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HPLC and purity: how purity is measured

In short

HPLC is a separation method that pulls the substances in a sample apart. This shows how much of a sample is the target peptide and how much is other material. The result often appears as a purity percentage on a CoA.

What HPLC is

HPLC stands for high-performance liquid chromatography. It is a laboratory method that separates the different substances in a sample. Once separated, each substance can be measured. HPLC is one of the most common ways to check the purity of a peptide.

How an HPLC measurement works

The sample is dissolved and pushed through a long tube packed with fine material, called the column. Different substances move through the column at different speeds. A detector at the end records each substance as it comes out. The result is a graph with peaks. Each peak stands for one substance, and its size reflects how much of it is present.

How purity is calculated

Purity is worked out by comparing the peaks. The area of the main peak is compared to the total area of all peaks. If the main peak makes up X percent of the total area, the purity is reported as X percent. The remaining (100 − X) percent is made up of other substances in the sample.

What a purity percentage does and does not tell you

A purity percentage tells you the share of the target substance compared to other substances that the method can see. It does not confirm the identity of the substance on its own. It also does not measure things the method is not set up to detect. For identity, a lab often adds mass spectrometry.

HPLC on a certificate of analysis

The HPLC result usually appears on the certificate of analysis, or CoA. The CoA lists the measured purity and often includes the graph. Reading the CoA lets you see the reported purity and how it was measured.

Did you know

HPLC stands for high-performance liquid chromatography.
A purity of X percent means that (100 − X) percent consists of other substances.
HPLC measures the ratio between peaks, not the absolute weight of a substance.

Frequently asked questions

What does HPLC mean?

High-performance liquid chromatography, a method to separate and measure the substances in a sample.

What is a good purity for a research peptide?

The purity a study needs depends on the lab application; the batch CoA shows the measured value.

Does HPLC also measure identity?

No, HPLC shows purity. Identity is often confirmed with mass spectrometry.

Where do I find the HPLC result?

On the certificate of analysis (CoA) that comes with a sample.

Sources

For laboratory and research use only. Not for human or animal consumption, diagnostics or therapeutic use.