Quality education · 7 min read

How Research Peptides Are Tested: HPLC, Mass Spec and Beyond

The analytical methods behind a peptide COA: RP-HPLC for purity, mass spectrometry for identity, plus peptide content, water, counterions and endotoxin. What each test tells you.

A Certificate of Analysis is a summary of lab tests. Knowing which test answers which question makes it much easier to judge whether a report is complete. Here are the methods used on research peptides, from the two every COA should have to the extras that more thorough reports include.

1. RP-HPLC: how pure is it?

Reversed-phase high-performance liquid chromatography pushes the dissolved sample through a column packed with a hydrophobic material (usually C18). Different molecules stick to the column to different degrees and come out at different times. A UV detector, typically at 214 or 220 nm where peptide bonds absorb, records each one as a peak.

Purity is the area of the main peak as a percentage of all peaks. The impurities it reveals are mostly synthesis by-products: sequences missing one amino acid, sequences with a protecting group left on, or oxidized forms. A credible report shows the chromatogram, the column, the wavelength and the gradient.

2. Mass spectrometry: is it the right molecule?

HPLC cannot tell you what the main peak is. Mass spectrometry can. The sample is ionized, most often by electrospray (ESI), and the instrument measures the mass-to-charge ratio of the ions. Larger peptides pick up several protons, so a 3,000 Da peptide may appear at about 1,000 m/z as a triply charged ion. Software converts these back to a molecular mass that should match the theoretical value within about 0.1%.

Many labs run the two together as LC-MS, which confirms that the main HPLC peak is the expected molecule.

3. Peptide content: how much of the powder is peptide?

A lyophilized powder also contains water and counterions. Net peptide content, measured by amino acid analysis or nitrogen analysis, tells you what fraction of the weighed powder is actually peptide. It is commonly 70–90%. See net peptide content vs gross weight.

4. Water content

Karl Fischer titration measures residual water. Lyophilized peptides are hygroscopic, so a low water figure at release and good packaging both matter for stability.

5. Counterions: TFA or acetate

Peptides purified by HPLC usually come out as trifluoroacetate (TFA) salts, because TFA is used in the mobile phase. Some are exchanged to acetate or hydrochloride. Ion chromatography or ¹⁹F NMR can quantify TFA. For some cell-based work the counterion matters, so it is worth knowing which salt form you have.

6. Endotoxin

Bacterial endotoxin (lipopolysaccharide) is measured with the LAL or recombinant Factor C assays and reported in EU/mg. It is relevant for sensitive cell-culture work and is usually listed as a separate test, not part of a standard identity and purity report.

What a minimum COA should include

RP-HPLC purity, with the chromatogram and method.
Mass-spec identity, with observed and theoretical mass.
The laboratory name, test date and lot number.

Every Atlas lot is tested by an independent lab for HPLC purity and mass-spec identity before it's listed, and the reports are public in the COA library. To learn how to read one line by line, see how to read a peptide COA.

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For Research Use Only. Not for human or veterinary use. Not for diagnostic or therapeutic use. Not a drug, food, cosmetic or dietary supplement. Not for resale to consumers.