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

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.

Worth saying I have only my own numbers here, and n is small.

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.

I would want the raw data before agreeing with my own summary of it.

Post #14 put the caveat in the right place and I want to underline it.

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.

Building on post #17 rather than restating it.

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.

Take it as a starting point and not as a specification.

Answering the question post #17 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.

I would want a second opinion before relying on that.

The arithmetic in post #17 is right; the assumption feeding it is the part to check.

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.

Someone will know this better than I do and I hope they say so.

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 have separated what I observed from what I concluded, which does not always happen.

I had written a reply contradicting post #20 and deleted it. Here is what survived.

Careful with the language on impurities in solid-phase peptide synthesis. "Not detected" and "not present" are different findings and the first is a statement about the method.

That is the distinction I keep failing to hold on to. Written down now.

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.

That is the version I would defend. It is not the version I started with.

The practical version of impurities in solid-phase peptide synthesis is three sentences long. The rigorous version is three pages and reaches the same conclusion with the conditions attached.

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.

This follows post #26 rather than contradicting it.

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.

I would put this at better than even and not much better.

Worth separating two things that post #24 runs together.

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.

The conclusion is tentative; the arithmetic underneath it is not.

Building on post #26 rather than restating it.

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 have said this before in a thread nobody could find, so it is worth repeating.

Good question, well framed, and I would like to see it answered properly.