Chromatography Forum

Hello friends,


I'm developing a method for HPTAs-hydroxy pentacyclic triterpene acids.
My solutions are made in phosphate buffer so I'm using LC_ESI_MS for quantitation of betulinic acid.
I don't wanna use phosphate buffer with ESI but I don't know how to proceed in this case, regarding to mobile phase composition.
I would really appreciate some advices.

I wish you all the best!

The first option is to look for a literature method with LC-electrospray and use whatever solvents the authors used.

The other option is to choose your own, in which case I would start just by using something simple like 0.1% formic acid. It may look odd to use an acid when you're hoping to see an acidic compound in electrospray, because the acid should, theoretically, mean that the acidic analyte is less ionised in solution, but electrospray is actually quite good at seeing things at pH values at which they "shouldn't" be ionised (for betulinic acid you'll have to decide whether to look in negative or positive mode. It may work in both). The main point of the acid is to give you a solvent pH which isn't very close to a pKa of any compound in the mix, for the benefit of chromatography. Since you'll be doing reverse phase, probably on a silica-based column, you're doomed to acidic pH values. If you were to need a buffer, you can use ammonium formate and ammonium acetate buffers, but I wouldn't bother unless it's actually necessary/beneficial to chromatography.

Routinely, when presented with phosphate buffer methods for HPLC with a need to run in LC-MS, I just replace buffer with 0.1% formic acid and see what happens. Depressingly, 90% of the time, it works.

Thank you so much, I see your point, I'm worry about ion supression because I'm trying to quantify betulinic acid in two types of solutions (pH-5.5 and pH-7.5) and I'm using RP C18. I'm quite confused because, on the other side pH control is important to control retention and I don't know which part fits better......if I use ESI (-) and alkaline pH (I was thinking at ammonium bicarbonate pH-9.2), I should avoid ion supression but what about +/-1 pH units around the pka of the buffer.....? If I use 0.1%HCOOH could it provide good control over the retention of both acidic and basic analytes?


Sincerely,

Mermeth

You'll have no problem with lack of retention regardless of pH. This is a molecule that has plenty of hydrophobicity to interact with the column, even in its fully-ionised state.
As regards buffer versus acid, and suppression, I'd just run it and see. In some instruments, adding a buffer can actually make sensitivity worse even though the pH is theoretically a better choice.