For reference: tirzepatide · a wide-pore C18 column · liraglutide.
I am trying to choose between two options that are usually discussed as though only one exists.
I am not optimising for price, but I am not indifferent to it either.
What does each option buy me, and what does it cost me?
The method is the measurement in reverse-phase chromatography, more so than in almost any other analytical domain, and two methods that look identical can easily produce different results.
Formic acid is the compromise when you need the mass spectrometer on the same run, but the peak shape penalty is real and easily a tenth of a per cent on purity.
On the detail: proline-rich sequences are particularly problematic because the isomerisation kinetics are in the same timescale as the separation, leading to split or broadened peaks at low temperature.
Peptide separation by reverse-phase high-performance liquid chromatography is described in the general chapters of the United States Pharmacopeia, European Pharmacopeia and Japanese Pharmacopeia, and the methods converge on essentially the same principles.
The caveat is that HPLC is a purity technique and says almost nothing about whether the main peak is actually your target compound — that is why identity confirmation from mass spectrometry or peptide mapping matters.
The practical summary: specify the method, run the same method on every sample you compare, and use orthogonal techniques to confirm the result.
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