When a research peptide is described as "≥99% by HPLC," that figure comes from a specific analytical technique. Understanding how High-Performance Liquid Chromatography works makes the purity number on a Certificate of Analysis far more meaningful.
What HPLC Does
HPLC separates a mixture into its individual components. A liquid sample is pushed under high pressure through a column packed with a fine stationary material. Different molecules travel through the column at different speeds depending on how strongly they interact with that material.
As each component exits the column, a detector measures it and records a peak. The result is a chromatogram: a plot of detector signal against time.
Retention Time
The time it takes a component to travel through the column and reach the detector is its retention time. Under consistent conditions, a given compound elutes at a characteristic retention time. This helps confirm identity and distinguishes the target compound from impurities, which elute at different times.
How Purity Is Calculated
Purity by HPLC is a measure of relative peak area:
Purity (%) = (area of main peak / total area of all peaks) × 100
If the target compound's peak represents 99% of the combined area of every peak on the chromatogram, the purity is reported as 99%. The remaining 1% reflects impurities or related substances detected during the run.
Reading the Chromatogram
A high-quality result generally shows:
- One dominant, sharp peak for the target compound.
- Few and small secondary peaks.
- A flat, stable baseline between peaks.
A noisy baseline or many secondary peaks can indicate impurities or degradation.
What HPLC Does Not Tell You
HPLC purity is a powerful metric, but it has limits:
- It measures relative purity, not absolute quantity. It tells you the proportion of the sample that is the target compound, not the exact mass present.
- It confirms separation, not identity on its own. Mass Spectrometry (MS) is typically used alongside HPLC to confirm the molecular weight matches the intended compound.
This is why robust COAs pair HPLC purity with MS identity confirmation.
Summary
HPLC separates a sample into peaks, and purity is the target peak's share of the total peak area. Look for a clean chromatogram with a single dominant peak, and check that the COA also includes mass confirmation. Together, these give a complete picture of what a vial contains.