Reference
What an HPLC purity figure actually measures
“99% pure” is a narrower claim than it sounds. It is a statement about relative peak areas under one detection method — not about how much of the vial is peptide.
How the number is produced
Peptide purity is normally measured by reversed-phase high performance liquid chromatography. The sample is dissolved and pushed through a column that separates its components by how strongly each one sticks to the column packing. Components emerge at different times, and a detector records each as a peak.
The purity figure is then calculated by area normalisation: the area of the target peptide’s peak, divided by the total area of all the peaks, expressed as a percentage. A result of 99% means the main peak accounted for 99% of the total peak area the detector recorded.
The three words that do the work
Of the total peak area. Anything the detector does not register is not in the denominator, and so cannot reduce the percentage. This is where the gap between the number and the intuition opens up.
Peptide detection is usually at around 214 nanometres, where the peptide bond itself absorbs, or sometimes 280nm, where aromatic residues do. A substance that does not absorb at the wavelength being used is invisible to the measurement. It is present in the vial and absent from the arithmetic.
What a purity figure therefore does not cover
- Counterion salt. Synthetic peptides are usually isolated as a salt — commonly trifluoroacetate or acetate. That salt is real mass in the vial and does not appear as a chromatographic peak.
- Residual water. Freeze-dried powder retains some moisture. Also real mass, also invisible here.
- Anything non-absorbing at the detection wavelength.
- Sterility and endotoxin. Different assays entirely.
- Identity. Purity says the sample was mostly one substance. It does not say which substance. That is what mass spectrometry is for.
Purity and peptide content are different numbers
This is the practical consequence, and it is the reason the two fields are worth reading together:
- Purity answers: of the peptide-like material present, how much was the target?
- Net peptide content answers: of the powder weighed into the vial, how much is peptide at all?
A sample can be 99% pure by HPLC and still be substantially less than 99% peptide by mass, because the salt and water were never in the calculation. Both statements are true at once and neither is a trick — they measure different things. A report giving only the first is not wrong, but it is answering the easier question.
What a good result looks like
A purity figure produced by a named method, a mass spectrometry result confirming the molecular weight matches the sequence, and ideally a net peptide content figure. Those three together describe what is in the vial. Any one of them alone describes part of it.
The reports we publish are listed on the reference index, and each one can be opened on the issuing laboratory’s own site rather than only on ours.
