Retention Behavior for ion-pair HPLC

[jan]

Hello, Everyone,

Just thinking if one uses ion-pair HPLC to analyze ionic mixtures what will be retention behavior for different charged species? Will doublely charged ion elutes later than single charged ion? Thanks.

[Kostas Petritis]

Jan,

In ion-pairing chromatography, things are not as straightforward as in ion-chromatography. In additon of the charge of your analytes, their retention will depend on their hydrophobicity, the nature and concentration of the ion-pairing reagent.

Let's take the example of the 20 proteogenic amino acids.
In reversed phase chromatography with 100% water no ion-pairing reagents:
Everything is eluting in the void volume just some hydrophobic amino acids (i.e. Leu, Ile, Trp, Phe etc) are retained.
TFA at 2 mM, all polar amino acids including the basic ones (Lys, His, Arg) are eluting at the void volume, some of the hydrophobic ones that were eluted in the void volume are now retained just a little bit
HFBA at 2 mM, polar amino acids are retained a little bit, basic amino acids are eluted after the polar ones, hydrophobic ones even later the basic ones
PDFOA (pentadecafluorooctanoic acid) at 2 mM, polar amino acids first, followed by hydrophobic ones and basic amino acids are eluted at the end.

When TFA, HFBA and PDFOA are used I assume the use of a gradient elution with ACN.

[Chris Pohl]

Jan,

In general, all other things being equal (this is often a bit difficult to accomplish but formic and oxalic acid are reasonable examples of a similar pair of monovalent and divalent species) the divalent species will elute later and a trivalent species even later. Another interesting aspect of this is that selectivity in ion pair is best for monovalent species (the odds are pretty good that a given pair of similar monovalent species will be resolved while co-elution of divalent species is much more common). For example, formate and acetate are easy to resolve by ion pair but oxalate and succinate co-elute under most if not all conditions.

[Kostas Petritis]

Chris,

I do not think that it is always the case, I guess it boils down to what you call ion-pairing chromatography. When using TFA for the separation of cationic mono di and trivalent species I would guess that the selectivity would be first the tri then the di and then the mono (even if you increase you TFA concentration up to 100 mM). The more you increase your ion pairing reagent side chain the more you pass on something more similar to ion exchange chromatography and then you have the selectivity order that you mentioned.

Such selectivity differences have been previously discussed by several authors and Patthy is one of them, I have mentioned his reference in one of my previous postings (see below):

http://www.sepsci.com/chromforum/viewto ... light=#939

Some people describes it as ion pairs in the mobile phase or in the stationary. In the first case, the ion pair reagent will form a pair with the analyte, the formed ion pair is more hydrophobic an is adsorbed on the stationary phase. The other mechanism is that the ion-pairing reagent is absorbed in the stationary phase and the analyte is interacting the same way as if it analysed in an ion exchanger (in addition to other hydrophobic interactions of course). In the second case, the first mechanism exists as well but participates in the retention mechanism less than 10% (if I remember well)...

[Chris Pohl]

Kostas,

Indeed, there are operating conditions under which you won't observe the behavior I described but from my point of view, it isn't really ion pair chromatography unless you observe such behavior (i.e. retention for polyvalent species > retention for monovalent species). The observation you described below regarding TFA is one of the reasons that I wouldn't classify it as an ion pair reagent. But, I understand that the meaning of this term is applied in a wide variety of different contexts and undoubtedly many would not agree with my definition.

[Victor]

Kostas,

Your posts and that of Chris are very interesting and helpful. However, I would be grateful for a further explanation of your statement below:

"When using TFA for the separation of cationic mono di and trivalent species I would guess that the selectivity would be first the tri then the di and then the mono (even if you increase you TFA concentration up to 100 mM). "

Do you mean that the order of elution is first the tri and last the monovalent cation? If so, why did you guess this would happen, and which solutes in your experience behave in this way? This is not at all my experience with TFA, which is much as Chris describes i.e. that the tri species comes out last and the mono first. I would predict this order either from a hypothesis that ion pairs form in the mobile phase and then are sorbed on to the stationary phase (reason: the tri-species might form pairs with 3 TFA ions, giving increased hydrophobicity) or from a dynamic ion exchange mechanism (reason: the coulombic attraction between +3 and -1 is greater than that between +1 and -1)

Many thanks

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[Victor]

Oh- I guess you probably mean that if the ion pair effects of TFA are small, then retention will be dominated by the decreased hydrophobic interactions of a tri-charged species compared with a mon-charged, if we are just thinking in terms of normal interactions with the C18 ligands.

[Kostas Petritis]

Victor,

That is what I meant. As Chris also mentioned is difficult to have everything else equal and compare in terms only of mono di and tri charged species.

For such species, if you trace the capacity factor k' vs. chain length of the ion pairing reagent (i.e. from 1 up to 7... let's say TFA up to PDFOA) the curves won't be parallel and you will have changes of selectivity.

I have to dig in my ion-pairing chromatography papers to see if I can find an adequate example, but I do not think that suprices a lot of people in this forum what am I saying .