Chromatography Forum

I work with peptides, and I've found that analyzing at pHs where the net charge (positives minus negatives) or absolute charge (positives plus negatives) is nearest to zero results in the best separation. Triethylamine and ammonia both work great as basic modifiers up until about a few units below their pKas, and then the absorbance goes through the roof in the range I work (mainly 210-220).

I tried DIEA (diisopropylethylamine) since I had some on hand, and it was the same way. Once you get near the pKa, the absorbance skyrockets.

I've concluded that it's probably the uncharged ammonia/amines absorbing strongly at that range. Modifying 20mM ammonium bicarbonate to around pH 11.8 (the optimal pH for a particular peptide I'm working with) works, but the residual ammonia results in a very high baseline absorbance which kills the sensitivity.

I've managed to do relatively good separations with sodium phosphate adjusted to high pH, but the peak shape and resolution is not quite as good as it would be if I could use a better ion pairing agent. I've found some 1M solutions of tetraethylammonium hydroxide for sale, but I'm a bit weary of purchasing some without having any ideas about how well it will work. I can't find any absorbance information on the stuff, although I have seen that the far more expensive tetrabutylammonium solutions of the same grade have a reasonably low absorbance in the range I work.

Does anyone have any advice about other basic modifiers to try? Will tetraethylammonium, blended with citric or phosphoric acid be a good choice for a high pH analysis? What are the pros/cons of using shorter/longer chains on the ammonium (i.e. methyl versus ethyl versus propyl versus butyl, etc.)?

Thanks for your help!

It's actually the hydroxide counterion to the free base that absorbs at low UV wavelengths. So, if you can minimize the pH you will have the lowest background.

Chris

While searching for a separation method for peptides using these ion-pairing agents it seems to me that you on the wrong train, heading for trouble...

Why don't you use conventional RP and HILIC as your peptide LC techniques?

It's actually the hydroxide counterion to the free base that absorbs at low UV wavelengths. So, if you can minimize the pH you will have the lowest background.

Chris

I disagree with that, actually. I have no doubts that hydroxide does absorb, however, the absorbance of TEA or TEAP at a 20mM concentration adjusted to a pH of over 9 or so results in an extremely high absorbance at low UV wavelengths. My LC was maxing out at it's 2AU limit, and when I speced the solvents in a spectrophotometer, I saw an absorbance of around 3AU.

On the other hand, taking sodium phosphate or HCl at 20mM and adjusting to a pH of 11.5 with NaOH showed little (sodium phosphate) or no (NaCl) background absorbance at such a high pH. Adjusting ammonium bicarbonate with NaOH to a high pH showed very high background absorbance, but I could still produce a reasonably good chromatogram at 230nm (the baseline drift and sensitivity weren't sufficient for my needs, but the separation did work).

While searching for a separation method for peptides using these ion-pairing agents it seems to me that you on the wrong train, heading for trouble...

Why don't you use conventional RP and HILIC as your peptide LC techniques?

Zwitterion separation on RP favors some sort of modifer in the mobile phase to suppress or pair with ions. The traditional approach seems to be to use low pH modifiers at high concentrations, but with modern columns having a much wider pH stability and better sillanol capping, I don't see why exploring higher pH modifiers is a bad idea. Especially for peptides that contain a large number of basic residues, you'd need a very high concentration of modifiers in order to get good separation at a low pH (a 15mer or so with 6 lysines required a TEAP concentration of 200mM to acheive good separation!). That's hard on the system.

peptidemetdev,

If your amine is pure it does not absorb at low wavelengths. If it does it is due to impurities. All amines form a yellow-orange oxidation products upon exposure to air which absorb very strongly in the UV. The strong absorbance your report are most likely due to such to these oxidation products.

Chris