During method selection our chromatographists have options like NH4, TFA, Formic Acid. What's the point of choosing only between acid? Why not use the strongest that guarantees ionization? The sample contains small organic molecules.

And if the analyte is an acid, don't we want to have a base as a modifier? Otherwise how can we ionize it using ESI?

  • $\begingroup$ What are your analytes? And LC-MS is no method to "purify" anything. $\endgroup$ – Karl Aug 22 '19 at 8:34
  • $\begingroup$ Just guessing: Any free electron pair can accept a proton, finding one in your analyte that could be abstracted is often not so easy. $\endgroup$ – Karl Aug 22 '19 at 8:44
  • $\begingroup$ Added info about the analyte, thanks. We do use LCMS for purification. Sure, not all the sample is passed through MS if you allude to the fact that it would destroy the sample. $\endgroup$ – Stanislav Bashkyrtsev Aug 22 '19 at 9:12
  • $\begingroup$ I am more alluding to the fact that typical LCMS systems are not set up for preparative work. Especially your modifiers would get separated in the LC too. I assume you actually do MS on the fractions you collect from your LC system, right? $\endgroup$ – Karl Aug 22 '19 at 9:21
  • $\begingroup$ I never thought about this.. I know that during drydown the modifiers also participate in salt creation, so somehow they get into the fraction. But yeah - we run MS for QC after purification. $\endgroup$ – Stanislav Bashkyrtsev Aug 22 '19 at 9:46

Stanislav, that is a Nobel Prize winning question. In short, nobody knows nitty-gritty details of how the electrospray ionization imparts a charge to the analyte. When Fenn (reinvented) the electrospray phenomenon and used for ionizing large biomolecules he was shocked and gave an analogy that it is like making the elephants fly. He got a Nobel Prize as well and a lot of patent issues in the university. There are three electrospray ionization models, you can read on Wikipedia, https://en.wikipedia.org/wiki/Electrospray_ionization#Ionization_mechanism

The key point in electrospray is that you need an electrolyte (forget about acids or bases). All you need is a conducting liquid between the electrode to generate the electrospray. In that process, the ion is created. There are studies, I am sure, on the effect of pH on the efficiency of ionization. I can tell what to avoid in electrospray: TFA (because it forms ion pairs), hexanes, all other pure non-conducting organic solvents. So you you can get the point that ionization in the solution has little to do with ionization in the electrospray.

Now coming to your chromatography part, which is purely empirical. The reason for avoiding bases in mobile phases is that it they will eat (=dissolve) the silica columns. Silica is not pH stable above 8. Silica based HPLC columns can tolerate low pH like 2.5, and below that the bonded starts to peel off, e.g. if you use a C-18 column and use pH 1, it will become a bare silica in a few days.

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  • $\begingroup$ Huh.. Then what's the main purpose of the modifier if it doesn't impact ESP much? $\endgroup$ – Stanislav Bashkyrtsev Aug 22 '19 at 14:14
  • $\begingroup$ Modifiers are used for chromatographic purposes and you need some electrolytes in electrospray. Buffers do have an impact on the efficiency but solution ionization has little to do with electrospray ionization. I mean there is no simple correlation that can be generalized. $\endgroup$ – M. Farooq Aug 22 '19 at 23:16
  • $\begingroup$ It's worth noting that newer column derivatization chemistries are good enough that basic modifiers can be used, even with silica columns. $\endgroup$ – Curt F. Sep 19 '19 at 17:00

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