Chromatography Forum

Hi colleagues,

I have to run some analysis of a peptide (highly positively charge) that is not really stable under acidic conditions. Therefore I don't want to use water/ACN with TFA.

I thought in using ammonium acetate buffer, at pH 6.7. As far as I know one should avoid salts for the MS, since those could damage the instrument. So I think that ammonium acetate buffer is a good choice.

But then I am afraid about the resolution of the UV chromatogram. Do u think that I will get nice peaks (sharp peaks) using such conditons?

What about basic buffers for LC-UV-MS detection of highly positively charge peptides?

Thanks to all in advance,

magic_acetone

You are able to use 0,1% v/v TFA to your mobile phase and still inject to MS. But keep in mind that your signal will be severely reduced by the TFA. Alternate you can use 0,1% v/v Acetic Acid for some peptides. Acetic acid is nicer for your MS. I found acetic acid very useful to one of my developed methods of analyzing peptides on HPLC-MS/MS with ESI.

If you will use TFA: remember to clean your ion source now and then.

Hope it

can't really comment on chromatography, but volatile basic/neutral buffers, strangely, will not stop you from seeing positive-mode signals for basic compounds in electrospray. ESI is an electrochemical process, and it sees ions even at a choice of pH where a compound is essentially unionised. I'd keep the buffer concentration as low as I could get away with, but I'd definitely try it out. Non-volatile buffers simply block up the instrument! Chromatographic peak shape may be poor if any analyte group pKa is in the region of the selected pH.

Not sure what the chromatography for your compound is going to look like but can say I have used 0.1% TFA in an aqueous mobile phase with MS, (would probably try HFBA 1st but don't like that either). I Have also used ammonium acetate as well as ammonium formate buffers with LC-MS but do my best to avoid them too. Practically always have 0.1% formic in my aqueous mobile phase. I can say there have been methods where my compound may elute at 8 min and I send the first 4-5min to waste rather than through my MS which reduces greatly the amount of buffer the MS sees, especially when running a gradient that's highly aqueous to start and where all the buffer is. You can likewise send the flow back to waste at 9-10 minutes while the instrument is re-equilibrating with the aqueous buffer. If the only data you are collecting is from 5-9 minutes and that's where the instrument is pumping 80-90% ACN or MEOH you reduce the buffers entering your instrument greatly. Do you have something such as a Valco valve?