Chromatography Forum

Hi there.

I have a question and would like some advice.

I am experiencing retention time shifts for my compound peaks on my Agilent 1200 HPLC coupled to a MS system and I would like to ask if there is anything that I need to check:

i) Already re-prepare fresh batch of HPLC gradient - no avail
ii) Check the pump flow rate - suspecting there might be problem with the pump - however, flow rate of pump is ok
iii) Check on HPLC column, change to other column - also didnt solve the time shifts
iv) Running back the same samples that have been ran previously confirms the shifts

v) Will try to check and change the guard column cartridge , hopefully, this will help.

However, is there any other advice that can be given to me? I thought of checking the mixer; not sure this will works or not.

Please advise me and thanks a lot !!!

If your compound's pka is around the pH of the mobile phase you might experience retention time shifts...

Hi Kostas, thanks for the reply.

I dont think the sample has a problem because we ran the same sample again and we experienced a RT shifts.

Anyway, the latest runs seems to be fine now; not sure exactly where iy goes wrong in the runs previously. Thank God, I dont have to troubleshooting further.

Cheers,
Ken

Ken,
Could be the beginings of a pump seal going down (given that all else is correct.). Retention time shifts are usually due to flow rate or concentration changes during subsequent runs.

When you did your flow rate check did you have a column or some other back pressure device on your system or did you just disconnect the column and run without back pressure? If you didn't have back pressure on the pumps the flow may be correct but resume its flaky behavior when you reconnect to the column. That you got good results in subsequent runs doesn't mean you fixed the problem yet and troubleshooting is a constant.


should check the seals, check valves, plungers, frit filters that are inline to the system. The column could be the culprit as well. How many injections on the column? Calculate theoretical plates? Did you run standards to establish a baseline for this column when it was new?

Please also remember that Agilent pumps use a pulse dampener with a pressure dependent volume. This means that if you have pressure building up somewhere in your system (guard, inline filter, column, ...), the gradient delay volume changes and your peaks are shifting. Pumps that provide stable flow without pulse dampener are a more reliable solution.

Sounds more like an HPLC flow issue (pump issue, leak in pump, autosampler, connection), I'd start there. Use a graduated cylinder to make sure pump flow rate is at target; we just had a system which held pressure, delivered low flow rates, couldn't see any leaks, then found it was leaking purge valve, but couldn't see that at first because the waste was routed through tubing to a container. If you suspect a mixing valve, make up solvent in one reservoir, put all solvent leads into there, purge all, then see if problem gets better or stays the same. Start with the basics.

1) still could the pH(Pka ) issue;
2) gradient /mechanic?
3) temperature?
4) preparation? pre-mix or online mix?
5) column?
6) method robust?