Chromatography Forum

Folks,

What could be happening with a HPLC chromatograph whose same LC method (column, oven temperature, flow rate, mobile phase, gradient, tubing line) started to show:

1) smaller system pressure (reduction about 30% in kgf/cm2), this "new" pressure has been constant;
2) enlarging of all retention times, they have been repetitive, too;
3) broadening of all peaks including the first ones?


Thinking on pressure decreasing:
- all bobbles were removed from whole system
- degasser has working properly
- pump is producing correct flow rate (it was checked with cylinder and stopwatch)

Considering peak broadening with later assimetry (peaks with "belly")
- column was changed by a new similar one however the behavior was similar;
- all tubing line was cleaned with 30mL of Nitric Acid at 20% followed by hundreds of "liters" of water up to final pH 6-7 (without the column);

We are sure that smaller pressure dalayed all peaks.


Your suggestion(s) will be much welcomed.

Nice elutions!!

Carlos Teixeira

What type of pump? Running conditions? Mobile phase constituents? Flow rate?

Did you measure the flow delivery at the same pressure as the "normal" analysis pressure? If you have a leak, the pump may deliver well with little pressure, but may not deliver appropriately at all under a few thousand psi.

If all of the retention times (including t0) have increased by the same percentage, then you have a flow problem!

Smaller pressure will mean your flow is reduced meaning your peaks will take longer to come off the column and they won't come off as sharply which is exactly what you're seeing, all indicative as above poster said of a flow problem.

It could well still be air in the system as even if you've purged it something could be re-introducing air, e.g. a switching valve issue if you've more than one mobile phase etc. Agree with above as well on possible leak and the higher pressure may have a flow issue but your lower one with no column etc may not too, I'd systematically check all your tubing and see if you've got any leaks using some old fashioned e.g. skoosh some 10% decon on any suspicious bits to check for bubbles lol!

Hope you get it sorted!

lynz x

Hi Folks,

Thank you for your prompty comments.

Mobile phase is only Water:MEOH, with gradient that goes from 100% till 95% of MeOH at constant flow rate at 0.8mL/min.

We observed that peaks with similar increment on the tailing factor (more "belly peaks").

Any case, we will check for some internal leakings, and air presence.

Whether you have more recommendations, please, let me know.

All the best,

I should have been more explicit: the relationship between percent change in retention time and flow applies to isocratic systems only.

Dear Tom,

Since the method uses gradient mode the flow may be not the problem, in this case we should (re)evaluate if there are some bobbles inside the system.


Thank you and Folks for help us.


Nice elutions for all!

Carlos Teixeira

Flow rate problems (bubbles) are *always* a prime suspect when retention varies . The symptoms are less clear-cut in gradients, but the problems are there nonetheless.

Carlos,
You can try switching to the other HPLC (Similar brand) and perform your testing and observed whether the similar pattern of chromatogram observed (Band Broadening, tailing). I have came across with this problem where the retention time of my peak had shifted and low pressure and is due to leaking of the valves. The other might due to the column (void volumn). Hope this info can help u.