High-performance liquid chromatography is the standard way to measure how pure a synthetic peptide is. Nearly every certificate of analysis for a research peptide reports an HPLC purity, usually as a single percentage. This article explains how that number is produced, what it counts, and what it leaves out.
Principle
In reversed-phase HPLC, a dissolved sample is pumped through a column packed with small porous particles whose surface is hydrophobic. A mixture of water and an organic solvent, the mobile phase, carries the sample through. Peptides cling to the hydrophobic surface in proportion to their own hydrophobicity, so different molecules move through the column at different speeds and leave it at different times.
As each component leaves the column it passes a detector, which records a peak. The plot of detector signal against time is the chromatogram. The time at which a peak appears is its retention time; the area under the peak is proportional to how much of that component there is, as seen by the detector.
Column and mobile phase
Most peptide methods use a C18 column: silica particles of 1.7–5 µm carrying 18-carbon alkyl chains. Small peptides are run on columns with pores of about 100–120 Å; larger peptides need wide-pore (about 300 Å) packings so that they can reach the bonded surface inside the pores.
The mobile phase is usually two solvents mixed by the pump: A, water, and B, acetonitrile, each containing about 0.1% trifluoroacetic acid. The run starts with mostly A and increases B steadily, a gradient, so that peptides are released one after another as the solvent becomes more hydrophobic. The acid keeps the peptide's carboxyl groups protonated and pairs with its positive charges, which gives sharper, more symmetrical peaks.
When the column is connected to a mass spectrometer, formic acid is often used instead, because trifluoroacetic acid suppresses the electrospray signal. Peaks are then usually somewhat broader.
Detection
Peptides are detected by ultraviolet absorbance, usually at 214 or 220 nm. At these wavelengths the absorbing group is the peptide bond itself, which every peptide has, so the detector responds to the main peptide and to most peptide-related impurities in a broadly similar way. That similarity is what allows peak areas to be compared directly.
At 280 nm, only tryptophan and tyrosine absorb. Some methods record both wavelengths: the 280 nm trace can help tell aromatic and non-aromatic impurities apart, but it is not used for the purity figure.
Calculating purity
The chromatography software integrates each peak above a baseline. Chromatographic purity is the area of the main peak divided by the total area of all integrated peaks, expressed as a percentage:
Purity (%) = area of main peak ÷ total area of all peaks × 100
Peaks from the blank (the solvent with no sample) are excluded, and very small peaks below a set reporting threshold are often not integrated. Both choices are part of the method and affect the result slightly.
What the figure leaves out
Chromatographic purity is a relative measure of the UV-absorbing material that the column separates. Several things fall outside it:
- Non-peptide mass. Water, counter-ions such as trifluoroacetate or acetate, and inorganic salts do not absorb at 214 nm in the same way and are not counted. A material can be 99% pure by HPLC and still be only about 70–85% peptide by weight. That second figure is the net peptide content.
- Co-eluting impurities. An impurity that leaves the column at the same time as the main peptide is counted as part of it. Diastereomers (a single residue in the D-form) and some deamidation products elute very close to the parent peptide.
- Material that never elutes or never absorbs. Strongly retained aggregates, or impurities without a chromophore at the detection wavelength, are not seen.
- Identity. HPLC shows that one component dominates. It does not show that the component is the intended peptide. Identity is confirmed by mass spectrometry.
Common impurities
Most impurities in a synthetic peptide come from the synthesis itself or from slow chemical change in storage. Mass spectrometry of the impurity peaks, where it is done, helps identify them.
| Impurity | Origin | Mass change | Usual HPLC behaviour |
|---|---|---|---|
| Deletion sequence | An incomplete coupling step in synthesis | Minus one residue | Separate peak, often close to the main peak |
| Truncated sequence | Chain growth stopped early | Minus several residues | Usually well separated |
| Incomplete deprotection | A side-chain protecting group not removed | Plus the group's mass (for example +56 Da for tert-butyl) | Later peak (more hydrophobic) |
| Diastereomer | Racemisation of a residue during synthesis | None | Very close to, or under, the main peak |
| Methionine oxidation | Air, light or peroxides | +16 Da | Slightly earlier peak (more polar) |
| Deamidation | Moisture, pH and temperature acting on Asn or Gln | +0.98 Da | Close to the main peak; may show as a shoulder |
| Dimer or aggregate | Intermolecular association or disulfide formation | About twice the mass | Later peak, or retained on the column |
Why results depend on the method
The same lot can give slightly different purity results on different methods. A steeper gradient compresses peaks together and can hide small impurities under the main peak; a shallower one spreads them out. Column chemistry, temperature, sample load and integration settings all play a part. This is why a purity figure is most informative when it comes with the chromatogram, and why validated methods specify every one of these conditions.
Guidance such as ICH Q2(R2) describes how analytical methods are validated for specificity, linearity, precision and the limits of detection and quantitation. A validated method gives repeatable results, but it still measures only what the detector can see.
Reading a chromatogram
When a certificate of analysis includes the chromatogram, a few things are worth checking:
- One dominant, sharp, symmetrical main peak.
- The number, size and position of minor peaks, and whether they are listed with their areas.
- Shoulders on the main peak, which suggest a closely eluting impurity.
- A flat, stable baseline, and peaks that are integrated from it consistently.
- The method details: column, gradient, wavelength and run time.
- The lot number, which should match the lot on the container.
Frequently asked questions
- Does 99% HPLC purity mean the powder is 99% peptide?
- No. HPLC purity compares the main peak with other UV-absorbing peaks. Water and counter-ions are not counted, so the peptide's share of the weighed mass, the net peptide content, is lower.
- Can HPLC confirm which peptide is in the container?
- Not on its own. A retention time can be compared with a reference standard, but identity is confirmed by mass spectrometry, which measures the molecular mass.
- Why is detection set at 214 or 220 nm?
- The peptide bond absorbs strongly in that range, so every peptide and almost every peptide-related impurity is detected, with broadly similar response.
- Why might two laboratories report different purities for the same lot?
- Differences in column, gradient, wavelength, sample load and integration settings change how well small impurities are separated and counted. Differences of a few tenths of a percent are common.
- What does a shoulder on the main peak mean?
- A second component elutes almost at the same time. It is often a closely related impurity, such as a diastereomer or a deamidated form, and is better resolved with a shallower gradient or a different column.
Sources & Further Reading
- Aguilar MI. Reversed-phase high-performance liquid chromatography. In: Aguilar MI, ed. HPLC of Peptides and Proteins: Methods and Protocols. Methods in Molecular Biology, vol. 251. Humana Press; 2004:9–22. doi:10.1385/1-59259-742-4:9
- Mant CT, Chen Y, Yan Z, Popa TV, Kovacs JM, Mills JB, et al. HPLC analysis and purification of peptides. In: Fields GB, ed. Peptide Characterization and Application Protocols. Methods in Molecular Biology, vol. 386. Humana Press; 2007:3–55. doi:10.1007/978-1-59745-430-8_1
- Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. 3rd ed. Wiley; 2010. doi:10.1002/9780470508183
- D'Hondt M, Bracke N, Taevernier L, Gevaert B, Verbeke F, Wynendaele E, et al. Related impurities in peptide medicines. Journal of Pharmaceutical and Biomedical Analysis 2014;101:2–30. doi:10.1016/j.jpba.2014.06.012
- International Council for Harmonisation. ICH Q2(R2): Validation of Analytical Procedures. Adopted 2023.
See also
- HPLC — glossary
- Chromatographic purity — glossary
- Retention time — glossary
- C18 column — glossary
For in vitro laboratory research only. Not for human or veterinary use. This library describes chemistry, analytical methods and published laboratory research; it gives no guidance on use in people or animals.