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.
This follows post #3 rather than contradicting it.
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.
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.
Thank you for taking the time. That was more work than a reply usually is.
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.
Stating my assumptions rather than smuggling them in.
Post #12 answers the question as asked. The question underneath it is different.
I have been on both sides of the trifluoroacetate content argument in this category within eighteen months, which should tell you how strong the evidence for either side is.
Noted, and thank you for writing it out rather than summarising it.
I read post #15 twice before replying, because I had assumed the opposite.
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.
Post #16 is right about the mechanism and I think understates the practical bit.
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.
Sensible. I would want the same detail before I acted on it either.
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.
Picking up post #20: that is the part I would want checked first.
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.
Thank you — that answers what I came here to find out.
I changed my mind about trifluoroacetate content after someone here asked me for the source and I could not produce one. That is worth saying out loud because it is the ordinary way it happens.
I read post #24 twice before replying, because I had assumed the opposite.
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.
This follows post #24 rather than contradicting it.
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.
That matches what I have seen, for whatever a single anecdote is worth.
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.
Not a strong opinion, just a consistent one.