[SugarSkull]

I've been researching about why one might add an acid or a base to the mobile phase in HPLC (reverse phase in the example I'm looking at). I was hoping someone could help me resolve some conflicting explanations I've encountered.

Consider a mixture you would like to analyse by reverse phase HPLC - hence the stationary phase is non-polar and your mobile phase will be the more polar option*. This mixture contains acids (HA) and bases (B). Consider adding an acid, which I will call H-X, to the mobile phase.

The acidic mobile phase will mean the acid analyte, H-A, will remain largely protonated, and thus neutral. The basic analyte, will be deprotonated, B-, and thus ionic. Hence the neutral analyte, HA, will be retained on the non-polar column for longer than the polar/ionic B-, which will elute first.

This all makes sense to me. However other sources seem to say that if you were to add an acid, H-X, to a mixture with a basic compound, B, the two will make an ion-pair, i.e. (HB)⁺(X)^-, which will overall be electrically neutral and thus interact with the reverse phase column more than if the base were left alone.

So does it make sense to add acid when it seems to encourage both analytes to interact with the reverse phase column more?

And another thing: in the example above, there are reasons for the increased interactions. However, it seems to me that in general, a small percentage of acid is added to HPLc mobile phase as a matter of course. When I try to find out why I just get pages saying stuff like 'it improves peak shape' etc, but providing very little imformation on how adding acid to everything helps in all cases. Can anyone point me in the right direction on this?

I realize I may have asked the same questions 2 times without really understanding it. Well, I'm here to learn, right?






* I realize y'all would known this anyway, I'm just being clear for the benefit of less experienced readers who may be looking at this post.

[Babcock_Hall]

I just have time for an off-the-cuff answer for right now, and I don't claim expertise.  Trifluoroacetic acid is a common additive for protein and peptide RP-HPLC.  The conjugate base becomes the counterion for certain side chains, such as lysine.  Perhaps the way to think about it is that it is a question of which counterion the positively charged side-chains will have (chloride being another alternative), and which give the best chromatographic characteristics.

[Train]

The basic analyte, will be deprotonated, B-, and thus ionic.

Both the acid and base are protonated in an acidic environment.  The difference is the protonated acid is uncharged (HA), while the protonated base is charged (HB+), and vice versa.  Depending on the respective pKa's, as you raise the pH one or the other might lose the hydrogen first, resulting in either both neutral (HA and B) or both charged (A- and HB+) before you eventually have A- and B in a basic environment.

If the pKa of the acid is lower than that of the protonated base, then within that pH range you will have charged acid A- and charged base HB+ which can form ion pair in the mobile phase or on the column.