How to read a peptide HPLC chromatogram

A chromatogram is the graph an HPLC run produces. It is the evidence behind the purity percentage, so it is worth knowing how to read one. Five minutes is enough.

What HPLC does

HPLC stands for high performance liquid chromatography. The sample is dissolved and pushed through a narrow column packed with fine material. Different substances travel through the column at different speeds, so they come out one after another. A detector at the end records each one as it leaves.

The chromatogram is that record: time along the bottom, detector signal up the side.

The two axes

Along the bottom: retention time, in minutes. How long a substance took to pass through the column. Under the same conditions, the same substance always takes the same time. That is how identity is checked: the main peak of your sample has to appear where the reference standard's peak appears.

Up the side: detector response. How strongly the detector registered what was passing at that moment. The height of a peak matters less than its area.

Peaks and peak area

Each peak is one substance, or several that came out together. The area under a peak shows how much of the material it represents.

Purity is simple arithmetic on those areas. Add up the area of every peak. Divide the main peak's area by that total. A main peak with 99.31% of the total area means a purity of 99.31%.

On a Veritalys certificate the peak table under the graph lists each peak's retention time and area percentage, so you can check the arithmetic yourself. See it on the sample certificate.

Reading the small peaks

Small peaks around the main one are impurities. In peptides these are often incomplete versions of the same chain, pieces with one building block missing, or forms changed by oxidation or age. A few small peaks are normal. What matters is their total share, which is everything that is not the main peak.

Things worth noticing:

• One large peak, a few tiny ones. The usual picture for a high purity sample.
• A shoulder on the main peak. A bump on its side means something came out almost at the same time. It may be counted inside the main peak, which can flatter the purity figure.
• A second large peak. A major second substance. Purity will be low, and the identity result matters even more.
• Main peak in the wrong place. If it does not line up with the reference standard, the vial may hold a different compound.

What a chromatogram cannot show

The detector sees only substances that respond to it. Water, salts and leftover counter ions from manufacturing are mostly invisible. That is why a 99% pure sample can still hold less compound than the label says: the percentage describes only what the detector saw. Content, measured in milligrams against a reference standard, answers that question. Purity is not content shows the arithmetic.

It also says nothing about bacterial endotoxin or living microbes. Those need their own tests. See Which peptide test do you need?

How Veritalys draws the chromatogram

The laboratory reports a peak table: each peak's retention time and area. The certificate draws the chromatogram from that table, placing each peak at its measured time and sizing it to its measured share of total area. Peak widths are drawn schematically, and the certificate says so under the graph. The numbers in the table are the laboratory's.