Racemisation and why it is invisible to a mass measurement posts 91–120
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Noted, and thank you for writing it out rather than summarising it.
Post #89 and I disagree about the size of the effect, not about the direction.
Marking my uncertainty on racemisation explicitly. I am confident about the direction, much less confident about the size, and not confident at all that it generalises past the case in the first post.
Taking post #93 at face value and following it one step further.
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.
Speaking for myself and not for anyone else who has posted here.
My experience of racemisation contradicts the reply above. I am posting it as a data point rather than as a refutation, because one person's experience is exactly that.
Nobody has said the unglamorous part of racemisation yet, so: most of the variation is explained by things that are boring to write about and easy to check.
Worth separating two things that post #93 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.
Adding it in case it saves somebody the afternoon it cost me.
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.
Two sources, same conclusion, and I could not rule out that one copied the other.
The arithmetic in post #97 is right; the assumption feeding it is the part to check.
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 hold that lightly until someone with a larger sample weighs in.
Worth separating two things that post #97 runs together.
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 put the burden of proof on the interesting explanation, not the dull one.
This follows post #101 rather than contradicting 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.
Speaking only to racemisation as I have actually seen it, rather than as it is usually described: the effect is real, it is smaller than the thread suggests, and the variance between people is larger than the effect.
The useful distinction on racemisation 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.
Picking up post #104: that is the part I would want checked first.
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 to see it done twice before believing it once.
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.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
This is the sort of thing the wiki should carry and currently does not.
Before the thread moves on from racemisation — what is the sample size behind the claim? I am not being difficult; I have seen the same figure quoted from an n of four and from an n of four hundred.
Adding the measurement that post #108 says would settle it.
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.
I am not the right person to answer the follow-up to this.
Post #110 describes the usual case. This is about the unusual one.
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 mechanism is plausible, which is not the same as established.
Agreed on racemisation, with one qualification that I think matters. The reasoning holds for the case as described. Change the starting assumption and it does not, and the starting assumption is the part nobody states.
Answering the question post #114 raises rather than the one it answers.
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.
Agreed on all of that, and I have nothing to add to it.
I would call the community position on racemisation likely rather than established, and I would be comfortable defending that hedge.
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.