Chromatography Forum

hi,
I got my first UHPLC a few months back and I am unable to get a clean blank run (no injection). I always get 5 peaks with 5 mUA or below. The instrument manufacture insists they are solvent peaks, anyway we changed everything that can be changed (including the pump module) without success. I tried different UHPLC-MS solvent brands, including the manufacture 'best in the market' recommended, without success.

I really can't see how a brand new instrument from a reputable manufacture can have ghosts peaks. And, if it is the solvents, I can't believe I am the only one having this problem since, according to the manufacture, I am the only one complaining.

Does anyone else have this problem? How do you face it? Do we have to start working like in GC, where you just acknowledge and ignore the solvent peaks?

I'd really appreciate your thoughts.
Thanks,
Sergio

Hi Sergio,

This does not sound like a solvent peak issue, rather a contamination issue or an issue with your column. If you can, could you provide us more info on your matrix and analytes?

One test you could conduct in the mean-time is equilibrating your column at a low org % and performing washes after different EQ times. If this result ends up seeing, for example, a larger build-up of material after a 30 min EQ compared to a 20 min EQ, then you likely have some contaminant building up.

Personally, I would not ignore these peaks because they suggest something is wrong, but without more information it's hard to say. For example, you could be measuring a 5 mAU difference that occurs due to your mobile-phase absorbing slightly at low wavelengths (not sure what detection method you're using, I'm just assuming PDA because of mAU).
A picture, and more detailed description, will certainly help us help you as well as get your some more responses!
Good luck,
TS

Assuming you're running a gradient method with reverse phase chromatography, you can test whether solvent contamination (aqueous, additives, and organic) is responsible for neatly-shaped proper peaks in a blank chromatogram. What can happen is that contaminants that aren't eluted by the starting conditions of the gradient accumulate on the column while you're equilibrating it (either before the first run, or between runs). When the gradient gets high enough for them to elute, they elute.

If you think this is going on, try a method where you equilibrate the column for three times longer than normal. In an ideal world, if you're accumulating contaminants, the contaminant peak will now be three times higher than normal!

You may have the data already. If the contaminant peaks are the same size in each run of a batch, except the first run, where they're much bigger, it probably means that you are in the habit of pre-equilibrating your column while you're setting up the instrument, and equilibrating it a bit longer than it's getting between normal runs!

But another common problem with a brand new system is that some item of the tubing is still leaching stuff/contaminated with stuff. I had a huge argument with a manufacturer once about the brand of LC-MS-grade isopropanol I'd supplied for the installation of an instrument. The engineer found contaminant peaks and complained mine was rubbish compared to theirs. He got some of theirs, and it still made peaks, and everything went rather quiet and sheepish. A year later, when the instrument came up for its first PM visit, a different engineer told me they'd worked out that the tubing they were using leaches contaminants into isopropanol for the first few weeks!

Of course if you're using an ancient column of uncertain history, it may be contributing all sorts of humps and bumps in the chromatogram itself.

a little more more info:
- method is a gradient 5-95 ACN in H2O in 5 min and hold for 10 min, and 3 min post-run @ 0.8 ml/min on an C18 column 1.9u 2.1x100mm
- there is no injection, so no sample and no matrix effects
- columns are new (different columns, different brands)

increasing the equilibration time does increase the peak area, but the contamination can come from the solvent or from the instrument (by leaching, etc)

i think i wasn't specific in my question. What i would like to know is if anybody sees solvent impurities when using UHPLC-MS solvents (specifically ACN and H2O) when running fast reverse phase gradients in a UHPLC instrument at high acquisition rate (80 Hz)

thanks for your help,
sergio

ACN is known to leach things out of certain brands of glassware. Waters specifically recommends against using one brand (whose name I can't remember) with their Acquity systems... Try different reservoirs - corning, kimble, other brands. It could be the glassware.

I think new instruments can be a bit dirty as it is hard to keep entire production free from contaminants. Our installation engineers mentioned that new instruments are dirty and did very elaborate cleaning procedures of the new uhplc to get rid of most background, like acid and base washing. Hopefully it gets cleaner the more you use it. I compared different brands of ACN ms grade in ms full scan and they had very differen background. Not at all as clean as the methanol.

i tried Fisher, Aldrich, and Baker solvents. If you can suggest another good brand, i will try it too.

i washed the instrument with an organic mixture recommended by the manufacture (50% IPA, 25% ACN, 15% Hexane, 10% DCM).

I haven't tried any acid and basic mixtures, any recommendation?

thanks,
sergio

if yours is an inert system, do not wash it with anything apart from what the manufacturers recommend. Otherwise it will not be an inert system anymore.

Yes, that lovely mix suggested by the manufacturers will extract all sorts of lovely things from plastic tubing. I suppose if you do it long enough, there won't be anything else to extract...

Contaminants are really frustrating because what happens in practice is that you fiddle around trying every possible remedy, and eventually the problem disappears. But there is often little genuine certainty that the remedy solved the problem, rather than just a lot of time and usage. So finally, we believe that the last remedy we applied is what solved it - and message boards are full of people posting the equivalent of "I battled with this peak for months but finally it disappeared when I ran acetonitrile at exactly 0.4mL/min and 40 degrees C on a Tuesday while wearing black shoes and after coiling the tubing anticlockwise" and so they, and we, firmly believe that these conditions are the solution! I hope you manage to fix it soon.