High-performance liquid chromatography separates the components of a mixture by pushing it through a packed column under pressure. Components travel at different speeds depending on how strongly they interact with the packing material, so they arrive at the detector at different times.
The output is a chromatogram: a trace with peaks, each peak representing something that came off the column at a particular time.
Where the percentage comes from
Purity is calculated as the area of the main peak divided by the total area of all peaks, expressed as a percentage. A result of 99% means the largest peak accounts for 99% of the total detected signal.
Two consequences follow directly from that definition, and both are routinely glossed over in marketing.
It is relative, not absolute
The figure describes proportions of what the detector saw. Anything the detector cannot see does not appear in the denominator. Peptide detection is typically by ultraviolet absorbance at around 214 nm, which responds to the peptide bond. Substances without meaningful absorbance at that wavelength, including many salts and residual water, contribute little or no signal and are therefore largely invisible to the calculation.
This is why net peptide content and water content are reported separately on a thorough certificate. A sample can be 99% pure by HPLC peak area and still be a minority of the vial's mass.
It does not establish identity
HPLC reports that one thing dominates the sample. It does not report what that thing is. Retention time can be compared against a reference standard, which is suggestive, but compounds with similar physicochemical behaviour can elute at similar times.
Identity confirmation requires mass spectrometry, which measures molecular weight directly and compares it against the theoretical value for the intended sequence.
What good looks like
- A chromatogram image, not just a number, so the baseline and any secondary peaks are visible
- The wavelength and column conditions stated
- An MS trace alongside, with observed and theoretical mass
- Water content or net peptide content reported separately
- The whole set tied to a lot number that matches your vial
A supplier who provides the chromatogram rather than the summary figure is giving you the underlying data rather than their interpretation of it. That distinction is most of what separates documentation from marketing in this category.
Questions and answers
How is an HPLC purity percentage calculated?
As the area of the main peak divided by the total area of all peaks, expressed as a percentage. A result of 99% means the largest peak accounts for 99% of the total detected signal.
What does an HPLC purity figure fail to measure?
Anything the detector cannot see. Peptide detection is typically by ultraviolet absorbance at around 214 nm, which responds to the peptide bond, so substances without meaningful absorbance at that wavelength, including many salts and residual water, contribute little or no signal and are largely invisible to the calculation. A sample can be 99% pure by peak area and still be a minority of the vial's mass.
Can HPLC confirm which compound is in the vial?
No. It reports that one thing dominates the sample, not what that thing is. Retention time can be compared against a reference standard, which is suggestive, but compounds with similar physicochemical behaviour can elute at similar times. Identity confirmation requires mass spectrometry, which measures molecular weight directly against the theoretical value for the intended sequence.
What does a complete purity record look like?
A chromatogram image rather than just a number, so the baseline and any secondary peaks are visible. The wavelength and column conditions stated. A mass spectrometry trace alongside it, with observed and theoretical mass. Water content or net peptide content reported separately. And the whole set tied to a lot number that matches your vial.