How peptide purity is measured

A synthetic peptide is built one amino acid at a time. No synthesis is perfect, so the finished material contains the intended peptide along with small amounts of closely related by-products, such as chains that are missing one amino acid. Measuring purity means finding out how much of the material is the intended peptide.

HPLC: separating the sample

The standard method is reversed-phase high-performance liquid chromatography. A dissolved sample is pushed through a column packed with a material that holds on to different molecules for different lengths of time. Each component leaves the column at its own moment.

A detector at the end of the column measures how much ultraviolet light the passing liquid absorbs. Peptide bonds absorb strongly at wavelengths near 214 to 220 nanometres, so each component appears as a peak on a chart called a chromatogram.

What the percentage means

The purity figure is the area under the main peak divided by the total area under all the peaks, written as a percentage. It is a relative measure: the share of the detected material that is the intended peptide.

Because it depends on what the detector can see, the result is tied to the method. A report should name the column, the detection wavelength and the conditions used, so that the measurement can be repeated.

Mass spectrometry: confirming identity

HPLC shows how many components there are, but not what they are. Mass spectrometry measures the mass of the molecules in the sample. If the observed mass matches the mass calculated from the peptide sequence, the main component is the intended peptide.

The two methods answer different questions, which is why a certificate of analysis usually reports both.

Purity is not the same as quantity

A dry peptide also contains material that an ultraviolet detector does not register, mainly residual water and the counter-ions left from purification. For that reason the weight of peptide in a sample, sometimes called the net peptide content, is measured separately and is a different figure from HPLC purity.

Reading the result

A purity result belongs to the lot that was analysed. To see how these figures are laid out on a report, read our guide to the certificate of analysis.

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