Is acetate capable of Ion Pairing?

[mohan_2008]

Thanks Kostas.

[Kostas Petritis]

I agree with Tom that most of the time what we do in LC is not a "pure" mechanism.

Also, if memory serves, perfluoroalkyl aliphatic chains are more hydrophobic than their hydrogen homologues so it would make sense that TFA would be more hydrophobic than acetic acid (assuming that both are completely ionized).

Several years ago, I investigated different perfluorocarboxylic ion pairing reagents for amino acid and peptide separations. Among other, I studied all the breakthrough curves during column equilibration from TFA up to PDFOA (pentadecafluoro octanoic acid) at 100% aqueous mobile phases and different ion pairing reagent concentration and reversed phase columns (i.e. C18 or porous graphitic carbon) by using post-column conductivity detection. Once you do that, you can quantify the exact amount of ion pairing reagent absorbed in your column and it's potential for "dynamic ion exchange" behaviour.

You could imagine that you could do the same for acetic acid, although you will need to take into account the % of ionized acetic acid remaining at the pH of the mobile phase (the pka of perfluorocarboxylic acids is too low and thus it can be assumed that they are fully ionized).

[HW Mueller]

The logP of 0.5 is apparently estimated (EST), it says that TFA is more soluble in octanol than in water, a ridiculous proposition.
Kostas, the chemistry and physics of fluorine compounds is complex, here a quote from an "old bible" on "Chemistry of Organic Fluorine Compinds" by Milos Hudlicky, 1962, page 304:
"Higher polyfluoryl- and perfluoroparaffins are only slightly soluble in water (ref. 327). On the other hand, polyfluoro- and perfluoroaldehydes, ketones, and acids have very strong affinity for water. They easily form azeotropes and hydrates, which are dehydrated only with difficulty."

[danko]

Interesting discussion.
I wish someone had a HILIC system running, so that we could test potential differences in hydrophilicity. I have this feeling that it might be possible to separate Acetic acid and TFA on a HILIC column (10 % phosphate buffer pH 7 and 90 % ACN).

Best Regard

[sassman]

The logP of 0.5 is apparently estimated (EST), it says that TFA is more soluble in octanol than in water, a ridiculous proposition.
Kostas, the chemistry and physics of fluorine compounds is complex, here a quote from an "old bible" on "Chemistry of Organic Fluorine Compinds" by Milos Hudlicky, 1962, page 304:
"Higher polyfluoryl- and perfluoroparaffins are only slightly soluble in water (ref. 327). On the other hand, polyfluoro- and perfluoroaldehydes, ketones, and acids have very strong affinity for water. They easily form azeotropes and hydrates, which are dehydrated only with difficulty."

I agree that fluorinated acids can behave in ways that are not easily describable common terms such as hydrophobic and hydrophyllic. We do a lot of work with long chain (C4-C12) fluorinated acids and alcohols. They are hydophobic and oleophobic meaning that they don't like water or organic phases and tend to aggregate at interfaces. Something like TFA; however, I would expect to be highly water soluble.

[Kostas Petritis]

For perfluorocarboxylic acids, solubility in water decreases singificantly with increased chain length. I had to sonicate the PDFOA aqueous solution to get it dissolved...

[JA]

While the thread digressed into discussion of the properties of TFA vs. acetic acid, I think the underlying reason for our observations on the behaviour of the surfactant molecule may have been touched upon by contributions early on and I wanted to post a follow up to this.

The original method conditions were given in the opening post, however my colleague was working to simplify them for a purification on equipment requiring a significant scale up where isocratic elution must be employed. The column was switched to a Kromasil C18 and the mobile phase became a mixture of ammonium salt and methanol. The following observations have been made at analytical scale:

MP(A)= 0.1% w/v CH3COONH4 [natural pH ~ 6.7]
MP(B)= MeOH
10:90 v/v A/B, k' ~ 14, broad, heavy tailing

MP(A)= 1% w/v CH3COONH4 [natural pH ~ 6.8]
MP(B)= MeOH
10:90 v/v A/B, k' ~ 8, broad, tailing

MP(A)= 1% w/v CH3COONH4 [titrated to pH 4.7]
MP(B)= MeOH
10:90 v/v A/B, k' ~ 4, reduced tailing

MP(A)= 1% w/v HCOONH4 [titrated to pH 3.7]
MP(B)= MeOH
10:90 v/v A/B, k' ~ 3.5, identical to previous peak profile

MP(A)= H2O
MP(B)= MeOH
10:90 v/v A/B, k' > 20, not observed

Until the last run the colleague reasoned that retention decreased with decreasing pH as the ionic form of the "ion pair" agent was made less available.

- Can we summise instead that the dominant mechanism of retention is electrostatic interaction with SiO- ?
- If the above is true, are the secondary interactions responsible for the poor peak shape? That is, the hydrophobicity of the molecule causes it to tail?

[Uwe Neue]

Looks like standard stuff to me: quaternary amine interacts with ionized silanols, which become less ionized as you decrease the pH. You can use the changes in retention with pH of a quaternary amine to "titrate" the SiO-.

[HW Mueller]

The decrease in k with an increase in NH4OAc conc. at ~ the same pH (6. also supports this SiO- explanation. The SiO- interaction can be obliterated at high ion concentrations. The tailing behavior is typical as well.

Sassman might like this: another quote from the book I quoted above, also p. 304,
"The solubility of fluorocarbons in organic solvents is usually much lower than that of the corresponding hydrocarbons. Very often two phase systems are formed, which homogenize only at elevated temperatures."

[JA]

I was curious as to your thoughts on the reason behind the peak tailing even as the pH is decreased.

In a reversed-phase interaction with dispersive forces predominantly responsible for retention we often invoke the case of "secondary interactions", be them polar or ionic, with for example residual [ionised] silanols. Hence my query w.r.t the current situation where it is evident that retention is largely affected by those residual silanols.

Is the observed tailing therefore due to "secondary" dispersive (hydrophobic) interactions, as we might invoke in a traditional ion-exchange method, or the remaining "acidic" (ionised) silanols?

[Uwe Neue]

Not sure if the tailing is indeed increased, or if it is just more evident as the peak becomes wider. Do you have tailing factors or asymmetry factors?

The change in retention is normal: as the ionic interactions increase, retention increases. Actually, the retention is a multiplicative function of reversed-phase and ionic interactions, but this is not necessarily obvious in most experiments (U. D. Neue, A. Méndez, K. Tran. P. W. Carr, „The combined effect of silanols and the reversed-phase ligand on the retention of positively charged analytes“, J. Chromatogr. A 1063 (2005), 35-45).

There are multiple components in peak tailing. One that is simple to explain has nothing to do with silanols: as ionic samples are adsorbed on the reversed-phase surface, they repulse each other, which in turn creates tailing. This phenomenon can be manipulated by the concentration of ions in the mobile phase. The lower the ionic concentration, the more pronounced this type of tailing becomes.

[JA]

Unfortunately I don't have any data to hand at the moment.

Could you elaborate on the like-ion repulsion effect which causes tailing? I have trouble visualising it if I assume that the bulk of adsorbed sample ions prevent similar ions approaching the surface. In this case I guess that a smaller fraction of sample ions "run through" with decreased retention, giving a fronting peak.

Please correct my interpretation.

[Uwe Neue]

A small amount of adsorbed ions gives the highest retention, since there is only minimal repulsion. Now I increase the amount of ions, more repulsion occurs, and the retention of this larger concentration of ions is less than that of the smaller amount. Consequence: a tailing peak.

[reyoungs]

[quote="Vlad Orlovsky"]I don't expect acetate to be ion-pairing reagent as well as I don't 100% agree that TFA is ion-pairing reagent, you can argue this point but Hydrophobicity of both TFA and acetate are not that great. Fluorinated acid (C4) are used as ion-pairing reagents so I would expect unsubstituted carboxylic acids to show some IP properties at certain conditions, which are:
- IP needs to be at least C6 (butyric acid)
- low organic concentration

Vlad is correct his general summary of conditions required of ion pairing using actate and cationic analytes.

As Vlad states, ion pairing requires that the mobile phase be adjusted to a pH at which both the target analyte and the "Ion Pairing Reagent" be of the opposite charge to allow formation of a neutral ion pair that appears neutral to the surface of the bonded phase. Once you achive these conditions, you can form ion pairs.

Now the next part of the question is how much does the ion pair impact retention (and how). Vlad is also right that the lipophilic affect of acetate or a perfluoro acid is very low, but probably not zero.

Classical ion pair reagents consisted of short to medium sufactant types of molecules that remained charged at all pH ranges and bound tightly to the oppositely charged analytes. I won't get into whether they irreversibly change the surface of the phase and introduce an ion exchange effect as well, but the net result in reveresed phase is the increased retention of the target analyte, generally proportional to the chain length of the surfactant being used.

In this most general description, under the right pH condidtions, a wide variety of molecules can be considered to form ion pairs and enhance lipophilic retention, but will have limited impact due to their short alkyl chains. In this category are a variety of long chain carboxylic acids and amines that would only become ion pairing reagents when converted to the ionic form.

Yes, acetate can ion pair under the right conditions, but it will have poor lipophicity contributions. As was discussed earlier, the ammonium ion effect is probably also contributing. We looked at direct comparison of various buffers varying the cation and found that ammonium ion behaves very differently than any other cation, but the other striking effect we saw was that the cation also impacted peak shape and resolution under the same pH and ionic strength conditions.

[Kostas Petritis]

C6 is caproic (hexanoic) acid
C4 is butyric acid...