This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
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.
Bookmarking this. I will come back when I have something worth adding.
Answering the question post #31 raises rather than the one it answers.
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.
A weak preference rather than a position.
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.
I am aware this is the third time this month I have made this point.
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.
Everything in post #35 holds. The case it does not cover is the one I have.
The practical version of racemisation is three sentences long. The rigorous version is three pages and reaches the same conclusion with the conditions attached.
Narrowing post #35, because the general version has more than one answer.
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.
This is the version I would want a new member to read first.
What I would tell a new member reading about racemisation for the first time: the confident posts are not the reliable ones, and the reliable ones are longer.
This follows post #40 rather than contradicting it.
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.
For what it is worth, the same held on the two occasions I checked.
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.
I would not lead a decision with this, but I would not ignore it either.
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.
A guess, clearly labelled as one.
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.
Correct me on the arithmetic if it is wrong; I would rather know.
On post #43 — agreed on the reasoning, with one qualification.
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.
If the premise is wrong, everything after it is decoration.
Racemisation sits at the boundary between what this community can usefully discuss and what it cannot, and I think it falls on the discussable side, narrowly.
That is the distinction I keep failing to hold on to. Written down now.
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.
This is the sort of thing that ought to be settled and apparently is not.
Coming back to post #50, because the follow-up matters more than the original answer.
I read the earlier replies on racemisation twice before writing this, because I had assumed the opposite and wanted to be sure I was disagreeing with what was said rather than what I expected.
Post #50 is right about the mechanism and I think understates the practical bit.
The honest answer on racemisation is that it depends, and the useful part is the list of what it depends on. Four items, in rough order of how much they matter.
Most people get the first two right and then argue about the fourth.
Helpful, and short, which on this subject is harder than long.
Post #54 answers the question as asked. The question underneath it is different.
Worth stating the null on racemisation before we explain it: the observation may be nothing. That possibility deserves a sentence and usually does not get one.
Where the racemisation discussion usually stalls is that nobody wants to say "I do not know" and everyone is willing to say "it varies". Those are the same sentence with different clothes on.
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 is my reading. Someone else read the same page differently and was reasonable.
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.
Adding a source would improve this post and I do not have one to hand.
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.