Glossary
HPLC
HPLC separates a peptide from its impurities and gives purity as area percent at a fixed wavelength. What that figure includes, and what it overlooks.
In HPLC (high-performance liquid chromatography), a dissolved sample is forced through a tightly packed column at high pressure, and its components separate according to how strongly they are retained by the stationary phase. Peptides are routinely analysed by reverse-phase HPLC on a C18 column, running a water/acetonitrile gradient with 0.1% trifluoroacetic acid and UV detection at 214 nm — the wavelength at which the peptide bond absorbs.
Interpreting the purity figure
Purity is usually given as area percent: the area of the main peak divided by the summed area of every integrated peak. A value of 98.6% tells you that 98.6% of the UV signal detected came off the column as a single peak. It is relative, not a measurement of the amount of peptide in the vial — water, counter-ions and excipients that do not absorb go completely unseen.
Its blind spots
HPLC separates components but cannot say what they are. A deletion sequence eluting very close to the product may be concealed beneath the main peak, and an impurity lacking a chromophore may not show up at 214 nm at all. For that reason a trustworthy certificate combines the chromatogram with a mass spectrometry identity check on the same lot.
Related terms
certificate of analysis · third-party testing. Read what ≥98% actually means and how to read a COA.