Squalene detection in liquid chromatography with UV-Vis

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Good morning,

I have a problem with the detection of squalene with Liquid Chromatography. After a couple of injections (5th or 6th injection) new peaks appear in the chromatograph near the elution time of squalene, as a result the area of squalene is being affected. What can I do ?

The mobile phase is MeOH:H2O (98-2%) and the system follows an isocratic elution.

Thank you in advance !
If you run a blank afterwards do the peaks appear or are they only there when squalene is present?
After 5-6 injectios the peaks appear in blank too.
It seems your sample contains very late eluting compounds. One more experiment to make this sure: Extend the run time of your method by at least factor 6 (better: factor 10) and inject your sample. If it was 5 min long in the first place, you should then see the peaks between 25-35 min, depending how long the autosampler cycle takes.
Agreed - could be late eluters. If this proves to be the case and you require a shorter run time, you will have to employ a "clean out gradient" which in your case may mean adding a little THF or other eluent if you're actually running NP (and you probably should be - MeOH and water are both fairly polar but squalenes are so heavy and not so polar... this may be a mismatch of analyte and column/MP).

Tell us about your LC column. That should help us to help you.
Thanks,
DR
Image
Good morning and thank ypu for your help!

The mobile phase is MeOH:H2O (98-2%) with an isocratic elution and the column is C18 reversed phase..
98% MeOH - you're about at the end of the elutropic series for RP. That's not a great place to be. Squalenes are pretty non polar, aren't they?
Perhaps this would be better as a NP separation?
Thanks,
DR
Image
How wide are the new peaks (compared to squalene)? If they are late eluters (perhaps from the prior injection?) they should be much wider. If those widths are comparable to the squalene, I would expect them to be contaminants/degradants.

And I agree with DR that normal phase might be a better choice.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
The new peaks are wider than this of squalene..

We tried the same protocol in another HPLC with reversed phase and everything was ok, but in this instrument after 5 or 6 injections other peaks are eluted near squalene..

We follow the method described in many papers using reversed phase and isocratic elution..
If the problem occurs on more than one instrument, it's unlikely to be an instrument problem.

If this were my problem, I would start by verifying that it really is due to late-eluters (as opposed to sample degradation). Do a single injection and wait; continue acquiring data for 10 times the usual run time.

If there *are* late eluters, then changing either the method (e.g., to normal-phase) or the sample prep (perhaps with solid-phase extraction?) is the "best" approach. The "band-aid" approach would be to run 5-6 samples (however many you can get in before the late peaks elute), wait for all the late peaks to clear the column, and repeat. This would obviously kill the throughput, but if it's a one-off or infrequent analysis, it may be more effective than validating a whole new method.

If there are no late eluters, then it looks like it's a sample degradation issue (confirm by prepping a fresh sample). In that case, I would address the chemistry of the degradation.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
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