Follow-up: Comparing two chromatograms from different laboratories, properly

Comparing two chromatograms from different laboratories, properly — setting out what I have, and where I think it stops being reliable.

I would like to understand what this number means before I repeat it anywhere.

A PeptideMeter report on a semaglutide lot gives 96.6% purity. The supplier certificate for the same lot states 98%. Both documents name a reversed-phase method; neither states the same gradient.

My question is not "who is right". It is: given that those two figures were produced by different methods, what is the largest difference I should expect from method alone, and at what point does a gap stop being explainable that way?

Column temperature affects retention and selectivity and is omitted from most certificates. Two runs at twenty-five and forty degrees are not the same method.

I would want to see it done twice before believing it once.

Post #7 answers the question as asked. The question underneath it is different.

Retention time is only comparable within a laboratory on a given method. Quoting a retention time across two reports as evidence of identity is not a valid comparison.

The short version is the first sentence; the rest is why.

Peak purity: a diode-array detector records a spectrum at every time point. If a peak contains two co-eluting species with different spectra, the spectrum changes across the peak. A passing peak-purity result says the spectrum is constant; it is weak evidence of homogeneity if the impurities have similar spectra.

This is the sort of thing that ought to be settled and apparently is not.

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.

Worth reading the earlier posts in this thread before acting on mine.

Post #37 put the caveat in the right place and I want to underline it.

Area percent is not mass percent. It is a proportion of absorbance, weighted by each species' extinction coefficient. For closely related impurities the approximation is usually good. For structurally dissimilar impurities it can be poor.

Confirming post #48 from a second method, which matters more than confirming it from a second person.

Particle size and column dimensions determine what resolution is achievable at all. A 5 micrometre 250 millimetre column and a sub-2 micrometre 100 millimetre column are different instruments in practice.

One case, stated as one case.

Post #50 and I disagree about the size of the effect, not about the direction.

Where the baseline is drawn is the least documented and most consequential choice in the whole determination. Valley-to-valley, tangent skim and forced-to-zero can span a couple of percentage points on the same chromatogram.

Area percent is a proportion of absorbance, not a proportion of mass. Two species with different extinction coefficients at the detection wavelength contribute unequally to the total, and nothing on the certificate corrects for that.