High-performance liquid chromatography, almost always written as HPLC, is the method behind the purity percentage on a peptide certificate of analysis. It is a separation technique: it pulls a mixture apart so that the components can be detected one at a time, and it reports how much of the detector response each component accounted for.
Knowing what the instrument does makes the reported number easier to interpret, and makes the limits of that number easier to see.
How the separation works
A small volume of dissolved sample is injected into a stream of solvent, the mobile phase, which is pumped at high pressure through a packed column, the stationary phase. Components in the sample interact with the column material to different degrees. The more strongly a component is retained, the later it leaves the column. That difference in retention is what separates the mixture.
Peptides are usually run in reversed-phase mode. The column packing is nonpolar, commonly a C18 material, and the mobile phase is a mixture of water and an organic solvent such as acetonitrile, typically with a small amount of acid added. More hydrophobic components are retained longer.
Gradient and isocratic runs
Most peptide methods use a gradient: the proportion of organic solvent increases through the run, releasing progressively more hydrophobic components in turn. An isocratic run holds the mixture constant. A certificate that names the gradient is describing the conditions under which the separation was achieved, which is part of what makes the result reproducible.
What the detector records
As each separated component leaves the column it passes a detector. For peptides this is normally an ultraviolet detector, and the output is a chromatogram: detector response plotted against time, with each component appearing as a peak.
Two axes carry different information. The horizontal position of a peak is its retention time, which relates to how the component interacted with the column. The area under the peak relates to how much detector response that component produced.
Why the wavelength matters
Peptide methods commonly detect at 214 nm or 220 nm. At those wavelengths the peptide backbone itself absorbs, so the detector responds to essentially any peptide present rather than only to those containing particular residues. Detection at 280 nm relies on aromatic side chains, which many peptides do not have.
The consequence is that the reported percentage is an area percentage at a stated wavelength. It describes the share of response among species that absorbed at that wavelength, and a certificate that states the wavelength is telling you the scope of the figure.
How the percentage is calculated
The area of the main peak is divided by the summed area of all integrated peaks in the run, and the result is expressed as a percentage. It is a relative figure: it compares the main component against the other things the detector saw.
- Material that does not absorb at the detection wavelength does not contribute. Water, many salts and counterion residues fall into this category.
- Material retained so weakly that it elutes with the solvent front, or so strongly that it does not leave the column during the run, is not counted either.
- Integration decisions, such as where a peak is judged to start and end and how a shoulder is integrated, affect the arithmetic.
An HPLC purity percentage is a statement about relative detector response under one method. It is not a measure of the mass fraction of the vial contents, and it does not establish the identity of the peak that produced it.
What HPLC does not answer
Chromatography separates by physical behaviour, not by identity. A peak tells you that something eluted at a particular time and produced a particular response; it does not name the molecule. Two different compounds with similar interactions with the column can appear at similar retention times.
That is why certificates pair chromatography with a mass measurement. Mass spectrometry addresses which molecule is present, and purity and identity testing covers why the two results are reported separately. Where the two techniques are coupled, written as LC-MS, components are separated by chromatography and then measured by mass in the same run.
Reading an HPLC section on a certificate
- Which wavelength was used for detection?
- Was the run a gradient or isocratic, and over what time?
- What column and mobile phase are named?
- Is a chromatogram included, so the summary figure can be checked against the trace?
- Does the baseline return cleanly between peaks, or are peaks merging?
How to read a certificate of analysis covers the surrounding sections of the document, and published certificates for compounds in our catalogue are on the certificates of analysis page.
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