A purity figure on a certificate of analysis is a summary of a chromatogram. Being able to read the chromatogram itself tells you far more than the number alone, including whether you should trust that number.
The set-up
Most peptide purity testing uses reverse-phase HPLC. The column is packed with silica particles coated in long hydrocarbon chains, typically C18. The mobile phase starts mostly as water and gradually shifts towards an organic solvent, usually acetonitrile. Both typically contain about 0.1% trifluoroacetic acid to keep peaks sharp. More hydrophobic molecules stick to the column longer and come off later in this gradient.
Reading the axes
- X-axis: retention time, in minutes. This is when each component reached the detector.
- Y-axis: detector response, usually UV absorbance in milli-absorbance units (mAU), measured at about 214–220 nm.
What you'll typically see
- The solvent front: a disturbance near the start where unretained material comes through. It isn't counted in purity.
- The main peak: the target peptide, ideally tall, narrow and symmetrical.
- Impurity peaks: smaller peaks either side. Deletion sequences often elute close to the main peak, and more hydrophobic by-products elute later.
- Baseline drift: a gentle rise as the organic content increases. It's normal, but it has to be handled carefully when peaks are integrated.
How purity is calculated
Software integrates each peak, measuring the area between the peak and the baseline. Purity is the main peak's area as a percentage of the total area of all integrated peaks:
Purity (%) = main peak area ÷ total peak area × 100
This is an area percent measure. It assumes every component absorbs UV about equally, which is a reasonable approximation for closely related peptide impurities but not an exact one.
Things that affect the number
| Issue | What it looks like | Why it matters |
|---|---|---|
| Co-elution | A shoulder on the main peak, or none at all | An impurity hidden under the main peak inflates the purity figure |
| Integration settings | Small peaks below a threshold are ignored | Excluding small peaks raises the reported purity |
| Peak tailing | A main peak with a long trailing edge | Can hide late-eluting impurities and suggests column or method problems |
| Gradient too steep | Peaks bunched together | Poor separation; impurities can merge with the main peak |
| Short run time | The trace stops soon after the main peak | Late-eluting impurities may never be seen |
What good practice looks like
- A blank injection run alongside, so system peaks aren't mistaken for impurities.
- A clear statement of the column, gradient, wavelength and run time.
- The full chromatogram shown, not just a table of numbers.
- Ideally, confirmation by an orthogonal method, such as LC-MS or a second HPLC method with different selectivity.
Purity tells you how clean a sample is. Identity is a separate question, answered by mass spectrometry. See Mass spectrometry explained.
Related articles
- How laboratories validate analytical methods
- D-amino acids and peptidomimetics
- Why some peptides are hard to make
Sources and further reading
- Mant CT et al. HPLC analysis and purification of peptides. Methods Mol Biol 2007;386:3–55. doi:10.1007/978-1-59745-430-8_1 · PMID: 18604941
- International Council for Harmonisation. Quality guidelines, including Q2(R2) Validation of Analytical Procedures. www.ich.org/page/quality-guidelines