Purity Terminology: Purity, Content, Counterions and Impurities

Peptide certificates and catalogues use several related terms that are easy to mix up. Mixing them up leads to wrong concentrations and misplaced confidence. This guide defines each one.

The terms

Purity and content are different

Imagine 10 mg of powder with 98% HPLC purity, 75% net peptide content. It contains about 7.5 mg of peptide material, of which about 7.35 mg is the target sequence. The remaining 2.5 mg is counterion and water. If you assume 10 mg of peptide, your solution is about 25% weaker than you think.

Counterions

Synthetic peptides are usually purified by HPLC with trifluoroacetic acid (TFA) in the solvent, and positively charged residues leave the column paired with trifluoroacetate. The final powder is therefore a TFA salt unless it is exchanged for acetate or chloride. TFA is not inert in every experiment: Cornish and colleagues reported that trifluoroacetate inhibited proliferation of osteoblasts and chondrocytes in culture at concentrations that can be reached from peptide preparations. For sensitive cell work, consider asking for an acetate or HCl salt form.

Peptide-related impurities

D’Hondt and colleagues reviewed the impurities found in peptide medicines. The common types are:

• Deletion sequences: one residue missing, from incomplete coupling.
• Truncated sequences: chains that stopped growing.
• Incomplete deprotection: protecting groups left on side chains.
• Racemization: a residue in the wrong D/L configuration.
• Degradation products: oxidized, deamidated, isomerized or hydrolyzed forms that can also form during storage.

Deletion sequences can behave very similarly to the target on HPLC, so a high purity figure does not rule them out entirely.

Grade labels

Terms such as “research grade”, “high purity” or “>98%” are not standardized across suppliers. Treat them as marketing until a lot-specific COA with a chromatogram backs them up. Regulatory impurity thresholds, such as those in ICH Q3A, formally exclude peptides, but the vocabulary of reporting, identification and qualification thresholds is a useful reference.