Purity vs. peptide content.

Two numbers that get confused

HPLC purity compares the main peak with the other UV-absorbing peaks. It answers: of the peptide material in this vial, how much is the target sequence?

Peptide content, also called net peptide content, is the target peptide's share of the total weight of the powder. It answers: of everything in this vial, how much is peptide?

The two are measured in different ways and are rarely the same. For freeze-dried synthetic peptides, peptide content often lands somewhere between 60 and 90%.

What else is in the powder

• Counter-ions. Peptides are purified and freeze-dried as salts. Basic groups, such as the N-terminus and the side chains of arginine, lysine and histidine, carry a counter-ion, most often trifluoroacetate (TFA) or acetate. A peptide with more basic residues carries more of it.
• Water. Freeze-dried peptides pull moisture from the air, and some water stays bound even in a dry cake.
• Residual salts and solvents from synthesis and purification, in small amounts.

None of these absorb UV the way peptide bonds do, so HPLC purity can't see them.

How peptide content is measured

• Amino acid analysis. The peptide is broken down into its amino acids, which are then counted. It is the most direct method.
• Nitrogen analysis. The nitrogen in the sample is compared with the amount the sequence predicts.
• Quantitative HPLC or UV against a reference standard, or UV at 280 nm for sequences that contain tryptophan or tyrosine.

Why it matters at the bench

When you weigh out powder and dissolve it, the real peptide concentration is the weight times the peptide content. A stock made from 5 mg of powder at 75% peptide content holds 3.75 mg of peptide, and that 25% gap carries into every concentration you calculate from it.

Our solution calculator works from the mass you enter. If you know the peptide content, multiply the mass by it first.

A note on TFA

Trifluoroacetate is left over from cleavage and purification. At high enough levels it can affect some cell-based assays. If that matters for your work, check the counter-ion on the report, or look for the peptide as an acetate or hydrochloride salt.