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 Selank certificates
Selank certificates report HPLC-UV purity: HPLC with an ultraviolet detector. Current lots report 99.77% 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.