A vial labelled "100 mg" contains 100 mg of lyophilized powder — not 100 mg of peptide. The peptide fraction of that powder is the net peptide content, and for a typical lyophilized preparation it is commonly 70-90%. The remainder is counter-ions, bound water and residual salts. This is normal chemistry, not short-weighting, and it is the single most misread number in peptide documentation.
Three numbers that are not the same thing
| Number | Question it answers | How it is determined | Typical value |
|---|---|---|---|
| Gross weight | How much powder is in the vial? | Gravimetric fill | As labelled, e.g. 100 mg |
| Chromatographic purity | Of the peptide present, what fraction is the target sequence? | HPLC, area % | 98-99.9% |
| Net peptide content | Of the total powder mass, what fraction is peptide? | Amino acid analysis or nitrogen determination | 70-90% |
A vial can legitimately be 99.5% pure by HPLC and 78% peptide by content at the same time. The two figures are measuring different things: purity is about which peptide, content is about how much.
Where the missing mass goes
Counter-ions
Synthetic peptides are purified by reversed-phase HPLC, usually with trifluoroacetic acid (TFA) in the mobile phase. Basic residues — lysine, arginine, histidine, and the N-terminal amine — end up paired with a counter-ion. The peptide is isolated as a salt, not as the free base.
- TFA salt: each TFA counter-ion adds 114 g/mol. A peptide with several basic sites can carry several, adding meaningfully to total mass.
- Acetate salt: each acetate adds 60 g/mol. Acetate is often preferred because it is lighter and less likely to interfere in downstream assays; converting from TFA to acetate is an additional processing step.
Bound water
Lyophilized peptides are hygroscopic. Even after secondary drying, residual moisture in the low single-digit percent range is standard, and the powder will pick up more from ambient air once the vial is opened.
Residual salts
Buffer components from the final purification and lyophilization steps can remain in small quantities.
Why it matters for calculations
If a working concentration needs to be accurate, the calculation runs on peptide mass, not powder mass:
peptide mass (mg) = gross vial weight (mg) × net peptide content (%)
A 100 mg vial at 80% net peptide content contains 80 mg of peptide. Reconstituted in 2 mL, that is 40 mg/mL of peptide — not 50 mg/mL. Using the gross figure overstates concentration by 25% in this example. For qualitative work the difference may not matter; for anything quantitative it does.
Reconstitution arithmetic is covered in the reconstitution guide.
How to read the label and the COA together
- Vial label: gives gross weight and compound name.
- COA, HPLC section: gives chromatographic purity and, ideally, the detection wavelength and method.
- COA, MS section: confirms identity by observed vs theoretical mass. Note whether the theoretical mass quoted is for the free base or the salt — they differ.
- COA, content section: gives net peptide content and salt form. If this section is absent, net peptide content has not been measured for that batch.
Full walkthrough: how to read a peptide COA. Method detail: what 99% purity actually means.
Questions worth asking a supplier
- Is the vial weight gross powder mass or net peptide mass?
- What salt form is the material supplied in — acetate or TFA?
- Is net peptide content reported for this batch, and by what method?
- Is the theoretical mass on the MS report for the free base or the salt?
A supplier who cannot answer these is selling on the label rather than on the analysis.
Summary
Gross weight, chromatographic purity and net peptide content are three independent figures. High purity does not imply high peptide content, and neither is inferable from the number printed on the vial. Batch documentation is published on every product page in the HolyPeps catalog.
All products are intended for laboratory and research use only. They are not medicines, are not for human or veterinary use, and are not for diagnostic or therapeutic application.