HPLC vs mass spectrometry: what each test tells you about a peptide

What HPLC measures

High-performance liquid chromatography separates the components of a sample as they travel through a column. A detector, usually UV absorbance near 214 nm where peptide bonds absorb, records each component as a peak. Purity is the area of the main peak as a percentage of total peak area.

What HPLC cannot tell you

Purity percentages therefore say less than they appear to. Method details (column, gradient and wavelength) affect the result.

• Whether the main peak is the intended peptide.
• How much water, salt or counter-ion is in the powder.
• Impurities that co-elute with the main peak or do not absorb at the detection wavelength.

What mass spectrometry measures

Mass spectrometry measures the mass-to-charge ratio of ions. For a peptide, the observed molecular mass should match the mass calculated from the sequence. Matching masses support identity; a mismatch is a serious flag.

Why both are needed

HPLC without MS can certify a clean peak of an unknown substance. MS without HPLC can confirm the mass of something that is only a small fraction of the sample. Together they show a pure sample of the correct molecule.

Checking the numbers yourself

Compute the theoretical mass from the sequence with the peptide mass calculator, then compare it with the CoA. Monoisotopic and average masses differ, so check which one the lab reports.

Independent versus in-house testing

In-house results are useful, but third-party testing removes the conflict of interest. Where a supplier uses an outside lab, the lab's name, date and report number should be on the document.