What is HPLC (high-performance liquid chromatography)?
HPLC is a laboratory technique that separates the components of a sample by pumping it through a packed column; for peptides it is the standard method for measuring purity.
How HPLC works
A liquid sample is pumped at high pressure through a column packed with fine particles. Each component interacts with the packing differently, so components leave the column at different times. A detector at the column outlet records each one as a peak. The plot of detector signal against time is the chromatogram.
Peptides are usually analyzed by reversed-phase HPLC, in which the packing carries hydrophobic chains (commonly C18) and the solvent becomes progressively more organic over the run. More hydrophobic molecules are retained longer.
HPLC on GLP-3 RT certificates
GLP-3 RT certificates report HPLC-UV purity: HPLC with an ultraviolet detector. The main peak elutes at about 14.4–14.9 minutes on current certificates. Current lots report 99.67%–99.98% purity by area.
What HPLC does not tell you
- It does not identify the peak. Identity requires a second method such as LC-MS.
- Impurities that elute at the same time as the main peak can be hidden inside it.
- Water, salts and counterions produce little UV signal and are not counted.