Knowledge base
HPLC purity: what do 98% and 99% mean?
Almost every research peptide comes with a purity, such as 98% or 99%, measured by HPLC. But what does that number mean exactly, and what does it not tell you? This article explains how an HPLC measurement works, how to read a chromatogram and why you also look at the mass and the content.
How does HPLC work?
HPLC is high-pressure liquid chromatography. The sample is pumped under high pressure through a column packed with very fine particles. Substances that bind more strongly to that material leave the column later than substances that bind more weakly. This separates the peptide from related substances.
For peptides, reversed-phase HPLC is the usual choice, with a UV detector measuring at a wavelength where the peptide bond absorbs light (around 210–220 nm).
Reading the chromatogram
The result is a chromatogram: a graph with time on the horizontal axis and the detector signal on the vertical axis. Each substance gives a peak. The largest peak belongs to the peptide; smaller peaks beside it are impurities.
The purity is the area of the main peak divided by the area of all peaks together. At 99%, 1% of the measured signal comes from other substances.
What are the impurities?
These substances closely resemble the peptide itself, which is why good separation matters. How peptides are made is explained in What are peptides?.
- Chains with a missing or doubled amino acid, formed during synthesis.
- Chains from which a protecting group was not fully removed.
- Breakdown products, such as oxidised or deamidated forms.
What purity does not tell you
- Which substance it is: that needs a mass measurement (MS). A pure substance can be the wrong substance.
- How much peptide is in the vial: purity is a ratio, not an amount.
- How much water and counter-ion (such as acetate or TFA) the powder contains: they do not count in the purity percentage.
98% or 99%: does it matter?
The difference between 98% and 99% is one percentage point of related substances. More important than the exact number is that the purity was measured per lot, that the certificate also includes a mass measurement and that the lot number matches your vial. A round number without a chromatogram, lot or laboratory says less than an exact number from a report you can check.
Certificates at BrabantLabs
BrabantLabs certificates are on Lab results, with the measured purity per lot. How to read a full report, including MS and measured content, is in Reading a lab report. See for example the certificates for BPC-157.
Frequently asked questions
- What does HPLC stand for?
- High-pressure liquid chromatography: a separation technique a lab uses to measure which part of the signal comes from the peptide and which from related substances.
- Is 99% purity the same as 99% peptide in the vial?
- No. Purity is the ratio between the peaks in the chromatogram. A powder also contains water and counter-ions, which do not count in that percentage.
- Why is MS needed alongside HPLC?
- HPLC shows how pure a substance is, MS which substance it is. Together they confirm purity and identity.
- What purity is usual for a research peptide?
- Many suppliers state 98% or higher. Above all, check that the measurement was made per lot and that you can verify the report.
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For laboratory research only. BrabantLabs products are intended for laboratory research and not for human or animal use. This article describes what has been published in the scientific literature and is not a health claim or advice on use.