Trimethyl Ammonium Acetate as a Modifier

[Andy Alpert]

Strong acids like nitric and hydrochloric dissociate completely in water. Weak acids do not. Acetic acid is about 1% dissociated in water.

[Jimi]

We're almost there. If you don't mind, I would like to belabor this a bit, as I really want to get it clear in my head.

So acetic acid has a pka of 4.75. Suppose that we throw some ammonium acetate in water. The resulting pH of this solution will usually be about neutral. Therefore, if I am correct, the acetate ions will mostly stay as ions. Then if we add more acetic acid - or any acid I guess - and bring the pH to maybe 3 or 3.25 the acetate should mostly protonate (convert to the acetic acid), and by this mechanism the salt will dissolve more because we have made the anion of the salt neutral.

Is there anything I've said here that is off. Please let me know at your convenience.

Thanks again.

[adam]

Jimi

I think the key, that you may be missing, is that it comes down to equilibrium. It is LeChatlier's principle. If we add acid we shift the equilibrium so more of the ammonium dissolves.

But then I have a question myself: Which acid to add. It needs to be something strong, but phosphoric, sulfuric, and hydrochloric are all corrosive to the mass spec (and phosphoric is also not volatile enough). Also it has to be something that won't be problematic to the HILIC separation (causing undesirable ion pairing and such, as was described in a previous post). Can anyone tell us what acids generally work well in this capacity?

[Andy Alpert]

This won't be a concern if you follow my preferred practice of starting with a known amount of the acid in water and then adding the base (here, presumably ammonium hydroxide or triethylamine) until you reach the pH you want. This is definitely the best way to proceed if you want to get a pH that's below the pKa of the acid in question. In one paper I peer-reviewed, someone wanted to get an ammonium acetate solution that was at pH 3.7, so they dissolved the desired molar amount of ammonium acetate in water and then added acetic acid until they got to pH 3.7. I pointed out that to get to pH 4.7, which was the pKa, they would have to add one mole of the acid per mole of the starting salt. To get to pH 3.7, they would have to add 10x the molar quantity of the acid! That threw off the assumptions in their manuscript so much that I instructed them to redo the work by adding base to a solution of the acid to get to pH 3.7, redo the experiment, and then resubmit their manuscript.
Now, if you are starting with the salt and have a solution at pH 7, and want to get to pH 6 or so, then the easiest way would be just to add a small amount of the acid (here, acetic acid). That's fine.

[adam]

Thank you Andy! Can I follow up again, just on one aspect.

I am wondering which acids are recommended to use with HILIC. I think some of the strong acids are problematic if using mass spec. But also, I'm thinking about your previous point regarding the potential of undesirable ion pairing, that could reduce the HILIC retention time. Given this, I think which acid can be important because the counterion of the acid may have some effect. Given these concerns, can you recommend a few acids that would be good to use.

[Andy Alpert]

I expect you mean which anions are good to use, since that would pertain to their salts as well as the unbuffered acids. If you want an additive that's volatile because you're going to a mass spectrometer (or some other detector that requires a volatile mobile phase, such as CDA or ELSD), then ammonium acetate or formate (or unbuffered formic acid) are fine. That explains their current widespread use. Exceptions might include the following situations:
1) Absorbance detection: If you want to monitor absorbance < 235 nm, then it's more important for the additive to be transparent in that region than to be volatile. Good additives here would include triethylammonium phosphate or sodium methylphosphonate. Notice that in order to get a nonorganic ion into a predominantly organic solvent in reasonable concentration, you have to pair it with an organic counterion such as methylphosphonate. Which of these salts to choose? Read my papers from 2008 and 2018!
2) Optimizing selectivity instead of detection: This is relevant if you intend to proceed to a second dimension of chromatography or are willing to desalt fractions collected during the run. In that case, it might be best to use an unusual additive such as a magnesium salt or triethylammonium sulfate (cf. my paper from 2018).

Keep in mind that pairing a charged analyte with a well-hydrated counterion promotes its retention in HILIC while pairing it with a poorly-hydrated one promotes its elution.

[Jimi]

Hello All

I was just reading through this thread. I think there was a lot of great feedback throughout this discussion. But I noticed one detail that I think we still didn't resolve.

At the risk of belaboring this discussion too long, let me just ask the remaining question: Is there a relatively strong acid which is mass spec friendly and which will generally not cause a problem with the HILIC method (in other words the counter ion of the acid is not problematic in HILIC).

Thanks in advance for any feedback. And I appreciate everyone's patience on this one.

Thanks again
Mark

[Andy Alpert]

You can have an acid additive that is:

1) Volatile (= compatible with mass spec analysis);
2) Relatively strong;
3) Transparent at wavelengths < 240 nm.

Pick two. You have done so, having chosen properties 1) and 2). Formic acid seems to meet your criteria. If that's not strong enough for your purposes, then you could consider trying difluoroacetic acid, which is in the early stages of evaluation as an alternative to TFA. Its properties are intermediate between those of TFA and formic acid. The ion pair between a basic compound and difluoroacetate ion will result in somewhat earlier elution in HILIC than would formate ion but not as early as trifluoroacetate ion.