Trifluoroacetate content and its consequences posts 121–135

Picking up post #120: that is the part I would want checked first.

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.

Trifluoroacetate content: I would want to see the raw numbers rather than the summary before agreeing. Summaries lose exactly the information that would settle this.

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.

If that is already documented somewhere, ignore me and link it.

The documentation on trifluoroacetate content is better than this thread and I say that as someone who has posted in the thread.

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.

My understanding of trifluoroacetate content is a few years old and may have been superseded. If it has been, I would genuinely like to know rather than keep repeating it.

Where I part company with post #126, and it is a narrow parting.

Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.

That is where I would start, not where I would stop.

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.

Happy to be the one who is wrong here if it settles the question.

Answering the question post #126 raises rather than the one it answers.

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.

Adding the caveat now so it does not have to be extracted later.

Trifluoroacetate is a counter-ion rather than an impurity, and it appears in the mass balance rather than in the chromatogram. Conflating the two accounts for several confused threads here.

For what it is worth, the same held on the two occasions I checked.

Thank you for the correction. I would rather find out here than later.

On post #129 — agreed on the reasoning, with one qualification.

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.

The failure mode on trifluoroacetate content is boring rather than dramatic. It is almost always the step everyone assumes was done correctly because it is too simple to get wrong.