Setup, so nobody has to ask: orforglipron · a C18 column · survodutide.
Both of these get recommended confidently by different people, which suggests neither is obviously right.
My constraints are cost, measurement resolution and how much handling I am prepared to do — in roughly that order.
What is the actual trade-off, and does it matter at the scale I am working at?
On the detail: reverse-phase HPLC is the workhorse for peptide purity work, but it is almost universally run under conditions that are not optimal for a peptide of this chain length.
Reverse-phase stationary phases use C18 or C8 chains bonded to silica, and the pore size of the silica matters more for a peptide of this chain length than the ligand length does.
Sample preparation is almost always under-appreciated — a reconstituted peptide in strong solvent will distort its own peak on the gradient.
Peptide impurities from solid-phase synthesis include deletion sequences, truncations from premature cleavage, racemised residues from epimerisation and oxidised variants, each of which may have different chromatographic behaviour.
If two labs give different numbers, the method difference is the first hypothesis, not lab quality.
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Browse resultsMechanically, the chemistry of peptides at low pH and high organic-solvent concentration is not something most users have intuition for, which is why published methods exist.
Trifluoroacetic acid at 0.1 per cent is the standard ion-pairing agent because it suppresses tailing by neutralising the basic residues, but it also suppresses mass spectrometry signal.
Wider-pore phases — 300 Angstrom rather than 100 Angstrom — have faster mass transfer and narrower peaks for peptides above three kilodaltons, which is almost every peptide you will see.
The practical summary: specify the method, run the same method on every sample you compare, and use orthogonal techniques to confirm the result.
edited 15 Apr 2025 by deamidation_watch — expanded the table to cover the lower concentration
On the detail: start by understanding what the detector is measuring and what that means about how the molecule needs to be prepared and handled before injection.
Temperature affects both the viscosity of the mobile phase and the dynamics of molecular interactions, and a method developed at 25 degrees and run at 40 degrees will not behave identically.
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.
One qualification: the limit of detection on a reversed-phase HPLC is set by the noise on the baseline, which for these molecules is usually in the range of a tenth of one per cent or less, and anything smaller is not reproducibly detectable.
Ask for the chromatogram and the system suitability data, not just the number.
Mechanically, high-performance liquid chromatography is a separation technique, not an identification technique, and it answers the question "how much of the detected signal is a single species" not "which species is it."
Detector linearity is why a heavily loaded peak can give false purity by compressing the main peak height while leaving impurity shoulders unchanged.
If two labs give different numbers, the method difference is the first hypothesis, not lab quality.
On the detail: coupling the chromatography to mass spectrometry adds identity information but trades chromatographic quality for ionisation efficiency.
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.
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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