How to read an HPLC chromatogram on a certificate of analysis
Informational guide for research samples — not medical advice. QLD Analytical arranges independent testing; we do not sell or endorse any product.
A purity figure on a certificate of analysis is only as good as the chromatogram behind it. This guide walks through the parts of an HPLC chromatogram you are likely to see on a peptide COA, what each one means, and the questions worth asking when the trace and the headline number do not line up.
What a chromatogram shows
When a certificate of analysis (COA) quotes an HPLC purity figure, that number comes from a chromatogram: a trace recorded by the detector as the sample passes through an analytical HPLC column. Being able to read the trace yourself is the best way to check whether the headline percentage is supported by the data behind it.
A typical peptide chromatogram has two axes:
- Horizontal axis: time, usually in minutes from injection. Components of the sample leave (elute from) the column at different times.
- Vertical axis: detector response, usually UV absorbance (often shown in mAU, milli-absorbance units) at a stated wavelength. For peptides this is commonly 210–220 nm, where the peptide bond absorbs.
Each peak is something the detector saw coming off the column. A clean sample gives one dominant peak and, often, a few much smaller ones. The flat line between peaks is the baseline.
The main peak and retention time
The time at which a peak reaches its maximum is its retention time. In reversed-phase HPLC, the standard method for peptides, the column packing is non-polar (commonly C18-modified silica) and the mobile phase starts mostly aqueous, often with 0.1% trifluoroacetic acid (TFA), before the proportion of acetonitrile is gradually increased. As Bachem’s purification guide describes, polar components elute first and more hydrophobic ones later, so retention depends on how hydrophobic each molecule is.
Two practical points follow. First, retention time is only comparable under the same method: a different column, gradient, flow rate or temperature will shift it, so a peak at 12.4 minutes on one lab’s report and 9.8 minutes on another’s is not, by itself, a discrepancy. Second, retention time on its own does not prove identity. Bachem lists co-elution with a reference sample by analytical HPLC as one identity check for peptides, alongside molecular weight by mass spectrometry, but a single unlabelled peak at some retention time tells you nothing about which molecule it is.
Peak area and area-percent purity
Most chromatograms on a COA come with a peak table. Common columns are peak number, retention time, area, height and area %. Purity is usually the area % of the main peak: its integrated area divided by the total area of all integrated peaks. Bachem describes exactly this approach, with UV detection at 210–220 nm, as the standard purity assessment for peptides.
When you read the table, check that:
- The main peak is identified, either labelled with the compound name or clearly the peak whose area % matches the purity on the front page of the COA.
- The area % values add up to about 100%. If they do not, ask which peaks were excluded and why.
- The purity on the summary page matches the main-peak area % in the table, to the same rounding.
Integration choices matter. The software draws a baseline under each peak and ignores peaks below a set threshold; laboratories also normally exclude system or solvent peaks that appear in a blank run. These are legitimate practices, but a good report states the method conditions so a reader can see what was and was not counted.
What the smaller peaks can be
After solid-phase synthesis, crude peptide contains the target alongside related impurities. Bachem’s purification guide lists deletion peptides, truncated peptides, incompletely deprotected peptides, modified peptides and residual by-products. Purification removes most of them, but closely related molecules can remain at low levels and show up as small peaks near the main one.
The chromatogram alone usually cannot tell you which impurity a small peak is. Assigning peaks needs extra information, typically a mass for each peak from mass spectrometry. What you can read from the trace is how many peaks there are, roughly how large they are relative to the main peak, and whether any sit so close to it that they partly overlap (a “shoulder” on the main peak is worth noting).
What a chromatogram cannot tell you
- Identity. A clean single peak shows a clean profile, not that the material is the compound on the label. Identity needs a separate test, such as mass spectrometry.
- How much peptide is in the vial. Lyophilised peptides also contain water and counter-ions such as TFA or acetate. Bachem notes these are not measured by the same methods used to determine purity, so a high area % does not mean the powder is that percentage peptide by weight. See HPLC purity vs net peptide content.
- Hidden impurities. Anything that co-elutes with the main peak, or does not absorb at the detection wavelength, can be invisible in a UV chromatogram.
- Other lots. A chromatogram describes the sample that was injected on the test date. It says nothing about other batches or how a vial has been stored since.
A quick checklist for any COA chromatogram
- Is a chromatogram provided at all? A purity figure with no trace and no peak table is a claim, not data.
- Can you read the axes? Time and detector response should be visible, with the detection wavelength stated somewhere on the report.
- Is the method described? Look for the column type, mobile phases or gradient, and wavelength.
- Does the sample ID or lot match the vial and the COA front page?
- Does the peak table agree with the headline purity?
- Does the trace look original? Be cautious of heavily cropped images, missing peak tables, or what looks like the same trace reused across different products.
- Is the laboratory named and the report dated?
None of these points proves a document is wrong, but each one that fails is a good reason to ask the supplier for the full report or to get the vial independently tested. Our guide to what a COA should show covers the other fields to check.
Chromatograms on QLD Analytical certificates
QLD Analytical is an independent testing intermediary: samples are analysed offsite by Apex Labs Pty Ltd. Our standard panel reports purity by HPLC, identity by mass spectrometry and net content for the vial. As our HPLC page explains, the downloadable certificate PDF includes the underlying chromatogram pages from the laboratory, so you can review the peak assignments yourself rather than relying on a single number. Published records are listed on the results page, and you can order a test online.
FAQ
Why is peptide purity measured at around 210–220 nm?
The peptide bond absorbs UV light strongly in that range, so the target peptide and most peptide-related impurities are detected. Bachem describes UV detection at 210–220 nm as the standard approach for peptide purity.
Should two labs report the same retention time for the same peptide?
Not necessarily. Retention time depends on the column, gradient, flow rate and temperature, so it is only directly comparable between runs that used the same method.
Does one sharp peak prove the sample is what the label says?
No. It shows a clean chromatographic profile under that method. Identity needs separate confirmation, for example by mass spectrometry.
Is a chromatogram enough to judge if a product is safe for people?
No. A chromatogram is analytical data about one research sample. It is not medical advice or evidence that anything is suitable for human use.
Sources
- Bachem — Quality control of amino acids & peptides: a guide (analytical HPLC with UV at 210–220 nm, main peak area vs total area; co-elution and mass spectrometry for identity; water and counter-ions measured separately).
- Bachem — Peptide purification process & methods: an overview (RP-HPLC on C18 silica, 0.1% TFA/acetonitrile gradient, polar contaminants elute first; types of synthesis-related impurities).
- QLD Analytical — HPLC purity testing.