# How do I explain the existence of these 2 peaks in this mass spectrometry?

Question about HRMS:Phosphatidylcholines and phosphatidylethanolamines are common types of glycerophospholipids found in biological membranes. Because of the high amount of heteroatom content, electron impact mass spectrometry is a bad idea because the ions fragment too fast. For biological molecules, electrospray ionization is the most common method. A compound in the glycerophospholipid class was analyzed by low-res electrospray ionization. It produced peaks: 773 Th(100 percent abundance), and 795 Th(about 20% abundance). The question says these are not isotope peaks. The same compound was analyzed by high-rest EI mass-spec to have peaks at 772.5856 Th and 794.5680 Th. The question says to explain the 2 peaks’ presences in the high-resolution mass spectrometry.

For question #6, I came up with the molecular formula for C40H80NO8P for both classes of lipids but I was unsure of how to use that to come up with the actual molecular formula. The first lipid I found was dipalmitoylphosphatidylechloline with a molar weight of 734. But it's smaller than either of the 2 fragments. Then I added 6 methylene groups to come up with a molecular formula of C46H92NO8P. But I'm not sure how to get the molecular formula for both fragments(or if we actually need them). Can you help with this question or let me know of what I'm missing?

• I don't understand... – Mohamed Sep 24 '19 at 18:28
• Never mind, I thought the mass was different by 2, but it is different by 22. – Karsten Theis Sep 24 '19 at 19:43
• So the second species could have two carbons more (+24) and two hydrogens less (-2). Making a fatty acid chain longer by two would add two carbon atoms and four hydrogen atoms. For each single bond turned into a double bond, you would get to remove two hydrogen atoms. – Karsten Theis Sep 24 '19 at 19:52
• But how do you figure out the molecular formula for the first and second species? – Mohamed Sep 25 '19 at 0:05
• You already had a reasonable molecular formula, C40H80NO8P, with a molar mass of 734 g/mol. It ionizes by adding an H+, so 735 g/mol. That is 38 g/mol off from the 773 g/mol you need. You mentioned methylene groups at 14 g/mol. Three of those (not six) brings us to an additional 42 g/mol (overshooting a bit), so make two C-C bonds into C=C double bonds to take away 4 g/mol worth of hydrogen and you are there. – Karsten Theis Sep 25 '19 at 1:41

In electrospray ionization, a molecule can end up with an extra proton or an extra cation. Either one of these +1 charged species is accelerated and detected in the mass spectrometer (negative charged particles or neutral species are not; if there are multiple cations, they would supposedly repel each other once the solvent is gone, and not fly together).

So a molecular formula that fits the data is $$\ce{C43H82NO8P}$$. The lighter species detected in the spectrometer would be $$\ce{C43H82NO8PH+}$$, and the heavier species would be $$\ce{C43H82NO8PNa+}$$. The masses given by https://www.sisweb.com/referenc/tools/exactmass.htm for these species match the given values pretty well. The difference in mass is not due to fragmentation, but to a cation piggy-backing with the molecule of interest (instead of the usual proton).

The data does not distinguish between phosphatidylcholines and phosphatidylethanolamines. You can, however, infer the number of C=C double bonds in the fatty acids attached to the glycerol.

I had to look up the unit "Th" on wikipedia:

The thomson (symbol: Th) is a unit that has appeared infrequently in scientific literature relating to the field of mass spectrometry as a unit of mass-to-charge ratio. The unit was proposed by Cooks and Rockwood naming it in honour of J. J. Thomson who measured the mass-to-charge ratio of electrons and ions.

HRMS stands for high resolution mass spectroscopy, I think.

• Hey, it would be great if you could answer this question: So you're saying that if I add an Na+ ion instead of H+ ion to the molecular formula of the first fragment, I can then get the molecular formula of the second fragment and that molecular formula for the second fragment would be the molecular formula for the whole compound? – Mohamed Sep 25 '19 at 3:54
• I edited my answer accordingly. The covalent compound is $\ce{C43H82NO8P}$ and it forms two distinct positively charged species in the gas phase, one with a covalently attached H+ and one with a sodium ion (not covalently bound). – Karsten Theis Sep 25 '19 at 9:49
• Why did you not use the values from the high-resolution mass spectroscopy? – Mohamed Sep 25 '19 at 20:02