What "99% purity" does and does not tell you
Everyone in this industry prints the same number. Very few explain what it is measuring — and the thing most buyers assume it means is not what it means.
We are going to argue against our own marketing for a moment, because you are better served knowing this than not.
How the number is produced
Peptide purity is determined by reverse-phase high-performance liquid chromatography. A small amount of the sample is dissolved and pushed through a column packed with a hydrophobic stationary phase, while the solvent composition is gradually changed. Molecules exit the column at different times depending on how strongly they interact with the packing. A detector at the far end — typically ultraviolet absorbance at 220 nm, where the peptide bond itself absorbs — records what comes off and when.
The output is a chromatogram: a baseline with peaks on it. Purity is calculated as:
purity % = (area of the main peak ÷ total area of all peaks) × 100
So "99.4% purity" means the main peak accounted for 99.4% of the total absorbance area detected. That is a genuinely useful number. It is also a narrower claim than it sounds.
Four things the purity figure does not tell you
1. It is not the percentage of peptide by mass
This is the big one. A vial that is 99% pure by HPLC is not 99% peptide by weight. Lyophilised synthetic peptides also contain:
- Counter-ions — usually trifluoroacetate (TFA) or acetate left from purification, bound to basic residues. Depending on sequence this can be anywhere from a few percent to well over 20% of the dry mass.
- Residual water — lyophilised cakes are hygroscopic and typically retain several percent.
- Excipients, where a bulking agent such as mannitol has been used.
The measurement that answers "how much peptide is actually in this vial" is called peptide content, determined by amino acid analysis or quantitative nitrogen determination. It is a different test and it is not what a purity figure reports. This is completely normal and expected across the entire peptide industry — but it means a 5 mg vial contains 5 mg of lyophilised material, and the peptide fraction of that is a separate question.
2. It only counts what the detector can see
UV detection at 220 nm sees the peptide bond. Anything present that does not absorb meaningfully at that wavelength — inorganic salts, many solvent residues — contributes no peak and therefore does not appear in the denominator at all. It is invisible to the calculation rather than counted as an impurity.
3. It cannot see what co-elutes
If an impurity happens to interact with the column almost identically to the target peptide, it comes off at the same time and is counted inside the main peak. Closely related impurities are exactly the ones most likely to do this — a deamidated variant, or a diastereomer from partial racemisation, may differ from the target by a hydrogen atom's worth of chemistry and sit right on top of it.
This is why a single chromatogram at a single wavelength on a single method is weaker evidence than it appears, and why identity confirmation by a second, orthogonal technique matters.
4. It says nothing about identity
Here is the point that ought to change how you read a certificate. A 99.8% pure sample of entirely the wrong molecule will produce a beautiful chromatogram. Purity measures homogeneity, not correctness. On its own it tells you the contents of the vial are consistent, not that they are what the label says.
Which is why mass spectrometry is the more important line
Mass spectrometry ionises the sample and measures mass-to-charge ratio, giving the molecular weight of what is actually present. Compared against the theoretical mass calculated from the intended amino acid sequence, it answers the question purity cannot: is this the right molecule?
It also catches specific failure modes that HPLC may hide — a deletion sequence missing one residue shows up as a mass shift, as does incomplete removal of a protecting group.
If you are comparing suppliers, our honest advice is to weight the presence and quality of the mass spec confirmation more heavily than whether the purity figure reads 98.6% or 99.4%. The difference between those two numbers is far less consequential than whether anyone confirmed the identity at all.
Reading a chromatogram, briefly
- One dominant, symmetrical peak is what you want. Pronounced tailing can indicate secondary interactions or column overload.
- Shoulders on the main peak suggest a closely related impurity that is partly co-eluting — worth asking about.
- A cluster of small peaks just before the main one is a common signature of deletion sequences, which are usually slightly more polar than the full-length peptide.
- A flat, low baseline matters. A noisy or drifting baseline makes integration unreliable and can inflate the reported purity.
- Check the run time. A gradient that ends early can leave strongly retained impurities on the column, where they never get counted.
What we do, and what we would say if we were you
Every batch we release is tested by RP-HPLC at 220 nm, confirmed by mass spectrometry against theoretical mass, and screened for heavy metals. Certificates are available before you order and again against your specific batch number after you receive it — that is what batch lookup is for.
What we would tell you if you were a colleague rather than a customer: treat a purity figure with no supporting data as marketing, from anyone, including us. Ask for the chromatogram. Ask for the mass spec. A supplier who is comfortable handing those over is telling you something real; a number on a website is not.
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