Understanding HPLC Results

High-performance liquid chromatography (HPLC) is the method most often used to put a purity figure on a peptide sample. This guide explains how the technique works, what the chromatogram and peak table on a report show, and which details printed beside the figure tell you how it was obtained.

How HPLC separates a sample

A small volume of the dissolved sample is carried by a liquid, the mobile phase, through a packed column, the stationary phase. Each component interacts with the column to a different degree, so components leave the column at different times. A detector at the column outlet records a signal as each one passes.

For peptides the usual mode is reversed-phase HPLC (RP-HPLC). The column packing is non-polar, commonly silica bonded with C18 chains, and the mobile phase is a mixture of water and an organic solvent such as acetonitrile, usually with a small amount of acid. Over the run the share of organic solvent is raised, which is called a gradient, and components leave the column roughly in order of increasing hydrophobicity.

The detector and its wavelength

Most purity methods for peptides use an ultraviolet (UV) detector, often a diode-array detector (DAD) that records many wavelengths at once. The peptide bond absorbs strongly at low UV wavelengths, which is why peptide methods commonly read at around 214 to 220 nm. A component that does not absorb at the chosen wavelength gives little or no signal, so the wavelength printed on a report is part of what its figure means.

Reading the chromatogram

The chromatogram plots detector response against time.

• Retention time is when a peak appears, measured from the start of the run. Under one method, the same compound appears at about the same time.
• Peak area is the area under a peak. Under a given method it rises with the amount of that component reaching the detector, but the response differs from one compound to another.
• Resolution describes how cleanly two neighbouring peaks are separated. Overlapping peaks are harder to measure, and a component hidden under the main peak cannot be counted separately.
• The baseline is the signal when nothing is leaving the column. Drift or noise in it affects how small peaks are measured.

The peak table

Many reports list each integrated peak with its retention time, area and area percentage. The main peak is the named compound; the others are impurities, related substances or artefacts of the method. Integration, the software's decision about where each peak starts and ends, can change the areas of small peaks, which is one reason a method is validated before it is relied on.

From peak areas to a purity figure

The figure most often reported is area-percent: the main peak's area divided by the total area of all integrated peaks, multiplied by one hundred. As a hypothetical example, a main peak of 980 area units in a total of 1,000 gives an area-percent of 98.0. The figure describes what the detector saw under that method. It is not a count of molecules, and it is not the amount of compound in a vial; What Does Purity Percentage Mean? explains the difference.

What a method can miss

• Impurities that do not absorb at the detection wavelength.
• Impurities that leave the column at the same time as the main peak.
• Components that never leave the column, or that pass straight through without being retained.
• Water, salts and counter-ions, which a UV purity method does not measure.

This is why HPLC purity is paired with a separate identity test such as mass spectrometry; see Understanding Mass Spectrometry.

Method validation in brief

ICH Q2(R2) describes the characteristics a purity method is expected to demonstrate, among them specificity, range, accuracy, precision and the limits of detection and quantitation. USP General Chapter <621> sets out chromatography terms and system suitability checks, such as resolution and repeatability, that confirm a system is performing before samples are run. A report rarely shows this work, but the method details it does print, such as the column, mobile phase, gradient and wavelength, are what make its figure interpretable.

Where this site fits

Where a batch report is published, it is in the COA Library with the method printed beside the purity figure, and the product page links the report for the vial size it covers. For a section-by-section walk through a report, see How to Read a Certificate of Analysis.

Sources and references

• ICH Q2(R2), Validation of Analytical Procedures, International Council for Harmonisation, 2023.
• USP General Chapter <621>, Chromatography. United States Pharmacopeia.
• USP General Chapter <1225>, Validation of Compendial Procedures. United States Pharmacopeia.
• L. R. Snyder, J. J. Kirkland and J. W. Dolan, Introduction to Modern Liquid Chromatography, 3rd edition. Wiley, 2010.
• D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of Instrumental Analysis. Cengage Learning.

Research use

All materials on this site are supplied for laboratory research use only. They are not for human or veterinary use, and this guide gives no instructions for preparing or using them.