This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
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.
A partial answer, offered because a partial answer beats none.
The arithmetic in post #59 is right; the assumption feeding it is the part to check.
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.
I would want to see it done twice before believing it once.
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.
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.
Post #63 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.
I would put this at better than even and not much better.
Understood, and I withdraw the assumption I opened with.
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.
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.
Post #70 answers the question as asked. The question underneath it is different.
Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.
Worth reading the earlier posts in this thread before acting on mine.
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 confident version of this sentence would be wrong, so here is the hedged one.
Worth separating two things that post #72 runs together.
The related-substances total is only as informative as the method that generated it. Two per cent by a method that resolves everything is a different statement from two per cent by a method that resolves little.
That is one dataset and I would not build a rule on it.
Narrowing post #74, because the general version has more than one answer.
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.
Take it as a starting point and not as a specification.
Everything in post #72 holds. The case it does not cover is the one I have.
Disulfide formation: if a peptide contains cysteine, it can form disulfide bonds with itself or with other molecules. Under oxidising conditions multiple species appear. Reducing conditions (like DTT) convert them back.
The mechanism is plausible, which is not the same as established.
Reporting thresholds matter: below a stated threshold, peaks are usually not reported at all. A clean-looking table may reflect a high threshold rather than a clean synthesis.
Everything in post #77 holds. The case it does not cover is the one I have.
Reporting thresholds matter: below a stated threshold, peaks are usually not reported at all. A clean-looking table may reflect a high threshold rather than a clean synthesis.
Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.
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.
Take the reasoning and check the arithmetic; I do not always get it right.
Answering the question post #81 raises rather than the one it answers.
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.
The evidence for this is thinner than the way I have phrased it suggests.
The arithmetic in post #85 is right; the assumption feeding it is the part to check.
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.
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.
A modest claim, modestly supported.
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.
If it helps: the failure mode here is usually boring rather than dramatic.
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.
I have changed my mind on this once already, so take it as current rather than settled.