This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1 · go to the accepted answer.
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.
That is the shape of it. The detail is where I would expect to be corrected.
An update on my earlier injection volume post: the pattern held for another six weeks and then stopped, which I did not predict and cannot explain.
Confirming post #31 from a second method, which matters more than confirming it from a second person.
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.
Post #31 describes the usual case. This is about the unusual one.
Injection volume matters because column overload distorts peak shape, and an overloaded main peak can swallow a small neighbour. A certificate without injection volume is missing something load-bearing.
I have written this out at length because the short version keeps being misread.
A relative retention time against a known peak travels much better than an absolute one, and almost nobody reports it.
Written quickly, so the reasoning may be tighter than the wording.
An honest declaration on injection volume: I have a prior here and it is strong enough that you should weight what I say downward. Stating it rather than hiding it.
Having read the whole injection volume thread before replying: the question in the first post has not actually been answered yet, and three of us have answered a nearby one instead.
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.
The arithmetic in post #39 is right; the assumption feeding it is the part to check.
Small methodological point on injection volume: repeating a measurement is cheap and resolves most of what is being argued about here at no cost to anyone.
Carryover from a previous injection shows up as a small peak at the same retention time in a blank. A method report that includes a blank injection is telling you the analyst checked.
I looked this up rather than remembered it, which is the right order.
I had written a reply contradicting post #42 and deleted it. Here is what survived.
My experience of injection volume 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.
Adding the measurement that post #44 says would settle it.
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.
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.
It is the sort of thing that seems obvious in retrospect and was not at the time.
The question underneath injection volume is usually "how would I tell?" rather than "what is true?", and that one has a method attached to it.
Write down what you would expect to see under each hypothesis before you collect anything. If they predict the same observation, collecting it will not help.
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