HPLC and mass spectrometry for peptides

Two questions sit behind every research peptide: is this the right molecule, and how much of the sample is that molecule? They sound alike, but laboratories answer them with different instruments. Knowing which is which makes certificates much easier to read.

HPLC answers "how much"

High-performance liquid chromatography pumps a dissolved sample through a column packed with fine particles. Components interact with the packing to different degrees and leave the column at different times, each recorded as a peak by a detector, usually measuring ultraviolet absorbance. For peptides the standard form is reversed-phase HPLC, in which more hydrophobic molecules are retained longer.

Purity is then calculated as the area of the main peak divided by the total area of all peaks. Impurities from synthesis, such as a sequence missing one amino acid or carrying a leftover protecting group, usually show up as small neighbouring peaks. UPLC follows the same principle with smaller particles and higher pressure, which sharpens the peaks and shortens the run.

Mass spectrometry answers "what"

A mass spectrometer converts molecules into ions and measures their mass-to-charge ratio. For a peptide, the measured mass is compared with the theoretical mass calculated from the sequence. Agreement within the instrument's tolerance supports the identity of the substance. A shift can reveal a modification: oxidation of a methionine residue, for example, adds 16 daltons.

Why one method is not enough

HPLC can show a single clean peak for the wrong molecule, if that molecule happens to be pure.
Mass spectrometry can confirm the right molecule in a sample that also contains a lot of something else.
Combined, often in one instrument as LC-MS, they cover both questions.

Reading the numbers

A figure such as "99.51% (HPLC)" is a statement about one lot, analysed on one day, by one method. A figure written as ">99.8%" usually means the result is above the reporting limit of the method. Figures from different laboratories and methods are not directly comparable, which is why the UKPeps certificate library lists the laboratory and method for every row.

Where to see it in practice

Every UKPeps product page links to the certificates for that substance, newest first, with the laboratory and the measured purity. The full library, searchable by substance or lot number, is open to everyone.

Sources

Mant CT et al. HPLC analysis and purification of peptides. Methods Mol Biol, 2007. PubMed 18604941 · doi:10.1007/978-1-59745-430-8_1
A methods chapter on HPLC, including reversed-phase modes, for analysing and purifying peptides.
Sharma N et al. Synthetic pharmaceutical peptides characterization by chromatography principles and method development. J Sep Sci, 2022. PubMed 35460196 · doi:10.1002/jssc.202101034
A review of chromatographic principles and method development for characterising synthetic peptides.
Lian Z et al. Characterization of Synthetic Peptide Therapeutics Using Liquid Chromatography-Mass Spectrometry: Challenges, Solutions, Pitfalls, and Future Perspectives. J Am Soc Mass Spectrom, 2021. PubMed 34110145 · doi:10.1021/jasms.0c00479
A review of LC-MS approaches for characterising synthetic peptides.
Guthals A et al. Peptide identification by tandem mass spectrometry with alternate fragmentation modes. Mol Cell Proteomics, 2012. PubMed 22595789 · doi:10.1074/mcp.R112.018556
A review of peptide identification by tandem mass spectrometry using different fragmentation modes.

Published laboratory and preclinical research, cited for scientific context only. It is not evidence of safety or effect in people.

HPLC and mass spectrometry: how peptide purity and identity are measured