A vial described as 99 percent pure can be roughly 75 percent peptide by mass. Both figures are accurate. They measure different things, and almost no supplier explains the gap.
The two measurements
HPLC purity is a proportion of detected material. The sample is separated on a column, a detector records what emerges, and the purity figure is the area under the main peak as a percentage of total integrated peak area.
Net peptide content is a proportion of mass. It is what fraction of the powder in the vial is actually peptide.
The first is about composition of the peptide fraction. The second is about the contents of the vial. They are not interchangeable and one does not imply the other.
Where the rest of the mass goes
Three things, none of which appear in an HPLC purity figure.
Water
Peptides are hygroscopic. Lyophilised material retains bound water and continues to take on more from the air once opened. Five to ten percent by mass is normal.
Counterion
The largest contributor and the one most often overlooked. Peptides are purified by reverse phase HPLC using trifluoroacetic acid, and the peptide comes off as a TFA salt. Depending on how many basic residues the sequence contains, TFA can be ten to twenty percent of the mass.
Acetate salt forms exist and carry less mass, which is one reason the salt form is worth knowing.
Residual solvent
Small quantities remaining from synthesis and purification. Usually a minor contributor but not zero.
Doing the arithmetic
Take a 10mg vial at 99 percent HPLC purity with a net peptide content of 78 percent.
You have 10mg of powder. About 7.8mg of that is peptide. Of that peptide fraction, 99 percent is the target compound, so roughly 7.7mg is what you intended to buy.
Not 9.9mg. The difference is not a defect and nobody has misled you, but if your calculation assumed 10mg you are running about 23 percent below your intended concentration.
When it matters
It matters when your work depends on accurate mass. Dose response curves, binding affinity determinations, anything where the concentration is the measurement rather than a means to it. Getting this wrong shifts an entire curve.
It matters when comparing across suppliers. Two vials both labelled 10mg at 99 percent can contain measurably different quantities of peptide. A price comparison on the label figure is not a like for like comparison.
It matters less for qualitative work. If you are asking whether a compound produces an effect at all, a 20 percent discrepancy rarely changes the answer.
How it is measured
Usually by amino acid analysis: the peptide is hydrolysed into its constituent amino acids, which are quantified against standards. Nitrogen determination is an alternative.
Both are more involved than running an HPLC trace, which is why net peptide content is supplied less often. It is a real analytical cost rather than an omission.
What to ask
Three questions:
- Is net peptide content available for this batch?
- What is the counterion, TFA or acetate?
- Was the HPLC purity figure measured at 214 nanometres?
A supplier who can answer all three is running real analysis. One who does not recognise the first question is passing on someone else’s paperwork.
Working around it
If net peptide content is unavailable and your work needs accurate mass, the options are to have it measured independently, to quantify your reconstituted solution directly by UV absorbance where the sequence contains a suitable chromophore, or to design around it with a standard curve rather than an absolute concentration.
None of those are free. The point of knowing about the gap is being able to decide whether it matters for what you are doing, rather than discovering it after the fact.
Related reading
The purity guide covers what an HPLC figure includes and excludes. The certificate guide covers where each of these appears on the document.