Peptide purity and HPLC: what the number means

A peptide purity figure, such as 98%, is the share of the total peak area that is the target peptide when a sample is separated by liquid chromatography, usually HPLC. The rest is related material: shorter chains, altered chains and other by-products of synthesis. Purity does not tell you how much peptide is in the vial by weight, and it says nothing about whether a product may be sold in Canada.

This page explains how the number is made and how to read it, using the wording of the European Medicines Agency's guideline for synthetic peptides in medicines. That guideline is a European standard for medicines. We quote it to explain the terms, not to say any product meets it.

Why purity is the headline number

The EMA guideline, which took effect on 1 June 2026, counts purity among the most important quality attributes:

"Purity is one of the most important critical quality attributes (CQAs) for synthetic peptides."

European Medicines Agency, Guideline on the Development and Manufacture of Synthetic Peptides, section 4.3.2, retrieved 12 October 2026.

In plain words, the guideline ranks purity among the properties that matter most. The reason is in how peptides are made: the guideline describes by-products such as chains with an amino acid missing, which come from the synthesis itself. Our page on what is in a synthetic peptide besides the peptide walks through the kinds of by-product.

How HPLC turns a sample into a number

HPLC stands for high-performance liquid chromatography. The sample is dissolved and pushed through a packed column. Different molecules leave the column at different times, and a detector draws a peak for each. The purity figure compares the area of the main peak with the area of all the peaks.

Chromatography is also how peptides are cleaned up after they are made. The EMA guideline says:

"Synthetic peptides are generally purified using chromatographic techniques"

European Medicines Agency, Guideline on the Development and Manufacture of Synthetic Peptides, section 4.2.2, retrieved 12 October 2026.

So the same family of methods is used twice: once to purify the peptide, and again to measure how pure it is.

How small a peak counts

For one case, a synthetic peptide developed against a biological reference medicine, the guideline sets a floor for what the purity method must be able to see:

"A quantification limit (QL) of 0.1% for LC purity testing is required."

European Medicines Agency, Guideline on the Development and Manufacture of Synthetic Peptides, section 6, retrieved 12 October 2026.

It also sets the points at which impurities have to be reported, identified and qualified:

"peptide-related impurities should be reported above 0.1%, identified above 0.5% and qualified above 1.0%"

European Medicines Agency, Guideline on the Development and Manufacture of Synthetic Peptides, section 4.4.1, retrieved 12 October 2026.

In plain words: under that guideline, a medicine's peptide-related impurities are listed above a tenth of a percent, identified above half a percent, and taken through a further step the guideline calls qualification above one percent. In the biological-reference case, the method must also measure down to a tenth of a percent. A purity figure with no word on what the method can see is harder to judge.

When two things share one peak

A single peak is not always a single substance. Two molecules can leave the column at the same moment and look like one. The guideline deals with this case directly:

"When co-eluting impurities are observed as one peak the qualification threshold of 1.0% applies"

European Medicines Agency, Guideline on the Development and Manufacture of Synthetic Peptides, section 4.4.2, retrieved 12 October 2026.

The guideline also asks, in its section on synthetic peptides developed against a biological reference medicine, for more than one way of looking at purity:

"Characterisation of purity should be addressed using an orthogonal approach"

European Medicines Agency, Guideline on the Development and Manufacture of Synthetic Peptides, section 6, retrieved 12 October 2026.

"Orthogonal" means a second method that separates molecules on a different basis, so an impurity hidden under the main peak in one method shows up in the other. Identity is checked separately, usually by mass spectrometry; see mass spectrometry and peptide identity.

What a 98% figure does not tell you

Three things are easy to read into a purity number that it does not say.

1. How much peptide is in the vial. Purity compares peaks. A powder also holds water and a counter-ion, which do not show in that comparison. That is a different number, explained in peptide content vs purity.
2. That the main peak is the right peptide. HPLC shows how clean a sample is; it does not by itself prove what the main peak is.
3. Anything about the law. A pure substance is not an authorized one. Health Canada's check for that is a different one:

"Check the label before you buy. Authorized prescription drugs in Canada have an eight-digit Drug Identification Number (DIN). You can also check whether a drug has been authorized for sale by searching Health Canada's Drug Product Database."

Health Canada public advisory, 9 Apr 2026, retrieved 4 October 2026.

What to look for on the certificate

The international guide for active ingredients, ICH Q7, says what a certificate of analysis should show for each test:

"The Certificate should list each test performed in accordance with compendial or customer requirements, including the acceptance limits, and the numerical results obtained (if test results are numerical)."

ICH Q7, section 11.42, retrieved 12 October 2026.

For purity, that means three things on the line: the method (for example HPLC with its wavelength), the limit the batch had to meet, and the number it got. A fuller certificate also shows the chromatogram or lists the largest single impurities.

In Canada, a performance claim to the public needs a test behind it. The Competition Act describes reviewable conduct as making a representation:

"in the form of a statement, warranty or guarantee of the performance, efficacy or length of life of a product that is not based on an adequate and proper test thereof, the proof of which lies on the person making the representation"

Competition Act, s. 74.01(1)(b), retrieved 4 October 2026.

A purity figure on a web page is a statement about a product. The certificate is where the test behind it should be visible. For the whole document, line by line, read how to read a peptide certificate of analysis.

More in the quality series: peptide content vs purity, TFA and acetate salts and mass spectrometry and peptide identity. Get the free library updates by email.