This is probably very basic, but I have been given the understanding that we subtract the mass of only one proton when looking for our compound in a mass spectrometry (negative mode). I dont really understand why we are only subtracting the mass of one proton when our compound could potentially loose more than one proton at higher pH?

In example, the amino acid glutamate, would it not loose or gain more than one proton is a negative (higher pH) or positive (lower pH) mode mass spectrometry (MS)? Perhaps I have misunderstood the concept of negative and positive mode for MS?

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  • $\begingroup$ Higher pH is rarely used in LCMS due to possibility of damaging column packing usually made of $\ce{SiO2}.$ $\endgroup$ – andselisk Jun 9 '20 at 9:07
  • $\begingroup$ This question concerns the MS, not the column, but some columns do tolerate higher pH as far as I understand it? $\endgroup$ – CuriousTree Jun 9 '20 at 9:10
  • $\begingroup$ I concluded from "mass spectrometry chromatogram" that you are asking about LCMS meaning the backend is a chromatographic column. And from from I heard, there is rarely a need to go with higher pH for amino acid assays and switching to other packing material like $\ce{ZrO2},$ which are usually considerably more expensive and have their own quirks. $\endgroup$ – andselisk Jun 9 '20 at 9:14
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    $\begingroup$ ah okay, thanks for the input! I also assume the higher or lower pH is relative (i.e. not always extreme), but in general sticking to a neutral pH range is more common. Im not all that familiar with this topic, so I will remove the 'chromatography' word to clarify my question. $\endgroup$ – CuriousTree Jun 9 '20 at 9:34

Interesting question, which originates from a major misconception related to pH in LCMS. Nowadays, there are dozens of ionization techniques so in the question always mention the ionization method.

Anyway, LCMS is usually done with electrospray ionization (ESI) technique and so are amino acids. First of all nobody completely knows the exact process in electrospray ionization as yet. This is not unusual in mass spectrometry where even a common ionization technique like ESI and electron impact, the picture is not 100% clear.

Second important point is the pH in ESI does not determine whether a negative ion or a positive analyte ion will be formed. I also used to have this misconception a decade ago about pH. The final ionization process in ESI takes place in a gas phase. However it is well known that certain ion can suppress ionization very badly. Don't try to connect mobile phase pH with the state of ionization of the molecule in the gas phase.

In negative ion, you are simply observing a net negatively charged species. Nothing

For details, I'd encourage you to read "pH Effects on Electrospray Ionization Efficiency" in the Journal of the American Society for Mass Spectrometry· Go to the journal website and locate the paper with this title. I am not providing a link so that students develop the habit of searching themselves.

  • $\begingroup$ Probably worth adding that in negative mode Mass Spectrometer doesn't change ionization - it changes whether the negative ions are captured. $\endgroup$ – Stanislav Bashkyrtsev Jun 9 '20 at 21:36
  • $\begingroup$ Thanks! This was very clarifying on a few misconceptions that I had - particularly the whole charge of molecule. I also found the reference article! $\endgroup$ – CuriousTree Jun 18 '20 at 7:51

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