by
lmh » Wed Jul 28, 2021 2:25 pm
(1) Compounds with carboxylic acid groups might show up in positive mode (and some instruments just seem to be biased towards better sensitivity in positive mode, so they may even show up better in positive mode), but the natural thing for an acid to do is to deprotonate to a negative ion RCOO(-), so it makes sense to use negative mode, not positive mode, for a carboxylic acid.
(2) If you're in positive mode, the fact there are 6 COOH groups doesn't mean you'd expect six charges.
(3) In negative mode, the fact you have six acid groups means you could potentially get up to six charges, but it doesn't mean you should expect six charges. It is highly likely that a number of the COOH groups will retain a counter-ion, so you should expect several of them to be found attached to hydrogen or sodium (or other positive ions). This means you may see multiple peaks in the spectrum, corresponding to different combinations of counter ions.
(4) But this does not affect retention, since in reverse phase conditions, the counter-ions will exchange rapidly during chromatography...
(5) ... but remember that electrospray is an electrochemical process, and the actual expected extent of ionisation of an analyte in the mobile phase at whatever pH it is is really rather irrelevant to the electrospray process. Things will happily ionise in electrospray at a pH where, in simple free solution, they should be barely ionised at all.
Summary: for a compound with multiple carboxylate groups, try negative mode, and don't worry about how many charges you see; expect multiple masses (coeluting as a single peak), with various combinations of counter-ions.
(general comment about post-column teeing: sometimes it works, sometimes it doesn't. Because there is electrochemistry going on in the electrospray spray needle, sometimes adding a base doesn't improve the ionisation of negative compounds even though it should increase the extent to which they're ionised in free solution. It's not just a matter of pH. If you're working in negative mode, you've attached a positive voltage to the spray-chamber, negative to the needle. When RCOOH deprotonates, the H(+) ion goes to the needle and collects an electron, and this is vital for the electrospray process to continue. If you add a base, you now have a protonated base instead of a proton, and if this protonated base is less able to get itself reduced at the spray needle than a bare proton, it may actually decrease the efficiency of the electrospray process. So be aware, try it out, and see what happens).