Chromatography Forum

Hello,
i need a volatile buffer pH 10.5 or slightly more, my compounds have pKa of 9.5, i would like to use a TEA 0.05%-acetate buffer pH10.5. My peaks are separating perfectly, peak shape is brilliant, resolution is brilliant, the only problem is a i'm getting a huge broad peak in the middle of the gradient in chromatograms.
Obviously TEA, i have tried adding it to mobile A and B at 0.05%, but i still see this massive peak. Is this a problem with TEA equilibrating? Has anybody any experience with TEA at this pH?

Temp 50oC, lambda 239nm, gradient 10-90%B over 20mins.

This method would be perfect otherwise!!

If you have already pumped a fair amount of TEA through, it may be time to try another brand (whose TEA are you using - for that matter, what's your acetate source?).

TEA is HPLC grade (Fisher)
Glacial acetic acid is analytical reagent grade (fisher)
I run a number of injections last night but this broad peak is still present, i thought after a number of injections running through the gradient it may equilibrate but to no avail!

Are you getting the same broad peak when you run a "dummy" gradient (no injection)?

If so, and if you have a PDA available, check to see if the peak is better or worse at a shorter wavelength (215 ?). If it's better at shorter wavelength, it may be a refractive index problem, which in turn might indicate a flow cell alignment problem in your detector. If it's worse at shorter wavelength, then it's time to try the old "three dummy gradients with varying equilibration time" test to see if the broad peak is coming from contaminated "A" mobile phase. If it is, then you are facing detective work to identify the source.

If the broad peak is not present in the dummy gradient, then it is coming from the sample or the injection process, so you need to inject mobile phase, your diluent, a matrix blank, etc. to track the source.

Finally, if the peak is broad enough compared to your analyte peaks, you might want to simply ignore it and let the data system deal with it.

Hi popnumber,
Any chance of posting a chromatogram so we can view the peak.

I've had trouble with using many high pH additives including TEA that give very poor baselines, especially with gradients! In fact the background noise level was so high that it totally obsured any chance of seeing are impurities at the 0.1% level. Nevertheless as you have observed, peak shape and retention is usually enhanced for basic compounds.

The new Waters X-bridge columns are purported to be stable up to pH 13. If this is the case then perhaps you can use phosphate buffer at this pH (it may give you better baselines in your gradient - (can anyone support this?) and still give good peak shape and retention...

We use Water XTerra column with ammonium bicarbonate buffer using ammnium hydroxide to adjust pH to 11.30. We will be able to get very smooth baseline for gradient elution. We use Waters Alliance 2695 HPLC system to run the gradient. We are runnig the application all the times.

Some of our customers have had success using Tetramethylammonium Hydroxide at high pH for LC/MS.