HPLC purity: what the percentage measures and what it does not

An HPLC purity tells you what share of the UV signal at one wavelength belongs to the main peak. It does not tell you how much peptide is in a milligram of powder: water, counter-ions and salts do not appear in that figure. Keep the two apart and you read a certificate of analysis correctly.

How the figure is produced

In high-performance liquid chromatography (HPLC), the dissolved sample runs through a separation column. A UV detector measures what leaves the column; for peptides, the manufacturer Bachem gives detection at 210 to 220 nm. Each separated substance shows up as a peak in the chromatogram.

Purity is then the area of the main peak relative to the total area of all peaks:

purity (area percent) = area of the main peak / sum of all peak areas × 100

This is called area normalisation. The figure is a relative share within the chromatogram, not a mass and not a content.

What the figure leaves out

A lyophilised peptide is not only peptide. Bachem says it usually supplies peptides as trifluoroacetate salts, so with TFA as the counter-ion; peptides made by liquid-phase synthesis usually come as acetate or inner salts. Residual water comes on top. Bachem measures these components with their own methods, not with the method used for purity.

Bachem puts it this way: research peptides are typically more than 95% pure by HPLC, yet the peptide content of the solid may range from 70 to 90%, because counter-ions and residual moisture add weight. For peptides with many basic amino acids, arginine in particular, a low peptide content is to be expected.

A certificate of analysis may therefore list several values that answer different questions:

Net peptide content counts peptidic impurities; purity does not. The two figures measure different things.

Why one sample can have two purities

Area normalisation counts everything inside the main peak. If the column does not separate an impurity from the main peak, it counts as main substance. Purity therefore depends on the method: column, mobile phase, gradient, wavelength.

An example from the literature: a research group at Ghent University re-measured a peptide for which the supplier had stated at least 95.0%, using a different column and 210 instead of 220 nm. They found 80.6%. In their assessment, the supplier's method did not separate well enough (Verbeke and others, 2015).

A purity value without its method is hard to judge. Which details a certificate should carry is covered in Reading a certificate of analysis.

Reading a peak table

A chromatogram usually comes with a table with these columns:

• Peak number: in the order in which substances leave the column.
• Retention time: when the peak appears, in minutes.
• Area: the integrated area under the peak.
• Area percent: the share of this peak in the total area.

Check four points as you read. First: are the wavelength and method stated? Second: do the area percentages add up to 100? Third: does the main peak's area share match the purity on the certificate? Fourth: from what size are peaks reported at all? The Ghent group set this reporting threshold at 0.1% of the main peak's area. Smaller peaks then do not appear in the table.

The peak table says nothing about which substance sits in the main peak. Only an identity test settles that.

What this means for your work

Read HPLC purity for what it is: the main peak's share under exactly these measurement conditions. For the amount of peptide in the powder you need the net peptide content; for whether it is the right substance you need mass spectrometry. At Novo Labo you can request the certificate of analysis for a batch by writing to hello@novolabo.com.