Revisiting: Aggregates and why a purity assay may miss them entirely posts 31–54

Answering the question post #27 raises rather than the one it answers.

Practical note on Aggregates: write down what you expect before you look. The number of times I have found what I went looking for is higher than chance would allow.

Storage-related degradation and synthesis-related impurity look different on a chromatogram. A growing oxidation peak over time is not a manufacturing finding.

A request rather than an answer: could whoever has the primary source for Aggregates post it? I have seen the claim three times this month and each version had lost a qualifier.

Everything in post #31 holds. The case it does not cover is the one I have.

Speaking only to Aggregates 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.

Narrowing post #35, because the general version has more than one answer.

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.

This has been discussed before and I could not find the thread, so, again.

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.

It is worth stating the boring hypothesis before the interesting one.

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.

Two claims get bundled together under Aggregates and they need separating. The descriptive one — this is what was observed — is usually well supported. The causal one — this is why — usually is not.

Almost every disagreement in threads like this one dissolves once you say which of the two you are making.

On Aggregates, the part that usually goes wrong is that the question is asked as though it has one answer. It has a range, and the width of the range is the interesting bit.

If you can post the two or three numbers you are working from, several people here will check the arithmetic rather than argue about the conclusion.

Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.

Nothing above should be read as advice about what anyone else should do.

I think the Aggregates question is answerable and has not been answered, which is a more optimistic position than most of this thread.

Fair, and the limits you put on it are the part I will remember.

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.