This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
The arithmetic in post #30 is right; the assumption feeding it is the part to check.
Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.
Worth saying I have only my own numbers here, and n is small.
Noted, and thank you for writing it out rather than summarising it.
Truncation products: fragments from incomplete synthesis or from degradation. They elute quite differently from the intact peptide because they are much smaller and have different hydrophobicity. They are usually well separated.
Post #33 put the caveat in the right place and I want to underline it.
Where an impurity is identified rather than merely counted, the certificate is telling you the manufacturer has characterised its own process. That is a meaningful difference in documentation quality.
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.
The right answer here may simply be that it has not been measured.
Deamidation at asparagine or glutamine adds approximately one dalton and frequently produces a close-eluting pair. It is the impurity most likely to be integrated into the main peak by accident.
The short answer was in the first line; everything after is the working.
Taking post #35 at face value and following it one step further.
Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage.
Post #36 and I disagree about the size of the effect, not about the direction.
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.
Stating my assumptions rather than smuggling them in.
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.
Speaking for myself and not for anyone else who has posted here.
On post #36 — agreed on the reasoning, with one qualification.
Related substances: compounds chemically related to the target peptide but not the target peptide itself. The standard method separates them and reports them as area percent. How related they can be before they exceed specification is a regulatory question.
Answering the question post #37 raises rather than the one it answers.
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.
I have written this out at length because the short version keeps being misread.
Peptide impurities that differ by a single residue are the hardest to resolve and the most likely to be biologically relevant, which is an unfortunate combination.
Written quickly, so the reasoning may be tighter than the wording.
Truncation products: fragments from incomplete synthesis or from degradation. They elute quite differently from the intact peptide because they are much smaller and have different hydrophobicity. They are usually well separated.
Noting that the question and the thing people usually mean by it are different.
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.
Two people can read the same figure differently here and both be reasonable.
Deamidation at asparagine or glutamine adds approximately one dalton and frequently produces a close-eluting pair. It is the impurity most likely to be integrated into the main peak by accident.
One of those cases where knowing the mechanism does not help the decision.
Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it.
I would treat that as a working assumption and revisit it.
Quietly grateful for the plain phrasing. Not every thread gets that.
Taking post #45 at face value and following it one step further.
Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage.
Oxidation at methionine or tryptophan adds sixteen per oxygen and typically elutes earlier. In aged material it is the modification that grows.
Filing this under things that are true until someone shows me otherwise.
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.
Small point, but it is the one that usually catches people.
Taking post #51 at face value and following it one step further.
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.
Scavengers and cleavage-cocktail residues can persist and appear as small early-eluting peaks. They are process-related rather than sequence-related, which is a useful distinction when reading a profile.
Written from notes rather than memory, which is why the numbers are specific.
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.
I have kept the units in throughout, for the obvious reason.
This follows post #56 rather than contradicting it.
A supplier that can tell you what its principal impurity is has answered a harder question than one that can tell you its purity, and the answer is more useful.
Worth separating two things that post #54 runs together.
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.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
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.
It is worth stating the boring hypothesis before the interesting one.