Chromatography Forum

Hi,

We have a 2695 separation module and a 2998 PDA detector, the column we use is C8. We don't use acetone as mobile phase anymore, except for one compound. The everyday mobile phase that we use is: acetonitrile and H2O.

Two days ago I had to analyse that compound for a whole day using acetone as mobile phase and I washed the column with MeOH (1ml/min, 20 mins) after the analysis. The other day I was running the normal routine analysis using acetonitrile/H2O, but I found that all chromatograms show minor acetone peaks. The worse thing is that one of the metal complexes starts to show dissociation from the chromatogram, for example, I repeated one of the old samples and the ratio of metal complex : ligand is different from before...

Is the acetone still stuck in the column? Those chemists are using D-acetone as NMR solvent when preparing NMR samples for those metal complexes, so I don't understand why acetone can cause dissociation to the metal complexes..

Any suggestions will be much appreciated! Thank you, and sorry for the rush to post this!

It is rather impossible to see previous mobile phase peaks in subsequent runs. How do you confirm the identity of acetone peaks. Any mobile phase trapped in the pores of silica should leach slowly leading to a drifting baseline rather than peaks. Peaks imply that something containing acetone was injected.

I agree with M Farooq. You don't get leakage problems in your system?.

Hi IUpuppy

I´ll try evaporating the acetone with nitrogen an dissolving the residue in a more suitable solvent. Acetone absorbs UV light and has many impurities even the p.a. quality.


Good luick.

Hello All, thanks so much for the responses.

I just wonder, could air bubble traped in the syringe be the cause of the peak height variation? Maybe acetone is not the reason of the peak variation...

Hi IUpuppy,

Yes, an air bubble in the syringe of the 2695 could affect both peak height as well as peak area upon performing injections. You could try carefully removing the syringe, filling it with degassed 50:50 MeOH/water, replacing it into the injector body and re-prime the needle using the keypad. It's been a bit since I've done this myself, you'd have to be careful to not displace the Teflon part at the top of the syringe (away from the plunger)...in essence you'd have to start a needle priming with the keypad, wait until the syringe plunger is pulled down as far as it goes in that routine, pause the prime, unscrew the knurled nut, then the syringe, take the syringe out (a little messy, have a Kimwipe handy), fill the syringe with the MeOH/water mix, replace the syringe into the injector in the reverse order of steps (again, a little messy), finish the prime...and finally repeat the prime and check for bubbles.

Hello All, thanks so much for the responses.

I just wonder, could air bubble traped in the syringe be the cause of the peak height variation? Maybe acetone is not the reason of the peak variation...

Yes, as MattM said, air bubble in the syringe will cause variations in peak height and peak area.

I recommend MattM's procedure, from my experience it also works with 100% MeOH.