This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Post #58 is the version of this I will quote in future. One addition.
Acetate content: counter-ion content. Trifluoroacetate or acetate from the salt form of the peptide. Affects mass calculations and should be stated on a complete certificate.
I looked this up rather than remembered it, which is the right order.
Where I part company with post #61, and it is a narrow parting.
The bit of racemisation that nobody enjoys is that the answer changes depending on what you are trying to decide with it. Say what the decision is and the thread will converge.
I have three months of notes on racemisation and the honest summary is that the trend is real and the week-to-week numbers are noise. I nearly drew the opposite conclusion from the first fortnight.
Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.
Reframing racemisation slightly, because I think the disagreement is about the question rather than the answer. If the question is "does it happen", yes. If it is "how often", nobody here knows.
I had written a reply contradicting post #64 and deleted it. Here is what survived.
The documentation on racemisation is better than this thread and I say that as someone who has posted in the thread.
Picking up post #66: that is the part I would want checked first.
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.
On reflection I would soften that slightly.
Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it.
A modest claim, modestly supported.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
Reading back through the racemisation threads from last year, the same three questions come up every time and only one of them has ever been answered properly. That seems like a documentation gap rather than a knowledge gap.
Off-target structures: if the sequence synthesis goes wrong, a completely different amino acid can be incorporated. The resulting off-target peptide is a structural isomer with the same mass but a different sequence. No chromatographic purity method detects this without a reference standard.
Seconded. It reads as careful rather than confident, which is the right register.
If someone has run racemisation properly I would rather read that than my own reconstruction of it. Posting mine only because the thread has gone quiet.
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.
Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage.
Not disagreeing with anyone above, just adding the bit I keep having to look up.
Adding the measurement that post #75 says would settle it.
The reason racemisation keeps being re-asked is that the answer is conditional and people quote it without the condition. It is not that the answer is unknown.
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.
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.
Genuinely open to being wrong about this one.
Confirming post #80 from a second method, which matters more than confirming it from a second person.
Something worth flagging about racemisation: the strongest-sounding claims in this thread are the ones with no source attached, which is the usual pattern and not a coincidence.
Racemisation has a well-known answer and a correct answer, and the interesting work is establishing that they are the same. Nobody has done that here yet.
That last point is the ceiling on what any purity figure can claim. A method that cannot see a species cannot exclude it, and no certificate says which species its method cannot see.
That holds under the stated conditions and I have stated them.
Post #82 describes the usual case. This is about the unusual one.
Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.
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.
The honest answer is that it depends, and here is what it depends on.
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.
That has held every time I have looked, which is not the same as always.
On post #86 — agreed on the reasoning, with one qualification.
My understanding of racemisation is a few years old and may have been superseded. If it has been, I would genuinely like to know rather than keep repeating it.
Freeze-drying itself does not generate impurities in any significant way. Handling before and after it does, which is why transit conditions belong in the discussion.
That holds for the case as described. Change the assumptions and it may not.
Truncation products are substantially lower in mass and usually well separated chromatographically, which makes them the easiest class to see and the least likely to be missed.
That is the version I use. It may not be the version that is correct.