Calibration matters: a high-resolution instrument out of calibration can report mass with ppm error large enough to be uninformative. Check when the instrument was last calibrated before trusting the reported accuracy. Nothing above should be read as advice about what anyone else should do.
On the subject in the title: Counter-ion content and its effect on measured mass Working notes rather than a conclusion.
Something about counter-ion content does not reconcile and I would like a second pair of eyes before I decide which half is wrong.
Two sources, both reputable, giving figures that cannot both be right unless they are measuring different quantities. My suspicion is that they are, and I cannot see how.
The opening post put the caveat in the right place and I want to underline it.
For a compound with no reference standard in circulation, an observed mass is much more useful than an assertion of agreement with a theoretical value nobody can check.
Quantitation by MS: most quantitation is done by LC-UV detection at 214 nm, not by MS, because extinction coefficients are better known. MS can quantify if an internal standard is used but that requires preparation.
Genuinely open to being wrong about this one.
Post #2 is the version of this I will quote in future. One addition.
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
If this contradicts something upthread, the upthread version may well be the better one.
The honest summary of what a mass result buys you: it narrows the field of what the material could be, considerably. It never closes it, and no certificate should be read as though it had.
The literature is thinner on this than the confidence in the thread implies.
I came in to disagree and I am leaving without a disagreement.
Post #4 describes the usual case. This is about the unusual one.
Offering a way to settle counter-ion content rather than another opinion about it. Two measurements, taken the same way, a fortnight apart. If the difference is within the noise, the question was not answerable at this precision.
Common adducts: sodium adds ≈22, potassium adds ≈38 compared to hydrogen. A [M+Na]+ peak is common and its mass is predictable from the base mass.
That is all I can say without guessing.
Calibration matters: a high-resolution instrument out of calibration can report mass with ppm error large enough to be uninformative. Check when the instrument was last calibrated before trusting the reported accuracy.
Nothing above should be read as advice about what anyone else should do.
What mass accuracy establishes: the measured mass is consistent with a specific composition. What it does not establish: purity, sequence order, stereochemistry, or the absence of an isobaric species. Every one of those requires something else.
Confirming post #10 from a second method, which matters more than confirming it from a second person.
Mass accuracy is expressed in parts per million. It is the difference between observed and theoretical mass divided by theoretical mass, multiplied by a million. A high-resolution instrument in good calibration achieves low single-digit ppm on a peptide of this size.
Post #12 describes the usual case. This is about the unusual one.
Tandem mass spectrometry: MS/MS fragments the molecular ion and uses fragment masses to confirm identity and detect modifications. A simple identity confirmation by LC-MS does not address post-translational modifications or impurities with the same or very close mass.
The general answer and the answer for your case may diverge here.
Purity and identity are different questions: LC-MS establishes that the species at a retention time has the expected mass. It does not establish how much of the sample is that species (that is what LC-UV purity answers).
Adding this to the thread rather than to the wiki, because I am not confident enough for the wiki.
Mass error in parts per million is (observed minus theoretical) divided by theoretical, times a million. On a high-resolution instrument a low single-digit figure is unremarkable and expected.
The short version is the first sentence; the rest is why.
A mass match establishes that the measured mass is consistent with the proposed composition. It does not establish purity, sequence order, or the absence of an isomer, and all three are frequently claimed from it.
Small methodological point on counter-ion content: repeating a measurement is cheap and resolves most of what is being argued about here at no cost to anyone.
Resolution and mass accuracy are different specifications. An instrument can resolve two species and still assign their masses imprecisely, and the reverse is also possible.
This is the sort of thing the wiki should carry and currently does not.
Taking post #20 at face value and following it one step further.
Positional isomers and epimers are mass-identical. Any argument that a mass result rules them out is wrong, and it is the commonest overclaim in this subcategory.
Anyone with a larger sample, please post it.
Monoisotopic and average mass are different numbers and both appear on documents. At this molecular weight the difference is a few daltons, which is more than the tolerance being claimed.
This is where my knowledge stops and I would rather mark the edge than blur it.
I read post #22 twice before replying, because I had assumed the opposite.
Tandem mass spectrometry with fragmentation gives sequence information that intact mass cannot. It is the analysis that would actually confirm identity, and it is rarely supplied.
That is all the detail I have. Someone else will have more.
Adducts — sodium and potassium especially — shift the observed mass in predictable increments. A report showing an unexplained plus twenty-two is usually showing you a sodium adduct.
Someone will know this better than I do and I hope they say so.
Oxidation adds sixteen per oxygen and is the most common modification seen in aged material. Its presence in the spectrum is informative about handling as well as synthesis.
That is a cleaner way of putting what I was circling around.
Coming back to post #26, because the follow-up matters more than the original answer.
For anyone finding this later: the short answer on counter-ion content is that it depends on one thing, and the rest of the thread is people identifying which thing.
I disagree with the framing of counter-ion content above, and I think it is a substantive disagreement rather than a terminological one. Setting out why, so it can be checked.
The reasoning depends on an assumption that is doing a lot of work and is never stated. If the assumption holds, the conclusion follows. I do not think it holds generally.
Where I part company with post #26, and it is a narrow parting.
Deamidation adds approximately one dalton and produces a species that frequently elutes very close to the parent. It is the hardest common impurity to see chromatographically and the easiest to see by mass.