Trimethyl Ammonium Acetate as a Modifier

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

21 posts Page 1 of 2
Hey Folks

Does anyone have any familiarity with the use of Trimethyl Ammonium Acetate as a modifier for HPLC.

I have used ammonium acetate many times over the years, but never the 'trimethyl variant'. A colleague of mine says he is seeing this recently, so I wanted to know if this was a new trend I should be aware of.

I guess the key question is: What unique benefits would it offer?

Thank You!
For analytes containing negative charge for which it would serve as a counterion, better retention in reversed-phase chromatography and worse retention in HILIC. it would probably suppress ionization somewhat in mass spec, since the more hydrophobic counterions don't dissociate as readily.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
Andy,

A delayed follow up, please.

In HILIC, would trimethyl ammonium acetate work as well as ammonium acetate, from the standpoint of mediating charged interactions (for the purpose of this question maybe let's assume that it's not ion pairing, because that would further complicate the matter).

Thanks
Mark
Andy,

A delayed follow up, please.

In HILIC, would trimethyl ammonium acetate work as well as ammonium acetate, from the standpoint of mediating charged interactions (for the purpose of this question maybe let's assume that it's not ion pairing, because that would further complicate the matter).

Thanks
Mark
I suspect that you're approaching the concept of ion pairing the way it's used in reversed-phase, where one might use something along the lines of octanesulfonic acid to effect a significant change in polarity or retention of something. The fact is that any charged analyte is going to acquire a counterion for its charged group(s), and if the cation that's present is trimethylammonium ion, then that's the counterion that the anionic groups will pair with, whether in reversed-phase or HILIC. Trimethylammonium acetate will be even more effective than ammonium acetate for forming ion pairs since the more hydrophobic (= less well hydrated) an ion is, the stronger are the electrostatic bonds it forms. That's why: 1) Ions of the same charge elute in order of less to more hydrophobic in ion chromatography, and ; 2) In mass spectrometry, why ionization of peptides and other analytes is suppressed when trifluoroacetate ion serves as a counterion. It doesn't dissociate as readily.

If this screed didn't address your concern, then please elaborate in detail.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Hello Andy

I would ultimately defer to you on such matters - as I know you understand these things quite well - but I had thought that TEAA would not act as an ion pair agent in HILIC. Here is my logic. Just as when we put NaCl into water and the Na+ and Cl- ions separate from one another, despite having an attraction. The fact that the Na+ and Cl- ions are "happy enough" (thermodynamically speaking) when solubilized by water, combined with a little good-old-fashioned entropy, keeps them separate. Similarly, I thought trimethyl ammonium cation and acetate anion would be "happy enough" in an environment of 95% ACN to exist as separate ions (without much driving force to pair up). But, again, I would defer to your understanding, and would appreciate your letting me know if I've gone astray in my thinking.

By the way, the big picture here is that I've been told one needs some kind of salt to help mediate interactions in HILIC, and ammonium acetate is only sparingly soluble in 95% ACN. This is what brought us to TEAA as an alternative (it dissolves quite readily in ACN).

Thanks
Whether or not the anion and cation of your added salt are dissociated in the HILIC solvent is irrelevant here. The important thing is the interaction with charged analytes.

If your analytes aren't charged and the stationary phase is more or less neutral, then you can sometimes get by in HILIC without added salt. If the analytes are ionizable, though, then if you don't add salt, then their counterions will be whatever they were exposed to before the chromatography. If a charged analyte has more than one type of counterion, then the resulting ion pairs will differ in polarity. Result: Elution in split peaks or even in two separate peaks connected by a continuum (cf. Fig. 14 of my paper from 2008 that introduced ERLIC). Adding salt helps to insure that all the molecules of the same type will share the same counterion(s). Usually you need at least 20 mM salt overall in the mobile phase to insure that. 20 mM is also enough to form a complete double layer of ions on the surface of the stationary phase, if it has any charged groups. That helps with reproducibility.

What analysis are you performing that requires you to start at 95% acetonitrile? That would presumably involve an analyte that has hardly any polarity at all.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Thanks for the feedback.

Lack of retention is one problem we sometimes face with HILIC. We typically start method development by running a scouting gradient from 5% water to 50% water (in ACN, and with whatever additive). My understanding is that 50% water in HILIC is about the same as 100% ACN in reversed phase.

Would you agree with my thinking to use TEAA as a modifier instead of ammonium acetate (not that it will work in all cases, but is it generally a reasonable a approach)
Yes, TEAA is a valid additive to use if solubility is a consideration. However, if your analyte has a negative charge, then supplying it with a triethylammonium counterion instead of ammonium ion will result in earlier elution. There's a tradeoff involved. In that case, then consider trying ethylenediamine acetate instead.

Which HILIC column are you using? If you're using one that's not one of the more retentive ones, then switching to one that is at the top of the retention scale would permit you to get better retention of marginally polar analytes with less organic solvent. Our PolyHYDROXYETHYL A and Tosoh's Amide-80 are at the top of this range, with the various ZIC- materials coming after that.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
OK Thanks Andy
One additional thought has occurred to me (not necessarily related to the initial focus of this thread).

I mentioned that I often start my HILIC runs at 95/5 ACN/Water (with whatever modifier). And Andy had mentioned that this might be too week. But it also occurs to me that it may not give enough "elbow room" for ion exchange process or if we want to get the analytes ionized. In other words, with such high organic maybe everything is locked in neutral form (or paired up, such that the ion pairs can't exchange). Generally speaking, maybe some of the things that need to happen in HILIC require a little more than 5% water.

I'm wondering if some of the problems I have seen, historically, might be related to this.

I realize this was vague, with no details, but any thoughts about the validity of my thinking?

Thanks in advance!
You seem to be speculating that an exchange of ions between the analytes and whatever's in the mobile phase might be frozen out if there's < 5% or so of water. That's a falsifiable hypothesis. If you're game to indulge in a little Truth-and-Beauty, then here's an experiment you could perform to test that hypothesis. Take a charged analyte that has one counterion (because that's the counterion in the sample solvent) and inject it into a HILIC column where the mobile phase contains a very different counterion of that charge. If there is counterion exchange, then your analyte peak will either be severely skewed or will elute in two separate peaks with a continuum between them. One of those peaks will be the ion pair consisting of the analyte with its initial counterion. The other will be the ion pair of the analyte with the counterion supplied by the mobile phase. Since the two counterions differ in polarity (since you chose them that way), the ion pairs elute at different times in HILIC. The continuum will be molecules of the analyte that started with the original counterion but which exchanged it for the counterion in the mobile phase during their migration down the column. There's an example of this pattern in Fig. 14 of my 2008 paper that introduced ERLIC, cf. the following link: http://pubs.acs.org/doi/pdf/10.1021/ac070997p . Here, the analyte was arginine, with an anionic counterion that either did or did not match what was in the mobile phase.
If no counterion exchange occurs at high % organic solvent, per your speculation, then you won't see this two-peak pattern.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
I have been watching this thread, and we struggle with this issue a lot lately, i.e. we want to use a higher concentration of buffer, in a HILIC mobile phase, but it's not soluble when there's a high percentage of ACN (forgive me, this may be a little redundant with previous discussion, but possibly can help to clarify). I've heard of two solutions for this problem:

One - use a more organic soluble buffer. For example, instead of ammonium acetate use trimethyl ammonium acetate. But do these 'more organic' salts work as well to mediate charged interactions?

Two - Use acid or base to make the salt more soluble. So, using ammonium acetate again as the example, I guess you would add acetic acid drop wise to put the acetate in the unionized form (not quite sure about this).

Andy (and all), would you kindly comment on the validity, or relative merits, of these two approaches. And I realize neither will work in all cases, but I am specifically focusing on the issue of 'the use of salts to mediate charged interactions'.

Thanks very much
My paper from 2018 (link:https://www.sciencedirect.com/science/article/pii/S0021967318300785/pdfft?md5=5a06f33c2cf39d6345b8b063b71f34ed&pid=1-s2.0-S0021967318300785-main.pdf) addressed these issues. Using triethylammonium as the cation will definitely permit you to get a higher concentration of the salt into a given % ACN compared with using ammonium ion. Either ion will suffice to deal with electrostatic effects too, such as shielding the analytes from (-) charged groups on the surface of the stationary phase. However, the ion pair resulting from triethylammonium ion pairing with a (-) charged analyte will be less polar than the ion pair resulting from ammonium ion, and the ion pair will elute earlier in HILIC.

Using acid or base to make a salt "more soluble": There's an example of that in the paper too. With 85% ACN, I was able to make a mobile phase containing 120 mM triethylammonium sulfate at pH 3 but only 80 mM at pH 6; at 120 mM, pH 6, there was phase separation. I might mention that I prepared the salts by adding triethylamine to an aqueous solution of sulfuric acid until I got to the pH I wanted.
PolyLC Inc.

(410) 992-5400

aalpert@polylc.com
Thanks Andy for the fast response. I got the paper and will read through it carefully.

One quick follow up. Something didn't seem right when I (in my last post) said "add acetic acid to a solution of ammonium acetate, to help it dissolve". I understand that if we add acetic acid we would generate more H+, and hence move the pH below the pka, so acetate would becomes unionized. What confuses me is that it also seems for every H+ we put in solution - from the acetic acid - that we would also form an acetate ion. Or does the lower pH "overwhelm" this effect (so to speak) such that the vast majority of acetate is unionized.

I know this is a Chem 101 question, but for some reason I am getting stuck on it at the moment. Would certainly appreciate a few sentences to push my thinking in the right direction.

Much Thanks!
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