Trifluoroacetate content and its consequences — setting out what I have, and where I think it stops being reliable.
What changes if the standard account of trifluoroacetate content is wrong? I ask because I have been treating it as settled and I noticed this week that I could not say why.
Working through the consequences rather than the evidence, since others here are better placed on the evidence.
On trifluoroacetate content, I would rather understate and be corrected upward than overstate and be quoted. That is a house style here and it is a good one.
I read post #3 twice before replying, because I had assumed the opposite.
Adding a small correction to the trifluoroacetate content summary above rather than a disagreement with it. The substance holds; one of the figures is out by a factor that matters.
Post #3 answers the question as asked. The question underneath it is different.
Trifluoroacetate content is a good example of a question where the honest answer is boring and the interesting answers are unsupported. I would go with boring.
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.
That is a description of practice, not a recommendation of it.
One caution on trifluoroacetate content: everything above assumes the underlying documentation is what it claims to be. That assumption is doing real work and is rarely stated.
Trifluoroacetate content: I have looked for the primary source twice and failed twice. Either it does not exist or it is somewhere I do not know to look, and I would like to know which.
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.
The thing about trifluoroacetate content that took me longest to accept is that a plausible mechanism is not evidence of an effect. It is a reason to look, not a result.
Narrowing post #12, because the general version has more than one answer.
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.
Everything in post #10 holds. The case it does not cover is the one I have.
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.
Post #14 put the caveat in the right place and I want to underline it.
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.
The arithmetic in post #16 is right; the assumption feeding it is the part to check.
Whatever the answer on trifluoroacetate content turns out to be, the method for getting there is the same: state the assumption, do the arithmetic in public, invite the correction.
Fine by me. I had wanted a stronger conclusion and there is not one available.
Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.
I am reporting what happened, not recommending it.
The related-substances total is only as informative as the method that generated it. Two per cent by a method that resolves everything is a different statement from two per cent by a method that resolves little.
One of those cases where knowing the mechanism does not help the decision.
Post #20 is the version of this I will quote in future. One addition.
The useful distinction on trifluoroacetate content is between what was measured and what was inferred from it. Both end up in the same sentence and only one of them has error bars.
What would change my mind on trifluoroacetate content is a second dataset collected by someone with no stake in the first. Until then I hold it loosely and I would rather say so than pretend to more.
Acknowledging rather than arguing. The reasoning holds as far as I can follow it.
I had written a reply contradicting post #23 and deleted it. Here is what survived.
A supplier that can tell you what its principal impurity is has answered a harder question than one that can tell you its purity, and the answer is more useful.
Filing this under things that are true until someone shows me otherwise.
Confirming post #23 from a second method, which matters more than confirming it from a second person.
I disagree with the framing of trifluoroacetate content above, and I think it is a substantive disagreement rather than a terminological one. Setting out why, so it can be checked.
The reasoning depends on an assumption that is doing a lot of work and is never stated. If the assumption holds, the conclusion follows. I do not think it holds generally.
Source for the trifluoroacetate content figure, since it was asked for. It is in the discussion rather than the abstract, which is why the version circulating is stronger than the paper is.
Reading the surrounding paragraph is worth the two minutes. The authors are more careful than their summarisers.
Following, with nothing to contribute beyond having asked the same thing elsewhere.