Zwitterions vs ampholytes

The former having charge separation, the latter not. See the abstract from J.E.A. Comer's reference here.

In particular where he says 'Both molecules have two pKa values. In clioquinol, the base pKa is lower than the acid pKa, so at pH values between the pKa values, the molecule is neutral. However, the acid pKa for ampicillin is lower than the base pKa, and the molecule is charged across the entire pH scale.'

Now, the part that I don't get is how one decides that the 'acid' pKa is this value and the 'base' pKa is that value, and therefore that there is in fact charge separation in the molecule or not (depending on whether intramolecular deprotonation from a hypothetical non-charged species is thermodynamically favoured or not).

Please correct me if I'm wrong. If you have a real substance in front of you and you want to know the pKa's, maybe you dissolve it in water and titrate it with a strong base, starting from very acidic pH, following the pH curve vs the equivalents of base added, and you should get the classic 'jumps' at the points corresponding to the pKa values.
Suppose that in the 0-14 pH range you find 2 pKa's, let's call them pKa1 and pKa2, with pKa1 < pKa2. At that point all you know is that this molecule is initially protonated N times; at pKa1 it will be half N-protonated and half (N-1)-protonated; at pKa2 it will be half (N-1)-protonated and half (N-2)-protonated.

In what was does that tell you where protonation takes place?

In the example from the reference, ampicillin and clioquinol are mentioned.
OK, I agree that from knowledge of chemistry we can say that the pKa of phenols is about 10 and the pKa of pyridinium is about 5, so I don't expect pyridine to deprotonate phenol, or clioquinol to be zwitterionic. But in fact this is an assumption - what do I know of what happens in a molecule where both groups are present, maybe in some particular arrangement that may favour reactions that wouldn't happen intermolecularly? And for ampicillin, OK, benzylamine is usually sufficiently basic to be protonated by an average carboxylic acid. But how do we know for sure that the ampicillin molecule exists (in solution? in the solid as well?) with a deprotonated carboxyl oxygen and a protonated amino group?

Based on what experimental evidence does one decide that a molecule is zwitterionic or ampholytic, in the sense defined in the above cited abstract?

And are there 'borderline' cases, meaning: can we find in one solution molecules (of the same substance, of course) with zero total charge, but some with charge separation and some not?

  • $\begingroup$ I will add this comment in case other people have the same question. I can't post an answer because the original post was closed down (...). It turns out that a molecule with overall charge z may indeed exist in all its allowed tautomeric forms, so the answer to the last question in the post is: yes. We currently have access to some software that allows to calculate the % of each species according to its charge and the % within a single charge class. $\endgroup$ Sep 17, 2019 at 15:43


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