When a peptide supplier states a product is 99% pure, they are almost certainly citing the result of an HPLC analysis. This figure is widely used, frequently cited without context, and regularly misunderstood. Understanding precisely what HPLC measures — and what it does not — is foundational to evaluating quality claims in peptide research.
The principle behind HPLC
High-Performance Liquid Chromatography works by forcing a liquid sample through a column packed with a stationary phase material under high pressure. Different components of the mixture interact with the stationary phase to different degrees, causing them to travel through the column at different speeds and exit at different times. A detector — typically UV absorbance — records the signal as each component exits, producing a chromatogram: a graph of signal intensity plotted against time.
In reversed-phase HPLC (the most common configuration for peptide analysis), the stationary phase is hydrophobic (water-repelling) and the mobile phase is aqueous. More hydrophobic compounds interact more strongly with the stationary phase and elute later. Peptides are separated on this basis, with each component appearing as a peak at its characteristic retention time.
How purity is calculated from HPLC data
The purity percentage reported in a CoA is calculated from the peak area data in the chromatogram. Each peak corresponds to a component in the sample. The area under each peak is proportional to the quantity of that component. Purity = (target peak area ÷ total of all peak areas) × 100. A result of 99.2% means 99.2% of the total detected material elutes at the target retention time.
Several factors affect the accuracy of this calculation: the wavelength at which detection is performed (different compounds absorb UV light differently, so a UV-based detector may over- or undercount certain impurities relative to the target peptide); the gradient used to separate components; and whether a response factor correction has been applied. These are technical parameters that should be considered when comparing purity figures from different laboratories or methods.
What HPLC cannot tell you: identity
HPLC retention time is not a definitive identifier. Two different peptides can have similar or identical retention times under a given set of conditions. This means HPLC alone cannot confirm that the compound you have is the compound you ordered. This failure mode is rare but real — particularly with closely related peptide sequences that differ by only one or two amino acids.
Identity is confirmed through mass spectrometry (MS). Mass spectrometry measures the mass-to-charge ratio of ions generated from the sample. For a peptide, the measured molecular weight can be compared to the theoretical molecular weight calculated from its amino acid sequence. If the two match within acceptable tolerance (typically <0.5 Da for peptides under 3 kDa), identity is confirmed. If they do not match, the compound is not what it is claimed to be, regardless of HPLC purity.
HPLC vs mass spectrometry: a practical comparison
✓ Detects impurity peaks
✓ Separates structurally similar compounds
✓ Fast, high-throughput
✗ Cannot confirm molecular identity
✗ May miss UV-transparent impurities
✓ Measures molecular weight precisely
✓ Detects sequence errors
✓ Definitive identification tool
✗ Does not quantify relative purity
✗ More expensive per sample
What to look for in a CoA
A CoA that contains only an HPLC purity figure is incomplete from a quality documentation standpoint. Full documentation should include: the HPLC purity result with the analytical method specified (column type, mobile phase, gradient, detection wavelength); the mass spectrometry result with the observed molecular weight, the theoretical molecular weight, and the observed vs theoretical comparison; and the appearance of the product (white lyophilised powder is standard for correctly processed peptides).
Some CoAs also include water content (Karl Fischer titration) and residual solvent data. These are additional quality parameters rather than minimum requirements, but their presence indicates a more thorough analytical process.