Posting this under the heading it deserves: Racemisation and why it is invisible to a mass measurement Everything below is what sits behind that.
A question about racemisation that I think has a definite answer, unlike most of what I ask here.
I have the reasoning below and I am fairly confident about the direction. I am not confident about the size, and the size is what the decision turns on.
Deletion sequences arise from incomplete coupling and are lower by one residue mass. Where they elute depends on the hydrophobicity of the residue that is missing, so they can appear on either side of the main peak.
Everything in post #2 holds. The case it does not cover is the one I have.
Incomplete deprotection leaves a protecting group attached, raising the mass substantially and usually pushing retention much later. A late-eluting peak on a peptide chromatogram is worth asking about.
That is all the detail I have. Someone else will have more.
The most useful reply I ever got about racemisation was a request to state my units. It sounds like pedantry and it has saved me twice.
Post #4 answers the question as asked. The question underneath it is different.
What I can speak to on racemisation is narrow, so I will keep it narrow rather than generalising from it. Beyond that boundary I do not know.
Storage-related degradation and synthesis-related impurity look different on a chromatogram. A growing oxidation peak over time is not a manufacturing finding.
Worth checking against a second source before it gets quoted onward.
Worth separating two things that post #7 runs together.
Summarising the racemisation thread so far, since it is long and the answer is buried: the first reply has the method, the fourth has the correction to it, and the rest is people agreeing at length.
Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis.
Building on post #9 rather than restating it.
Relative response factors mean impurities are not detected in proportion to how much of them is present. A one per cent peak is not one per cent by mass unless the response factors happen to match.
That is the version I would defend. It is not the version I started with.
Post #13 and I disagree about the size of the effect, not about the direction.
Aggregates may be a multiple of the monomer mass and may not elute at all under a standard method. What does not come off the column does not appear in the area percentage.
Reporting the observation and leaving the explanation open deliberately.
An impurity profile that changes between lots is more informative than the total. A stable profile suggests a controlled process; a shifting one suggests something is varying.
I would treat the number as indicative rather than as a measurement.
Careful with the language on racemisation. "Not detected" and "not present" are different findings and the first is a statement about the method.
Racemisation: I have looked for the primary source twice and failed twice. Either it does not exist or it is somewhere I do not know to look, and I would like to know which.
The question underneath racemisation is usually "how would I tell?" rather than "what is true?", and that one has a method attached to it.
Write down what you would expect to see under each hypothesis before you collect anything. If they predict the same observation, collecting it will not help.
Narrowing post #22, because the general version has more than one answer.
Dimer and higher-order multimers: two or more peptide molecules bonded together. They appear at double the mass and higher. They may or may not separate from the monomer on HPLC depending on the method.
The disagreement above is smaller than it looks once the terms are fixed.
Everything in post #20 holds. The case it does not cover is the one I have.
On racemisation: the maintained page in the documentation commons covers the general case with citations and a review date, which is more reliable than any reply here including this one.
Adding the boring version of racemisation, because the interesting version keeps getting posted and the boring one is usually right.
Check the ordinary explanations, in order, and stop when one of them accounts for what you are seeing. Most of the time the second one does.
Worth separating two things that post #24 runs together.
The honest reading of a purity figure: it is an upper bound on how much of what the method could see was the intended species, under one integration convention, on one sample.
Flagging that the sources on this are thinner than the confidence in the thread suggests.
Sensible. I would want the same detail before I acted on it either.
Racemisation produces a diastereomer that is mass-identical and chromatographically resolvable only on a method chosen for the purpose. Standard reversed-phase frequently will not separate it.
The strength of my opinion here exceeds the strength of my evidence.
Post #26 is the version of this I will quote in future. One addition.
Trifluoroacetate is a counter-ion rather than an impurity, and it appears in the mass balance rather than in the chromatogram. Conflating the two accounts for several confused threads here.
If this contradicts something upthread, the upthread version may well be the better one.
Where I part company with post #28, and it is a narrow parting.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
The literature is thinner on this than the confidence in the thread implies.