This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1 · go to the accepted answer.
An honest declaration on impurities in solid-phase peptide synthesis: I have a prior here and it is strong enough that you should weight what I say downward. Stating it rather than hiding it.
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.
It cost nothing to check and would have cost something not to.
I read post #30 twice before replying, because I had assumed the opposite.
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.
I will take the caveat as seriously as the claim, which is the point of putting it there.
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.
Post #36 is right about the mechanism and I think understates the practical bit.
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.
Coming back to post #34, because the follow-up matters more than the original answer.
I would keep impurities in solid-phase peptide synthesis and the decision it usually gets used for separate in this thread. They are related and they are not the same question, and merging them is why the last one went badly.
Building on post #36 rather than restating it.
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.
Post #38 put the caveat in the right place and I want to underline it.
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.
For what it is worth, the same held on the two occasions I checked.
Post #39 is right about the mechanism and I think understates the practical bit.
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.
I would be glad to be shown a cleaner way of putting this.
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.
This is the sort of thing the wiki should carry and currently does not.
Staff rationale: Hidden by community flags. The claim about a named supplier was not accompanied by a batch, a date, a method or a document, which R6 requires.
A methods point on impurities in solid-phase peptide synthesis rather than a substantive one: if the comparison is not like for like, the difference you are measuring is the difference in method.
The corresponding entry is in the public moderation log. Hidden posts are never deleted.
Post #43 and I disagree about the size of the effect, not about the direction.
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 confident version of this sentence would be wrong, so here is the hedged one.
Taking impurities in solid-phase peptide synthesis seriously for a moment rather than deflecting: the honest position is that the community has observations and no controlled comparison, and those two things support very different sentences.
Everything in post #47 holds. The case it does not cover is the one I have.
Summarising the impurities in solid-phase peptide synthesis 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.
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.
This follows post #50 rather than contradicting it.
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 part I am sure of is shorter than the part I have written.
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.
Saving this. It is the version I will quote when the question comes round again.
Picking up post #53: that is the part I would want checked first.
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.
Filing this under things that are true until someone shows me otherwise.
On post #52 — agreed on the reasoning, with one qualification.
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.
It took me longer than it should have to see that.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
The claim about impurities in solid-phase peptide synthesis upthread is stronger than its source supports. I have read the source. The source says "associated with" and the post says "causes".
What I would check first on impurities in solid-phase peptide synthesis is whether the thing being measured moved or whether the way of measuring it moved. Those look identical in a graph.
Post #56 describes the usual case. This is about the unusual one.
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.
Caveat: everything above assumes the paperwork is what it says it is.
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