How to Read an HPLC Chromatogram: Peaks, Retention Time and Area Percent

For in vitro laboratory research use only. This article explains how to read an analytical chart. It makes no claims about the effects of any material.

A purity result on a certificate of analysis usually comes from HPLC-UV, and the chart behind that number is called a chromatogram. Our HPLC purity explainer covers what the percentage means. This guide covers the chart itself: what the axes show, what the peaks are, and how the reported percentage is worked out from them.

The two axes

A chromatogram is a line plotted over time.

• The horizontal axis is time, usually in minutes, counted from the start of the run, when the sample enters the instrument.
• The vertical axis is detector response. For a UV detector this is absorbance, often shown in milli-absorbance units (mAU).

When nothing is coming off the column, the line stays flat. When a component reaches the detector, the line rises and falls again, forming a peak.

Retention time

The time at which a peak reaches its highest point is its retention time. Under the same method (same column, solvents, gradient, flow rate and temperature), the same compound comes off at about the same time. Retention time on its own doesn’t name a compound. Two different compounds can come off at similar times, which is why identity is checked separately by LC-MS. Our HPLC vs LC-MS guide explains how the two methods split the work.

The baseline and the main peak

The flat line between peaks is the baseline. A steady, level baseline makes peaks easier to measure. A baseline that drifts or is noisy makes small peaks harder to tell apart from background.

In a typical purity chromatogram, one large peak stands out. That is the main component. Any smaller peaks are other components the method detected under its conditions.

Peak area, not peak height

The software measures each peak’s area, which is the space between the peak and the baseline. This step is called integration. Area is used rather than height because a wide, low peak and a narrow, tall peak can represent similar amounts of material.

A results table often appears under the chart, listing each peak with its retention time, area and area percent.

How area percent is calculated

Area percent compares each peak with the total of all integrated peaks:

Area % = (peak area / total area of all integrated peaks) x 100

A simple example with made-up numbers: if a chart has three integrated peaks with areas of 9,800, 120 and 80, the total is 10,000. The main peak is 9,800 / 10,000 x 100 = 98.0%. The two smaller peaks are 1.2% and 0.8%.

On a certificate, the HPLC-UV purity figure is normally reported as the area percent of the main peak.

Things that affect the number

A few method details shape what the chart shows:

• Detection wavelength. Peptide bonds absorb UV light at low wavelengths, so peptide methods often detect at around 210 to 220 nm. The wavelength is normally stated in the method details.
• What the detector can see. Only components that absorb UV light at that wavelength appear as peaks. Water and some salts don’t show up, which is one reason net content is reported as a separate result.
• Integration settings. Very small peaks below the method’s threshold may not be counted.
• Overlapping peaks. A shoulder on the side of a peak can mean two components came off close together.
• Early peaks. Peaks right at the start of the run are often from the solvent or the system rather than the sample. Methods account for these, for example by running a blank.

What a chromatogram can’t tell you

• What the main peak is. That’s the identity result, from LC-MS.
• How much material is in the vial. Purity is a proportion, not a quantity. Net content is a separate result.
• Anything about other lots. A certificate reports results for the sample tested. It is not a guarantee for other lots.

For the terms used here, see our research peptide glossary.

Peptix certificates

The peptide lots with a published certificate on our peptide COA lookup page each have a third-party Certificate of Analysis showing the lot number, HPLC-UV purity and LC-MS identity. For example, you can view the TB-500 10 mg product with its lot certificate, or browse all research peptides.

For research use only. Not for human or animal use.