Ammonium Formate vs. Ammonium Acetate in HILIC

[Jimi]

So I'm thinking we can fit in one more 'HILIC Additives' discussion before we all break for Thanksgiving. And, if no one objects, I can just continue this thread rather than start a new one.

The issue I wanted to discuss is that of Ion Pairing in HILIC. And my basic question is, why doesn't it happen more often. It seems to me that in all the recent discussion we've had about HILIC additives, the only additive which has been noted to ion pair is TFA (or more specifically, the triflouroacetate ion). I guess I am just wondering why, under conditions of high organic, pairing doesn't happen more often. For example, why doesn't acetate pair, or formate. Or, with respect to cations, why doesn't ammonium pair. It just seems to me that pairing would be more common under such high ACN concentrations.

Any general thoughts or rules of thumb (what might pair...and under what conditions) would be of interest.

Thanks Very Much

[Andy Alpert]

Sure they do. Look up my paper from 2008 that introduced ERLIC (cf. the following link: http://pubs.acs.org/doi/pdf/10.1021/ac070997p). Fig. 14 shows the graphic consequences when the anion that's the counterion for a basic analyte doesn't match the anion that's in the mobile phase.

There's nothing magical about TFA in this respect. It's widely used because it promotes retention in reversed-phase chromatography, is volatile, and is transparent at low wavelengths. It happens to form a particularly tenacious ion pair, which is troublesome in mass spec since it doesn't dissociate readily from its cation.

[Jimi]

Thanks for quick response Andy.

Over the years, I have seen it stated (here and there) that it is best to ionize analytes in HILIC because ionized analytes are better retained (which makes sense). But if the reality is that - most of the time HILIC - an ionized analyte will pair with the counter ion, then it seems that logic may be flawed.

In other words, why bother to ionize something if it will become neutral anyway by virtue of pairing (or course, in any given situation it may or may not help selectivity to ionize the molecule; but it seems to me that, as a general rule, ionizing the analyte would not lead to increased retention - because the ion will generally pair and become neutral).

Am I right or am I still missing the boat.

[Andy Alpert]

Depends on the following:

1) How polar the counterion is. An ion pair with formate or acetate as the anion will be more polar than the ion pair with trifluoroacetate as the anion. See also that figure I just referred you to, in regard to the relative polarity of an ion pair involving phosphate vs. methylphosphonate.

2) What the concentration of the counterion is in the mobile phase. If it's less than 20 mM, then an ion is not guaranteed to find a counterion in the mobile phase and instead will interact with functional groups of the opposite charge on the stationary phase. There are some examples of that in the same paper as well (Fig. 17 in particular, showing retention of nucleotides as a function of the concentration of salt in the mobile phase. Look what happens between 10 and 20 mM).

That assumption that ionized analytes are more polar is not always the case. Basic solutes are the most hydrophilic ones. If you have a solute that has both basic and acidic groups in a position to form a zwitterion, then that's less polar (i.e., retention is less in HILIC) than the situation where the pH is low enough to uncharge the acidic group and leave the basic group's (+) charge unpaired within the molecule.