How HPLC purity is calculated

"Purity by HPLC" is a specific calculation with specific assumptions, and knowing them lets you judge whether a stated figure is robust or flattering. This note walks through how a chromatogram becomes a percentage: integration, baseline, peak thresholds, and the two ways a lab can quietly inflate the number. It is written for researchers who read COAs rather than run instruments, but the instrument details are here for those who do. All Alphex products are for laboratory and in-vitro research only.

From injection to number

1. Separation. The dissolved sample is injected onto a reverse-phase column and eluted with a gradient from mostly water to mostly acetonitrile, both usually containing 0.1% TFA. Components leave the column in order of increasing hydrophobicity.
2. Detection. A UV detector at 220 nm records absorbance against time. Peptide bonds absorb here, so every peptide-like species produces a peak roughly proportional to the number of peptide bonds present.
3. Integration. Software draws a baseline and computes the area under each peak above it.
4. Calculation. Purity = area of the main peak divided by the sum of all peak areas, times 100. This is "area percent" or "area normalisation". It assumes every component has the same response per unit mass at 220 nm, which is approximately true for peptides of similar composition and false for non-peptide contaminants.

The assumptions, and where they bend

• Equal response. A deletion sequence with one fewer peptide bond responds slightly less than the target; a non-peptide impurity that does not absorb at 220 nm does not appear at all. So area percent slightly overstates purity relative to true mass fraction, and says nothing about salts, water or non-UV-absorbing organics.
• Baseline placement. A generously drawn baseline under a rising gradient can swallow small peaks. Good practice is a consistent, documented integration method.
• Peak threshold. Most methods ignore peaks below a set area or height (say 0.05% of the main peak) as noise. A threshold set high enough excludes real impurities. The COA should state it, or the trace should show that minor peaks were integrated.
• Co-elution. An impurity that elutes under the main peak is counted as target. Better methods (slower gradients, longer columns, orthogonal conditions) reveal it. A single perfect peak on a fast gradient is less reassuring than a slightly imperfect one on a slow gradient with resolved shoulders.
• Wavelength. 220 nm sees peptide bonds and is the standard. 280 nm sees only aromatic residues and would miss impurities without them; 214 nm is more sensitive but noisier. See 220 nm versus 280 nm.

How the number can be inflated

• Reporting at 280 nm for a peptide with a single aromatic residue, where most impurities are invisible.
• Using a steep, short gradient so everything co-elutes into one peak.
• Setting the integration threshold high, or excluding the solvent front region where polar fragments elute.
• Simply typing a number with no trace attached.

The defence against all four is the same: insist on the chromatogram, with method conditions and the integration table, and read it yourself. See what a chromatogram is and how to read one.

What Alphex reports

Alphex batches are analysed by reverse-phase HPLC at 220 nm against a reference standard, with the acceptance threshold set at 99+% area purity. The COA carries the trace, the integration result, the batch number and the issuing laboratory. Method background is in how Alphex tests every batch; how the figure relates to identity and net content is in what 99+% HPLC purity actually tells you.

Sources and further reading

• ICH Quality Guidelines (Q1A stability, Q3A/Q3B impurities, Q6 specifications): the reference framework for purity, impurity and stability testing

Alphex research peptides are supplied for laboratory and in-vitro research by qualified researchers only. They are not authorised for human or veterinary use.

Research use only. Alphex products are supplied exclusively for laboratory and in-vitro research by qualified researchers. They are not authorised for human or veterinary use, and nothing in this note is advice on any such use.

Published 1 May 2026, last reviewed 16 August 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.

Alphex Research Team

Alphex Limited, UK research peptide supplier

The Alphex research team writes and maintains these notes for UK laboratories buying research peptides. Every batch Alphex lists is verified by reverse-phase HPLC with a batch-matched Certificate of Analysis. Content covers laboratory and in-vitro research only.