the column and without column and pre-column. The areas are approximately the same and are still 1/3 to previously obtained ones.
I must have missed something. What are the most common situations for an area reduction? Any suggestions?
Thank you in advance.

Possibilities include lamp alignment or dirty flow cell windows. That said, so long as your signal/noise ratio is still adequate, day-to-day differences in peak area should be cancelled out by the calibration process and should not be an immediate problem.

Thank you for the answer. The signal/noise ratio has changed. The noise is the same but the signal is about 1/3 of the original. I thought about cleaning the cell of the detector but then I thought that since the voltage is adecuate and the noise has not increased over time...that probably the cell is clean, but I may be wrong.

I think your sample might be sticks to the walls of the vial.There are some samples,even we prepare the solutions after some time they re-back to very slight solid layer on walls of the vials.due to that there is decrese in concentration of solution in the vial even though you inject same amount as previous you can not get the same area.
Regards
KIRAN REDDY MUNNANGI