An example would be two peaks typically seen for an aliphatic triamine (the amine groups are of different basicity) run at pH 2.0, MeCN-H2O, H3PO4 buffer, at RT, on a high quality C-18 column. The two peaks, for the same sample, can vary in proportion from run to run. On the other hand, at pH 4.0 (phosphate buffer) the compound produces one, sharp peak (of course at a different retention time compared with pH 2). Why two peaks and not one or three, or more? Can this phenomenon be associated in any way with the three ionization levels of orthophosphoric acid, or is this the result of pH-related equilibria of the triamine only?
What about this:
you have a poly -amine (or a cluster compound) with an amino group (A) and a different amino group (B). At a given pH an equilibrium exists between the two mono-protonated forms (A)H-B and A-(B)H. If this equilibrium is slow enough you could detect two different peaks in HPLC. Small variations from the equilibrium favor one product over the other. At pH 4 may be neither site A nor site B is protonated so you detect only one peak.
Does it sound?
Thanks Massimo Luigi. Okay, in general terms, it must be something like that... but I thought that acid - base equilibria are established rapidly. That way (with rapid equilibration) it would be hard to justify the multiple peaks... unless there is something in play that substantially slows down the equilibration. Also, at least one of these amino groups is quite basic (pKA >> 12) such that a homogeneous free base at pH 4.0 probably is not likely.
When you decrease pH and increase acetonitrile, decrease the buffer solubility, i think maybe you have salt peaks, i recommend regeneration wash and decrease the % acetonitrile in the technique. Best regards!
@ Jacek, if one site is always protonated pKA>12 then the "my theory" can be applied to the second protonation. Acid/base equilibria are usually fast in water, but as Cony pointed out the addition of an organic solvent can alter the system and the chromatographic separation can hamper the free movement of protons (some parts of the molecule are engaged with the stationary phase).
Cony - the buffer system that involves the multiple peaks uses H3PO4 as the buffer only, at a low concentration. This gradient does not show "salt peaks" when run with other compounds as well.
Massimo Luigi - the gradient is rich in water, beyond the time when the peaks elute. I agree that it is "something like your theory" however it is nice to really know what is going on. I feel so far you came the closest - thanks!
Jacek - I have seen the same problem with my compound which is a Mannich base diamino substitued (two dimethylamino groups) and at low pH (pH 2) I see one and sometimes two more peaks compared to pH 7.4. Did you find out about what was the cause of your problem related to triamines? Maybe, my problem could be related to.
Aline,
More recently we have had another thread on a similar topic, where we discussed it with professional chromatographers why would a single and pure compound in some cases give more than one peak (but unfortunately the structure of that compound was not disclosed). Still, the consensus has been that for some polyamines and other similar basic compounds that have a few centers of basicity differing in pKbs, at a given pH it is possible that, say, only one center (the most basic one) is fully protonated while the other one(s) are only partially protonated. This could result in more then one chromatographically distinct species, even though it is one compound.
Another possible scenario is when the compound has a chiral center in addition to a tertiary amine bearing three different substituents on nitrogen atom. After protonation, a new chiral center forms that in some cases will be conformationally prevented from equilibration (eg due to sterics) and thus a pair of diastereomers is formed upon protonation! Some other cases are still possible due to atropoisomers, etc. To my astonishment :-) , this means that, in principle, depending on the number of different centers of basicity, a single compound can give a multitude of peaks.
Regards,
Jacek
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