Chromatography Forum

Dear colleagues
I have trouble with an isocratic method (C18 column, 10 % ACN, 90 % Tetrabutylammonium hydrogen sulfate/phosphate), and in some (by far not all) injections I see massive interferences:



"normal" chromatogram:


I have cleaned all solvent lines with water, nitric acid, water, isopropanol, Acetone/acetonitrile and back to water. I have cleaned the syringe, changed the pre-column and flushed the UV flow cell.

Any other idea?

Thanks a lot
Jörg

Fully Degas mobile phase (use continuous degassing) and prime pump(s) with appropriate mobile phase. Flush column to remove any retained material (use stronger solvent), then fully equilibrate and monitor.

Fully Degas mobile phase (use continuous degassing) and prime pump(s) with appropriate mobile phase.

Done, extensively.

Flush column to remove any retained material (use stronger solvent), then fully equilibrate and monitor.

This is what I do on a regular basis. The method is not really good, retention time of the analyte peak is creeping towards dead time. Flushing with high acetonitrile for about an hour recovers retention time, but the mess I am worried about appears anyway .

If me, I might try the isocratic separation, then build a gradient after the peak of interest elutes with higher ACN for a few minutes, then build in plenty of re-equilibration time. Understand that your throughput will be less due to more time between injections, but your net result could be better.

The thing is, those "ghosts" don't look like chromatographic peaks, more like something building up in the flow cell and then suddenly breaking loose. That can happen with small air bubbles accumulating at the inlet end of the flow cell optical path.

You have already checked the degassing; if this were my problem I would check for any loose fittings or leaks on the *inlet* side of the pump; a leak there can aspirate some air and cause the kind of problem you're seeing.

Tom, your suggestion to tighten the inlet side of the pump seems to have helped. The fittings were not really loose, but it was possible to tighten them a little more without a wrench.
Strange that I did not notice any disturbance in the system pressure before.
Thanks anyway
Jörg

Tom, your suggestion to tighten the inlet side of the pump seems to have helped. The fittings were not really loose, but it was possible to tighten them a little more without a wrench.

That's loose !!