Understanding HPLC Analysis

HPLC, or high-performance liquid chromatography, is a common analytical technique used to separate the different components of a sample so they can be measured. It is widely used to characterise research materials. The explanation below is a neutral description of the method and what its results represent, not a claim about any material or any outcome of using one.

The mobile and stationary phases

In HPLC, a liquid sample is carried by a solvent (the mobile phase) through a column packed with a material (the stationary phase). As the sample travels through the column, its components interact with the stationary phase to different degrees. Components that interact more strongly move more slowly, and those that interact less move more quickly.

The chromatogram

Because components travel at different speeds, they leave the column at different times. A detector records each component as it exits, producing a chromatogram: a graph with peaks where each peak corresponds to a component that has separated from the mixture. The timing of each peak and the relative area it occupies are the primary data the chromatogram provides. For a detailed explanation of how peak timing works and what it reveals about a sample, see Understanding Retention Time in HPLC.

On a chromatogram, the size of a peak relative to the total is often expressed as a percentage of peak area. A purity figure that references HPLC is typically based on this kind of measurement. For how these percentages are presented and interpreted, see Understanding Purity Percentages.

When a research material’s specification references HPLC, it is identifying the analytical method associated with a stated figure. This is a factual description of how the figure was obtained. The wider set of specification fields is explained in Understanding Purity Specifications.

HPLC is often used alongside other techniques. Mass spectrometry, for example, helps confirm identity by measuring molecular weight, and the two methods provide complementary information. See Understanding Mass Spectrometry for an overview, and the Quality page for our general approach.

HPLC is valued because it is sensitive, reproducible and able to resolve closely related components of a mixture. For research peptides, this makes it well suited to indicating how much of a sample corresponds to the target material relative to minor by-products. Because the method is well established, a figure expressed “by HPLC” is widely understood, which is part of why it appears so often on specifications.

The conditions of an HPLC analysis, such as the column and the solvents used, are chosen to suit the sample. Different conditions can separate components in different ways, which is why a purity figure is reported together with the method rather than as a number on its own. Reading the value and the method together gives the figure its meaning.

An HPLC result describes a sample under the conditions of that analysis. It is a factual measurement, not a statement about how a material will behave in any experiment, and it should be read as one part of a fuller specification. For how a peak-area figure becomes a stated percentage, see Understanding Purity Percentages.