Peptide content versus purity — the distinction most buyers get wrong
Purity is a ratio of areas. Content is a fraction of mass. A 99 % pure vial can contain 78 % peptide, and the difference is the whole ball game.
Content and mass
Amino-acid analysis for peptide content
Acid hydrolysis followed by chromatographic quantification of the liberated residues — the reference method for content where no standard of the intact peptide exists.
Amino-acid analysis is the answer to the problem that makes peptide content hard: quantifying a compound normally requires a certified reference standard of that compound, and for most research peptides no such standard exists. AAA sidesteps it by hydrolysing the peptide to its constituent amino acids and quantifying those instead, against standards that are cheap and universally available.
Procedure
- Weigh 50–200 µg of the article accurately into a hydrolysis vial. Add an internal standard not present in the sequence — norleucine and α-aminobutyric acid are the usual choices.
- Hydrolyse in 6 M hydrochloric acid containing 0.1 % phenol, under vacuum or nitrogen, at 110 °C for 24 h. Phenol protects tyrosine from chlorination.
- Remove the acid under vacuum, reconstitute in the loading buffer.
- Separate and quantify. Either post-column ninhydrin derivatisation on a cation-exchange system, or pre-column derivatisation with OPA, AQC or PITC followed by reverse-phase separation with fluorescence detection.
- Compute peptide content from the recovered molar amounts against the expected sequence stoichiometry.
Residues that do not survive
| Residue | Behaviour under 6 M HCl, 110 °C, 24 h | Handling |
|---|---|---|
| Trp | Destroyed | Separate alkaline or methanesulfonic-acid hydrolysis, or exclude from the calculation |
| Cys | Largely destroyed | Performic-acid oxidation to cysteic acid before hydrolysis |
| Met | Partially oxidised | Report with a caveat, or oxidise deliberately and quantify the sulfone |
| Asn, Gln | Deamidated to Asp, Glu | Quantified as Asx and Glx — the pair, not the individual residues |
| Ser, Thr | Partial loss (5–10 %) | Correction factor from a control hydrolysis |
| Pro | Poor ninhydrin response | Detection at 440 nm rather than 570 nm |
Worked calculation
Peptide: 15 residues, M = 1 418.71 Da (free base) Expected composition includes 3 × Gly, 2 × Pro, 1 × Val Sample weighed: 142.6 µg Recovered Gly: 0.2874 µmol Expected Gly per mole of peptide: 3 moles of peptide = 0.2874 / 3 = 0.0958 µmol mass of peptide = 0.0958 µmol × 1 418.71 g/mol = 135.9 µg peptide content = 135.9 / 142.6 × 100 = 95.3 %w/w ← total peptide, salt-free basis Cross-check against Val: 0.0952 µmol / 1 = 0.0952 µmol (−0.6 % relative) ✓ Cross-check against Pro: 0.1889 µmol / 2 = 0.0945 µmol (−1.4 % relative) ✓ Agreement across three residues within 1.5 % relative supports the figure. A single-residue determination should never be reported alone.
Normative references
Cited because they are the documents the acceptance criteria above are taken from. The index applies them as written and states every deviation.
- Ph. Eur. 2.2.56 — Amino acid analysis
- USP <1052> — Biotechnology-derived articles: amino acid analysis
Related methods
Reference standards, and what "area percent" means without one
The difference between an assay and an area percent, why most research peptides have no available standard, and what that costs the numbers on this site.
Quality system
Water content by Karl Fischer titration
Coulometric KF on lyophilised peptides: sample handling, the two things that ruin the measurement, and what a high result actually tells you.
Content and mass