Abnormal Retention time shifht-Can you help?

[Roy]

Hi

The compound I am working with has 3 OH group attached to P and a phenolic OH group as well.

For the analysis I am using a SB C18 column, Mobile Phase A is Water w 0.1% TFA and Mobile phase B is ACN w 0.1% TFA with a gradient elution.

The problem is during the qualification run, the RT for the main compound gradually shifted from 25.1 min to 19. 1 min in a span of 8 hrs continuous run. I dont think instrument is a problem here. I also dont think equilibration is a problem as well.

Sample prep is in EtOH/Water.

Can you offer any explanation? and what can I do to prevent it?
Thanks a lot.

Lilly

[Kostas Petritis]

Generally speaking, it can be problematic when you are trying to re-equilibrate your column at 100% aqueous mobile phase. Maybe if you can start at 2% acetonitrile, it could be better...

[tom jupille]

I'll second Kostas's comment, but also ask what else is in the sample (i.e., what are the chances that you have irreversibly retained CRUD* building up on the column?

*CRUD = "Chromatographicall Retained Undesirable Debris"

[Roy]

First of all thanks for the replies.

My gradient starts from 30% organics, goes to 95% organics. So I dont think equilibration is a problem.

About the deposition in column; may be possible-- how can I avoid that?

Any other thoughts?

[tom jupille]

About the deposition in column; may be possible-- how can I avoid that?

Best way is not to let it get on the column in the first place (in other words, modify your sample cleanup). If that's not possible, you can use a guard cartridge to trap the crud (but you will have to change the cartridge periodically).

[MK]

how are your pressures? if it's dirt trapped in the column you should see some pressure increase. Is the temperature well controlled? are your mobiles premixed, if yes, can you loose some of the organic by evaporation? Is your compound stable or can it be changing to something else (can you look at the spectra of the shifted peak?)

[Roy]

I have been able to resolve the issue.

Thanks for all the help.

[rhaefe]

...and? How did you solve the problem?

curious in Reno...

Robert Haefele
Hamilton Company
rhaefele@hamiltoncompany.com

[Roy]

Just by increasing column equilibration time between runs.

Thanks all of you!

[Kostas Petritis]

It was an equilibration problem after all

[yaoguocan]

I think you would better disolve your sampl in mobile phase .it will decrease your equilibration time