Chromatography Forum

Hi,
I am running 20 mM potassium phosphate pH 5.6 (I didn't develop method) with ACN gradient. Method uses 225 nm wavelength. Problem - many many ghost peaks in the baseline. I switched to Fluka HPLC grade phosphate/phosphoric acid - didn't help. I tried Empore C18 extraction disks to filter, but did not correct the problem. I saw a post that someone's 0.45 um membrane caused degradation and ghost peaks with pH 2.5 phosphate but not at pH 4.0, and switching filter brands helped. I could try switching brands, but since my pH is higher I don't think that's the cause. Any suggestions would be greatly appreciated!

Susan

If you haven't already done it, do the "three blank gradients" test to confirm that your garbage peaks are actually coming from the eluant as described here:
http://www.lcresources.com/resources/TSWiz/hs400.htm

If the garbage peak size does *not* track the equilibration time, then you need to look a things like needle wash, etc.

If the garbage peak size *does* track the equilibration time, then it *is* garbage in the mobile phase. So the questions are "where is it coming from?" and "how to get rid of it?". You have already looked at one of the "usual suspects". The remaining ones:
- microbial growth in your buffer (do you see a difference with a fresh batch?)
- the filters (just because you're at a different pH doesn't mean that you might not be leaching something from your existing filters).
- the water (when was the last time the cartridges were changed?)
- residual detergent on the glassware (don't laugh; I've seen it happen)
- the pH electrode (*never* put a pH electrode in the bulk mobile phase; pull an aliquot, measure it's pH, then throw away the aliquot).

All of that said, I would expect the Empore disks to take out most of the hydrophobic CRUD, which is why something like needle wash solution is a suspect.