The purity figure on a data sheet usually originates from high-performance liquid chromatography. This page explains how that value comes about, what it states and where its limits lie.
Separation principle
HPLC is a separation method, not an identification method. A dissolved sample is driven by a solvent stream (mobile phase) under high pressure through a densely packed column (stationary phase). Each component interacts to a different degree with the packing material, therefore migrates at its own speed and leaves the column at its own point in time. A detector at the column outlet records the signal against time.
For peptides, reversed-phase HPLC is the usual variant: a non-polar stationary phase, typically C18-bonded silica gel, combined with a water-acetonitrile gradient and a small proportion of trifluoroacetic acid. Detection is usually via UV absorption at 210 to 220 nm, the range in which the peptide bond itself absorbs.
Reading a chromatogram
The result is a diagram with elapsed time on the horizontal axis and the detector signal on the vertical axis. Four features carry the information:
- Retention time – the point in time at which a peak appears. Under constant conditions it is characteristic of a compound and allows comparison between runs.
- Peak area – the integrated area under the curve. It scales with how much of the respective component has passed the detector.
- Peak shape – broad peaks, peaks showing tailing or shoulders can indicate co-elution, column overloading or a damaged column.
- Baseline – the section without a detector signal. Its quietness is a quality feature of the run.
In a well-resolved run one peak dominates and the others remain small. These smaller peaks are the related substances: truncation and deletion sequences, oxidised variants, residual reagents as well as by-products from synthesis and cleavage.
How the purity value comes about
The figure on a certificate of analysis is almost always an area-normalised value: the area of the main peak divided by the total area of all integrated peaks, expressed as a percentage. Two consequences follow from this that are easily overlooked.
The value is relative. It describes the proportion of the material the detector was able to see, not the proportion of the contents of the vessel. Water, inorganic salts, counter-ions such as acetate or trifluoroacetate and several common solvents absorb practically nothing at the detection wavelength and therefore contribute nothing to the total area. A high chromatographic purity value and a noticeable water content are not a contradiction.
The value is an approximation. Different compounds absorb ultraviolet light to different degrees. Two minor components present in equal amounts do not necessarily produce peak areas of equal size. Area percent is therefore an approximation of the composition, not a direct measurement of quantity.
What HPLC does not answer
- Identity. Chromatography separates, it does not identify. A peak at the expected retention time is consistent with the target compound, but consistency is not confirmation. The proof is provided by mass spectrometric analysis.
- Content. Which mass fraction of the vessel contents is actually peptide is answered by a quantitative content determination, not by the chromatogram. See Understanding peptide purity.
- Water and counter-ion content. Karl Fischer titration and ion chromatography are responsible for this.
- Microbiological quality. A separate question with its own methods.
Why the method details are decisive
Column chemistry, gradient profile, flow rate, injection volume and detection wavelength together shape what a chromatogram shows. A short, steep gradient can compress several minor components into something that looks like a single clean peak. A longer gradient separates the same sample and leads to a lower reported purity for exactly the same material. Neither of the two runs is dishonest; they answer the same question at a different resolution.
A document that states its conditions is therefore more informative than one that states only a result. Published conditions make a value reproducible, comparable between sources and verifiable by a second laboratory.
Common misinterpretations
- Reading the purity as a quantity. It is a ratio, not a mass.
- Comparing values from different methods directly without checking gradient, column and wavelength.
- Concluding the identity of the compound from a single dominant peak.
- Transferring a batch result to the product line. A value applies to the batch tested under the conditions tested.
- Accepting a figure without the associated chromatogram. Without the curve the integration remains unverifiable.
Documentation context
A chromatographic result only becomes verifiable once it is documented on a batch-specific basis and carries the method parameters with it. How such a document is structured is described in Reading a COA correctly. The assignment between document and container is covered by Batch testing and traceability. Batch-related documents are published under Analytics & Certificates insofar as corresponding real documents are available.
Related guides
- LC-MS in peptide analysis
- HPLC vs. LC-MS: differences in peptide analysis
- Understanding peptide purity
- Research Compound Library
For laboratory research only. This text serves as technical information and contains no medical, diagnostic or therapeutic statements, no dosages and no application instructions for humans.