What a reversed-phase purity number actually is posts 91–102

Practical experience of reversed-phase purity number, offered as one case with the conditions stated, not as a general finding. Conditions first, because they are what make it interpretable.

Before anything else: what was the gradient, and at what wavelength? Area percent at different wavelengths is not the same number even on the same sample because different species absorb differently at different wavelengths. With the method stated, I can tell you something useful. Without it, all I can say is that there is one large peak.

The reason reversed-phase purity number is hard to answer is that the obvious measurement and the relevant quantity are not the same thing, and substituting one for the other is silent.

This settles it for me, at least until somebody posts a reason it should not.

Confirming post #92 from a second method, which matters more than confirming it from a second person.

On integration: where the baseline is drawn matters more than most people realise. On a clean chromatogram with well-resolved peaks the choice is inconsequential. On a chromatogram with a trailing shoulder or a rising baseline it matters. Differences of one to two percentage points between defensible integrations are ordinary.

That is the shape of it. The detail is where I would expect to be corrected.

One caution on reversed-phase purity number: everything above assumes the underlying documentation is what it claims to be. That assumption is doing real work and is rarely stated.

Reversed-phase purity number is a good example of a question where the honest answer is boring and the interesting answers are unsupported. I would go with boring.

Reversed-phase purity number has a well-known answer and a correct answer, and the interesting work is establishing that they are the same. Nobody has done that here yet.

Everything in post #99 holds. The case it does not cover is the one I have.

Method validation is the demonstration that a method can separate the compound from its degradation products and impurities reliably. A method that cannot resolve an impurity from the parent peak will not detect that impurity.

Not a conclusion. A place to stand while looking for one.

Adding a small correction to the reversed-phase purity number summary above rather than a disagreement with it. The substance holds; one of the figures is out by a factor that matters.

Everything in post #100 holds. The case it does not cover is the one I have.

Reversed-phase separates on hydrophobicity. A peptide is retained on a non-polar stationary phase and eluted by increasing organic solvent. For peptides the mobile phase almost always contains an ion-pairing acid, typically 0.1% TFA, which suppresses secondary interactions and sharpens peaks.

That is what the documentation says. What happens in practice is usually close.