HPLC purity testing for peptides, explained

High-performance liquid chromatography (HPLC) is the standard way to measure peptide purity. It separates the components of a sample and reports how much of what it detects is the target peptide.

How it works

For peptides, the usual method is reversed-phase HPLC. The sample passes through a column, and different molecules leave it at different times. A UV detector, typically set around 210–220 nm where peptide bonds absorb, records each one as a peak [1][2].

Reading the chromatogram

• X-axis: retention time. When each component left the column.
• Y-axis: detector signal. How strongly it absorbed.
• The main peak should be the target peptide.
• Smaller peaks are impurities.
• Purity is the main peak's area as a percentage of the total area of all peaks [1].

What the percentage leaves out

HPLC purity only counts what the detector sees. Water, salts and counterions such as TFA don't absorb at those wavelengths, so they aren't in the number [1][2]. That's why purity and net peptide content are different measurements (see how to read a COA). Impurities that leave the column at the same time as the target peptide (co-elution) can also hide inside the main peak unless the method separates them [1].

Common peptide impurities

In synthetic peptides, typical impurities include deletion sequences (a missing amino acid), truncated sequences, incompletely deprotected peptides and modified forms [2]. Oxidized forms are also common in sequences containing methionine.

Purity isn't identity

HPLC shows how clean the main peak is, not what the main peak is. Identity is confirmed separately, usually by mass spectrometry [3].

Questions

How is peptide purity measured?

By reversed-phase HPLC with UV detection around 210–220 nm. Purity is the main peak's area as a percentage of all detected peaks.

Does HPLC purity include water and salts?

No. Water, salts and counterions such as TFA aren't detected by UV at those wavelengths, so they aren't counted. Net peptide content accounts for them.

Does HPLC confirm a peptide's identity?

Not on its own. It shows how pure the main peak is; mass spectrometry confirms that the main component has the expected molecular weight.