The headline on a certificate of analysis says something like “Purity: 99.1%”. The chromatogram underneath is where that number comes from, and it often tells you more than the number does. This guide walks through an HPLC chromatogram the way an analytical chemist reads one: the axes, the main peak, the small peaks, the baseline, and the details that separate a trustworthy report from a decorative one.
The short answer
- An HPLC chromatogram plots detector signal, usually UV absorbance, against time. Each peak is a component that left the column at its own retention time.
- Peptides are usually analyzed by reversed-phase HPLC on a C18 column, with a water-to-acetonitrile gradient containing a little TFA, and UV detection around 214 to 220 nm, where the peptide bond absorbs.
- HPLC purity is usually the main peak’s area as a percentage of all integrated peaks. It measures how much of the UV-visible material is one compound, not the compound’s identity and not how much peptide is in the vial.
- Small peaks near the main one are typically related impurities from synthesis or storage, such as shortened sequences or oxidized and deamidated forms.
- A useful report shows the trace, the integration table, the method conditions, the laboratory and the batch number. A bare percentage tells you little.
What HPLC does
High-performance liquid chromatography pushes a dissolved sample through a column packed with tiny particles under high pressure. In reversed-phase HPLC, the standard for peptides, the particles carry an oily coating, often C18 chains. The liquid flowing through starts mostly as water and gradually becomes richer in acetonitrile. Peptides stick to the oily coating and are released one by one as the liquid becomes strong enough to carry them off; more hydrophobic molecules hold on longer.
A detector at the column’s exit records what comes out. For peptides it is usually a UV detector set around 214 to 220 nm, where every peptide bond absorbs light. Some labs couple the HPLC to a mass spectrometer (LC-MS), which adds the mass of each peak; our guide to reading a peptide mass spectrum covers that half of the story.
Reading the trace
- The x-axis is time. The point where a peak tops out is its retention time. Under fixed conditions a given peptide elutes at a consistent time, but retention time alone does not prove what a peak is.
- The y-axis is signal. Taller, wider peaks mean more UV-absorbing material. The area under a peak, not its height, is what gets measured.
- The baseline is the flat line between peaks. A drifting or noisy baseline makes small peaks harder to measure.
- The solvent front is the disturbance near the start, from material that does not stick to the column at all. It is excluded from purity, and so are peaks that also appear when a blank is injected.
How purity is calculated
Software draws a line under each peak and calculates its area, a step called integration. HPLC purity is then usually reported as area percent: the main peak’s area divided by the total area of all integrated peaks. If the main peak covers 98.6% of the counted area, purity is 98.6%.
That makes the integration settings important. Where the software starts and stops each peak, and which small peaks it counts, both move the result, so a good report includes the integration table listing every peak with its retention time and area. Peak shape matters too: a sharp, symmetrical main peak is a good sign, while a shoulder on its side can mean an impurity hiding underneath. Our HPLC testing guide explains why results also depend on the column, gradient and wavelength.
See how our Janoshik certificates are listed, by batch.
View lab testingWhat the small peaks usually are
| Impurity | Where it comes from | Where it tends to appear |
|---|---|---|
| Deletion and truncated sequences | Incomplete steps during solid-phase synthesis | Close to the main peak, often slightly earlier |
| Oxidized forms | Methionine, cysteine or tryptophan oxidizing during synthesis or storage | Often just before the main peak |
| Deamidated and rearranged forms | Asparagine and aspartic acid reacting over time | Close to the main peak, sometimes barely separated |
| Protecting-group leftovers | Chemical groups not fully removed after synthesis | Often later, as they are more hydrophobic |
| Counter-ion and system peaks | TFA, buffer components or the solvent itself | Usually near the start; excluded when identified |
Our guides to how research peptides are made and how peptides degrade explain where these impurities come from. A re-test of a stored sample that shows new peaks next to the main one is usually a sign of degradation.
What a chromatogram cannot tell you
- Identity. A tall single peak proves the sample is mostly one thing, not that it is the right thing. Mass spectrometry confirms the molecule.
- Hidden impurities. Impurities that elute at exactly the same time as the main peak, including some mirror-image D/L forms, can sit inside it unseen. See our guide to D-amino acids.
- Invisible material. Water, salts and counter-ions barely absorb at 214 nm, so a 99% purity powder can still be well under 99% peptide by weight. See net peptide content.
- Biological contaminants. Endotoxin does not show up on an HPLC trace; see endotoxin in research peptides.
Red flags on a report
Reassuring signs
- The full trace and an integration table are shown.
- Method details: column, gradient, flow rate and wavelength.
- A named laboratory, a test date and a batch number that matches the vial.
- Identity confirmed separately by mass spectrometry.
Reasons for caution
- A purity number with no trace.
- Exactly 100.0%, which real methods rarely produce.
- Identical-looking traces, down to the noise, on reports for different batches or compounds.
- No batch number, or one that does not match the label.
For a complete walk-through of the rest of the certificate, read our peptide COA guide, and for the warning signs of poor-quality products more broadly, how to spot fake or underdosed peptides. The same report checks are worth running when you are choosing a research peptide supplier in Canada.
Practise on a real certificate
Janoshik test reports for the research peptides below are listed on our Lab Testing page. Open one alongside this guide, read the purity result, then compare its batch number with your vial.
BPC-15715-amino-acid research peptideView product
GHK-CuCopper tripeptideView product
TB-500Thymosin beta-4 research peptideView product
KPVLys-Pro-Val tripeptideView product
Research use only. Pure Peptides Canada supplies laboratory reagents that are not for human or veterinary use. This article is general education about analytical methods.
Frequently asked questions
What is an HPLC chromatogram?
It is the graph produced by high-performance liquid chromatography: detector signal, usually UV absorbance, plotted against time. Each peak represents a component of the sample leaving the column at its own retention time.
How do you read HPLC results for a peptide?
Find the main peak and its retention time, check that it is sharp and symmetrical, look at the small peaks around it, and read the integration table. Purity is usually the main peak’s share of the total integrated peak area, excluding the solvent front.
How is HPLC purity calculated?
Usually as area percent: the area under the main peak divided by the combined area of all integrated peaks, multiplied by 100. Integration settings and method conditions can shift the figure slightly.
What do the small peaks on a peptide chromatogram mean?
They are usually related impurities, such as deletion or truncated sequences from synthesis, or oxidized and deamidated forms that appear during storage. Peaks near the very start are typically solvent or counter-ion signals and are excluded.
Can HPLC confirm a peptide’s identity?
No. HPLC shows how much of the UV-visible material is one compound. Confirming that the compound is the intended peptide requires mass spectrometry, ideally reported alongside the HPLC result.
What wavelength is used for peptide HPLC?
Most peptide methods detect UV absorbance around 214 to 220 nm, where the peptide bond absorbs. Some methods also use 280 nm, where tryptophan and tyrosine absorb.
Certificates you can actually read
Janoshik certificates listed by batch on our Lab Testing page, research peptides priced in Canadian dollars and shipped tracked from inside Canada.
References
- D’Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2–30. DOI
- International Council for Harmonisation. ICH Q2(R2): Validation of analytical procedures. EMA
- US Food and Drug Administration. ANDAs for certain highly purified synthetic peptide drug products that refer to listed drugs of rDNA origin. Guidance for industry, May 2021.
For laboratory research use only. Not for human or veterinary use. This article is educational.