Purity, net content, net peptide content and salt forms
A certificate can report 99% purity for a powder that is only about three-quarters the intended peptide by mass. Neither number is wrong. They answer different questions. This page separates three numbers that are easy to confuse, explains why a peptide powder is never all peptide, and works through the arithmetic with an invented example.
Three numbers, three questions
- Purity, measured by HPLC with UV detection and reported as area percent, asks: of the UV-absorbing material the method separated, what share is the main peak? HPLC and how to read a chromatogram covers it in detail.
- Net peptide content, reported as percent by mass, asks: of the powder’s total mass, what share is peptide at all, as opposed to counterion, water and residual solvent? It is usually measured by amino acid analysis, in which the peptide is broken down into its amino acids, each is measured, and the total is converted back into a mass of peptide. UV absorbance can be used for some sequences. Net peptide content counts all peptide material, closely related impurities included, so it is a separate measurement from purity.
- Net content, reported as a mass per container, asks: how much is in the vial? Labs and sellers use this term loosely. It can mean the mass of powder filled or the mass of peptide in it, so read the unit and the method. A figure measured against a reference standard of the peptide is a measured amount of peptide; a figure taken from the fill mass is not.
Why a peptide powder is not all peptide
Counterions. Peptides carry charged groups. The free amino end of the chain, and basic side chains, are positively charged under the acidic conditions used in synthesis and purification. Each positive charge pairs with a negatively charged partner, the counterion, so the dried peptide is a salt. The counterion reflects the acid present at the last purification or exchange step. Cleavage and purification commonly use trifluoroacetic acid (TFA), so many synthetic peptides are supplied as TFA salts; a separate exchange step can replace the trifluoroacetate with acetate or chloride. Whatever the counterion, it is part of the powder’s mass.
Water. Freeze-dried peptides are hygroscopic: they draw water from the air, and some remains after drying. See Why research peptides ship as a powder.
Residual solvent. Small traces of the solvents used in synthesis and purification can remain.
Together these commonly leave a freeze-dried peptide powder somewhere around 70 to 90 percent peptide by mass, according to peptide manufacturers’ published guidance. Where a particular powder falls depends on the sequence, the salt form and how it was purified and dried.
The arithmetic of a salt
Take an invented peptide, Peptide A, with a molar mass of about 1,800 g/mol as the free peptide and three positively charged groups: the amino end and two basic side chains. As a salt, each charge pairs with one counterion molecule. Counting each counterion as the whole acid molecule (its proton sits on the peptide), the molar mass of the salt is the peptide plus three acid molecules:
| Salt form | Acid molar mass (g/mol) | Salt molar mass (g/mol) | Peptide share of the salt |
|---|---|---|---|
| Trifluoroacetate (from trifluoroacetic acid, TFA) | 114.02 | 1,800 + 3 × 114.02 = 2,142.06 | 1,800 ÷ 2,142.06 = 84.0% |
| Acetate | 60.05 | 1,800 + 3 × 60.05 = 1,980.15 | 1,800 ÷ 1,980.15 = 90.9% |
| Hydrochloride | 36.46 | 1,800 + 3 × 36.46 = 1,909.38 | 1,800 ÷ 1,909.38 = 94.3% |
The salt form alone moves the peptide’s share by about ten percentage points. Nothing about the peptide itself changed; only its partner ion did. This is chemistry and mass, nothing more. Real material can also depart from the ideal ratio, for example when extra acid remains, which is why counterion content is measured directly when it matters, for example by ion chromatography.
Putting the numbers together
Now take 10.0 mg of Peptide A powder as the TFA salt, and suppose that, some time after drying, it holds 6.0% water by mass. All of these numbers are invented.
- Water: 6.0% of 10.0 mg is 0.60 mg, so the salt is the other 94.0%, or 9.40 mg.
- Peptide: 84.0% of the 9.40 mg of salt is 7.90 mg. That makes the net peptide content 7.90 ÷ 10.0, or 79.0%.
- Counterion: the rest of the salt, 9.40 − 7.90, is 1.50 mg of trifluoroacetate.
- Purity: the HPLC figure is 99.1% by area, so the intended peptide is about 99.1% of 7.90 mg, or 7.83 mg. Closely related peptide impurities make up the remaining 0.07 mg or so.
So one powder carries a purity of 99.1%, a net peptide content of 79.0%, and about 7.83 mg of the intended peptide in 10.0 mg of powder: roughly 78% of its mass. Had the same peptide been an acetate salt with the same water content, the net peptide content would be about 85.4%, and the intended peptide about 8.47 mg.
Multiplying net peptide content by area percent gives an estimate, not a measurement. It carries the same assumption as area percent itself: that the related impurities absorb UV light about as strongly as the main peptide.
Example
Text version of this figure
| Salt form | Peptide (mg) | Counterion (mg) | Water (mg) | Net peptide content |
|---|---|---|---|---|
| Trifluoroacetate (TFA) | 7.90 | 1.50 | 0.60 | 79.0% |
| Acetate | 8.54 | 0.86 | 0.60 | 85.4% |
What to look for in a lab report
- Purity with its method: HPLC-UV, the wavelength, and “area percent.”
- Net peptide content with its method, if it is reported at all.
- The salt form, and the counterion content if it was measured.
- Water content, if it was measured; Karl Fischer titration is the usual method.
- For any “content” figure, the unit, and whether it means powder or peptide.
If a certificate gives only a purity figure, the peptide content of the powder is not 99%. It is unknown.