Chromatography Forum

Hi All,

During the validation of a new method, based on USP, I have been observing a reccurent, (but intermitent) problem. After the system suitability passes (precision NMT 2.0% Tailing NMT 2.0, Plates NLT 1,800, Accuracy (98-102%), the second or third standard braket drops about 3% of its area (limit 2%), and then after it remains during the rest of the run with the same dropped value . It had happen with two different Agilent HPLC systems with different detectors (VWD and DAD), as well different analysts, and with different standard concentration (in the linear range)

The method HPLC parameters are:
Column: 125 x 4.0 mm x 5µm (Discvery C18)
Mobile Phase: Methanol:Water:Acetic acid (80:20:0.5)
Flow rate: 2.0 mL/min
Temperature: 40C
Detection UV @ 353nm
injection 20µL
RT ~2 min
samples and standard are diluted with pH 8.0 boric acid buffer

Thanks in advance for any help to solve this apparent mistery

If it was me: first thing I'd do would be to repeat the sequence with the same vials, using 10ul injection size. Just because.

You need to tell us if your peak shape/integration/tailing/retention times change over the sequence. Also tell us if you're filtering your samples before injection.

Less than 2 minute RT is fast for non-UPLC procedures, and 2ml/min fast also. But you apppaently have selectivity with your wavelength, so no real issue there.

Thanks for your reply, Its happen with different equipment’s, system suitability passes ok, we filter the samples before injection, and no problem with the peak shape... I will perform a study injecting standards solutions for 24 hours to see if the error reproduces. I am suspecting of mobile phase it’s not de-aired properly... (10 minutes in ultrasonic) we will do this time through vacuum to discard this variable. Thoughts?