What HPLC measures

HPLC separates components of a dissolved sample on a packed column with a moving liquid phase. Compounds interact differently with the stationary phase and leave the column at different times (retention time). A detector, typically UV absorbance, records a signal over time. The result is a chromatogram: peaks whose areas are proportional to how much of each detected compound is present.

How purity is calculated

For peptides, purity is most often reported as area percent:

Purity (area %) = (area of the target peak ÷ sum of all peak areas) × 100

If the main peak is 98.5% of the detected area, the sample is reported as 98.5% pure by that HPLC method. That number is only as complete as the method behind it.

Important limits

• Area % is detector-dependent. If an impurity does not absorb at the wavelength used (often around 220 nm for the peptide bond), it does not enter the area total.
• Co-eluting impurities are invisible. If an impurity shares the target peak’s retention time, the two count as one peak. Method development matters.
• Solvent and salt are usually not in the chromatogram. Net peptide content after water, counter-ions, and residual solvent is a separate measurement (elemental or amino-acid analysis), not the HPLC percentage.

Reading a chromatogram

• Main peak: the target peptide, usually the largest.
• Smaller adjacent peaks: often synthesis-related impurities (deletions, oxidation, incomplete deprotection).
• Solvent front: early unretained material, normally excluded from the purity calculation.
• Baseline: detector signal with nothing eluting. A clean, flat baseline is a good method check.

What a usable COA should show

• Product name and sequence
• Lot or batch number
• HPLC method (column, mobile phase, gradient, detector wavelength)
• The chromatogram image, not only the final percentage
• Mass-spectrometry identity when used
• Issuing laboratory name and analysis date

Red flag. A “certificate” that is only a purity number, with no chromatogram, method, or lab attribution, is a claim rather than a report. Look at the trace — shape, baseline, impurity peaks — not just the headline percentage.

Other limits

• HPLC purity is not net peptide content. A sample can be high by HPLC area % and still contain less peptide by mass once water, salts, and counter-ions are counted.
• Different methods can give slightly different numbers on the same sample.
• Purity is meaningful only relative to a stated method.