HPLC purity, explained

HPLC is the standard test for how pure a peptide sample is.

What HPLC stands for

High-performance liquid chromatography. A liquid sample is pushed at high pressure through a narrow column packed with fine particles. Different molecules stick to the particles by different amounts, so they come out of the column at different times.

Reading the chart

A detector watches what comes out and draws a chart called a chromatogram. Each substance shows up as a peak. The peptide you want is the big main peak, and anything else shows up as smaller peaks.

How purity is calculated

Purity is the main peak's area as a share of all the peak areas added together:

Purity (%) = main peak area ÷ total peak area × 100

So if the main peak makes up 98 of every 100 units of area, the purity is 98%.

What HPLC can't tell you

HPLC shows how much of the sample is one substance, but not exactly what that substance is. That's the job of mass spectrometry, which weighs the molecule and confirms its identity. The two tests together are usually reported on a certificate of analysis.