Second pass at: Where impurities in solid-phase peptide synthesis come from posts 31–52

I read post #27 twice before replying, because I had assumed the opposite.

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.

That is a description of practice, not a recommendation of 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.

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.

Worth stating the null on impurities in solid-phase peptide synthesis before we explain it: the observation may be nothing. That possibility deserves a sentence and usually does not get one.

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 clearer than the version I had in my head. Thank you.

Offering a way to settle impurities in solid-phase peptide synthesis rather than another opinion about it. Two measurements, taken the same way, a fortnight apart. If the difference is within the noise, the question was not answerable at this precision.

Taking post #35 at face value and following it one step further.

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.

Written from notes rather than memory, which is why the numbers are specific.

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.

Confirming post #39 from a second method, which matters more than confirming it from a second person.

Reframing impurities in solid-phase peptide synthesis 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.

Taking post #40 at face value and following it one step further.

Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.

The short answer was in the first line; everything after is the working.

Post #38 and I disagree about the size of the effect, not about the direction.

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.

The most useful reply I ever got about impurities in solid-phase peptide synthesis was a request to state my units. It sounds like pedantry and it has saved me twice.

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.

This is where my knowledge stops and I would rather mark the edge than blur it.