What does "99+% HPLC-verified purity" actually tell you?

"99+% HPLC-verified" is the most repeated claim in the UK research peptide market, and one of the least understood. It is a real, measurable figure, but it answers a narrower question than most buyers assume. This note explains what a purity percentage on a Certificate of Analysis actually measures, what it does not measure, why the difference between 98% and 99+% matters more than one point suggests, and how to check that the number in front of you was measured rather than typed.

What HPLC measures

Reverse-phase high-performance liquid chromatography separates the components of a dissolved sample by how strongly they interact with a hydrophobic column as a solvent gradient runs through it. Each component leaves the column at a characteristic retention time and passes a UV detector. Peptides absorb strongly at 220 nm because of the peptide bond itself, so a detector set at 220 nm registers every peptide-like species in the sample as a peak.

The result is a chromatogram: a trace of absorbance against time. Purity is calculated by area. The area under the main peak, the target sequence, is divided by the total area of all peaks in the run. If the target peak accounts for 99.2% of total peak area, the COA states 99.2% purity. That is the whole calculation.

What "99+% purity" does not tell you

Because purity is a ratio of peak areas at one wavelength, several things sit outside it entirely:

• Identity. A chromatogram shows that one dominant species is present, not which species it is. A well-synthesised wrong peptide can also run at 99%. Identity is a separate test, normally electrospray mass spectrometry, where the observed mass must match the theoretical mass of the sequence within about 1 Da. Purity without identity is half a result.
• Net peptide content. Lyophilised peptides carry bound water and counter-ions (usually trifluoroacetate or acetate) left over from purification. A vial labelled 10 mg gross may hold 7-9 mg of actual peptide. HPLC purity says nothing about this, and it is the figure that matters when preparing a concentration-critical assay. Researchers who need it should ask whether the COA reports net content, or account for it in preparation.
• Non-peptide contaminants. Species that do not absorb at 220 nm, such as residual solvents or inorganic salts, are invisible to the detector and are not in the denominator.
• Sterility or endotoxin. HPLC is a chemical assay, not a microbiological one. Alphex does not run endotoxin or water-content assays; the COA reports HPLC purity, and the specification table on each product page states this plainly.

None of that makes the purity figure unimportant. It means the figure should be read alongside the identity result and, where relevant, the net content, rather than as a summary of everything about the vial.

Why 98% and 99+% are further apart than they look

The industry default threshold is around 98%. Alphex sets 99+%. One percentage point sounds trivial. Expressed the other way round it is not: 98% purity means up to 2% of the peptide content is something other than the target sequence, and 99+% means under 1%. That is a halving of the impurity fraction, and the impurities are the part of the vial that behaves unpredictably.

What sits in that impurity fraction for a synthetic peptide is fairly well characterised. Solid-phase synthesis builds the chain one residue at a time, and each coupling step is slightly less than perfect. The failure modes are deletion sequences (a residue skipped), truncated sequences (chain growth stopped early), incompletely removed protecting groups, and oxidation or deamidation of sensitive residues. Every one of these is a peptide, every one absorbs at 220 nm, and every one is close enough in structure to the target that it may still interact with the same assay system in an unrelated way. Halving that fraction is a meaningful reduction in noise for anyone trying to reproduce a result.

The other reason the threshold matters is what it says about the supplier's process. Reaching 99+% consistently across a catalogue requires either better synthesis or an additional purification pass, both of which cost money. A supplier that publishes 98% as its threshold has decided that the cost is not worth it. That is a legitimate commercial choice, but it is useful information when comparing UK research peptide suppliers.

How to tell a measured figure from a typed one

A purity percentage on its own is a claim. A chromatogram is evidence. The checks below catch most problem COAs, and they take a couple of minutes:

1. Is the chromatogram shown? A genuine COA includes the trace, with a visible baseline, retention time on the x-axis and a labelled main peak. A COA that states "Purity: 99.4%" with no trace should be treated as unverified. Ask for the trace; a real lab has it.
2. Do the numbers on the trace match the numbers in the table? The peak area percentage printed on the chromatogram should equal the purity figure in the results table. Mismatches usually mean the table was edited after the fact.
3. Is the batch number on the COA the batch number on the vial? A COA for a different batch, or a COA with no batch number at all, tells you about a batch you did not receive. Alphex prints the batch number on the label and issues the COA against that same number.
4. Is the testing laboratory named? An independent lab puts its name, address and a report reference on the document. Anonymised or supplier-issued COAs remove the one party with no incentive to round the number up.
5. Is there an identity result alongside the purity result? An observed mass from ESI-MS, next to the theoretical mass, with the difference stated. Purity with no identity is the most common gap we see.
6. Is the test date plausible? The analysis date should be after synthesis and reasonably close to it. A COA dated years before the batch could exist has been reused.

The full walkthrough, with the tells of a fabricated document, is in how to read a research peptide COA.

What Alphex reports and how to check it before you order

Every batch Alphex lists has been through reverse-phase HPLC at 220 nm against a reference standard, with an acceptance threshold of 99+% area purity, and the measured purity with its stated uncertainty. Both results go onto a Certificate of Analysis carrying the same batch number as the vial label, with the chromatogram included and the issuing laboratory named. The method is described step by step in how Alphex tests every batch.

You do not have to take that on trust. The in-stock batch COA for any product is available before purchase: use the COA request form or the "Request the in-stock batch COA" link on each product page, and we reply with the certificate and chromatogram. If a batch you receive tests below the purity stated on its certificate in your own independent analysis, we replace or refund it.

Summary

• HPLC purity is the main peak's share of total peak area at 220 nm. It measures how much of the peptide content is the target sequence.
• It does not measure identity, net peptide content, non-UV-absorbing contaminants or sterility. Read it with the MS identity result.
• 99+% versus 98% halves the impurity fraction, and the impurities are structurally similar peptides that add noise to reproducible work.
• A percentage without a chromatogram, batch number, named lab and identity result is a claim, not a measurement. Ask for the trace.

Sources and further reading

• ICH Quality Guidelines (Q1A stability, Q3A/Q3B impurities, Q6 specifications): the reference framework for purity, impurity and stability testing

All Alphex products are supplied for laboratory and in-vitro research by qualified researchers only. They are not authorised for human or veterinary use.

Research use only. Alphex products are supplied exclusively for laboratory and in-vitro research by qualified researchers. They are not authorised for human or veterinary use, and nothing in this note is advice on any such use.

Published 19 May 2026, last reviewed 16 August 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.

Alphex Research Team

Alphex Limited, UK research peptide supplier

The Alphex research team writes and maintains these notes for UK laboratories buying research peptides. Every batch Alphex lists is verified by reverse-phase HPLC with a batch-matched Certificate of Analysis. Content covers laboratory and in-vitro research only.