HPLC vs LC-MS for peptides: what each technique measures
Updated: October 8, 2026
HPLC separates the components of a sample and measures how much of each there is. LC-MS adds a mass spectrometer that says what each component is. They are used together because they answer different questions.
Reverse-phase HPLC
For peptides a C18 column is almost always used, with a water–acetonitrile gradient and an acid modifier (TFA or formic acid). The peptide bond absorbs UV light at 214–220 nm, so the detector records a signal as a function of retention time.
Purity is calculated as the area of the main peak divided by the sum of all peak areas.
Limits of HPLC
- An impurity that elutes at the same time as the peptide (co-elution) stays hidden under the main peak.
- It does not see what does not absorb UV, such as salts, water or some counter-ions.
- The result depends on the method: gradient, wavelength and how peaks are integrated.
- It does not identify the molecule: two different peptides can each give a clean peak.
LC-MS: identity by mass
After the column the eluent is ionised (usually by electrospray) and the mass-to-charge ratio is measured. Peptides appear with several charges ([M+nH]n+); from them the molecular mass is calculated and compared with the theoretical one. It also reveals impurities of a different mass, such as truncated sequences or oxidations (+16 Da on methionine).
Which combination to look at
HPLC for purity, MS for identity and, if the real amount of peptide per vial matters, the net content. A report that shows only a percentage with no chromatogram or mass gives the least information.