Chromatography Forum

Hello to all,
I am developing an HPLC method and not in each run an unexpected peak appearing in the void volume (around 3mins). Im using a gradient method with phosphate buffer pH 3.2 in A and a mix of MetOH and ACN in B in a Hypersil CN 250x4.6 column. My diluent is H2O and when I filled 5 different vials with water then in two of them appeared this peak whilst the rest three were clean of this peak. The area of this peak varies between positive vials and when i rerun a positive vial the area of this peak remains the same. I have different HPLC systems and different columns and the problem persists. So dont seem to be a carry over or a column or a system problem. Can you help with any ideas? Is there any possibility the HPLC vials or their septon closure system to be the reason of this problem?
Thnx in advance

Hello

Please paste chromatograms and overlaid pressure profile (good and bad) and give more details about method (pump parameters, gradient, detector).

Regards

Tomasz Kubowicz

I agree with Tomasz, we need more details. You inject water and you have buffer and MeOH/ACN in a gradient. It can be that regeneration time after the gradient is too short, when you have a ternary pump system one of the valves may work not properly. So, more details needed. Thank you.

I’ve seen it numerous times – contaminated HPLC vials.
Do a quick test: Inject water several times for the same vial testing some 3 – 4 different vials.
Could be like this vial 1 – 3 injections, vial 2 – 3 injection etc.
Then shake the very same vials and inject from them again.
Best Regards

I’ve seen it numerous times – contaminated HPLC vials.
Do a quick test: Inject water several times for the same vial testing some 3 – 4 different vials.

Another idea:
Inject from five empty vials and add water to each vial afterwards and inject it again.