Second pass at: Where impurities in solid-phase peptide synthesis come from

Second pass at: Where impurities in solid-phase peptide synthesis come from Writing it up because I had to work it out twice and would rather nobody else did.

Impurities in solid-phase peptide synthesis, from the point of view of someone who has read the documentation and is still stuck.

Saying which parts I have understood so that nobody wastes time re-explaining them, and where exactly the understanding stops.

Fine by me. I had wanted a stronger conclusion and there is not one available.

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.

Taking the opening post at face value and following it one step further.

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.

I read the opening post 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.

Post #5 answers the question as asked. The question underneath it is different.

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.

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.

One of those cases where knowing the mechanism does not help the decision.

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.

I would treat that as a working assumption and revisit it.

Helpful, and short, which on this subject is harder than long.