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Peptide purity testing normally combines two techniques. Reverse-phase HPLC with UV detection at about 210–220 nm separates the target peptide from related impurities and reports purity as the main peak's share of total peak area; mass spectrometry confirms identity by comparing the observed mass with the theoretical mass of the sequence.1 Neither measures how much of the powder is peptide, and neither on its own proves the material is exactly what the label says.
This guide explains how each method works, what the readouts mean, where the blind spots are, and what else a complete analysis includes.
| Method | Question it answers | Typical readout | Blind spots |
|---|---|---|---|
| Reverse-phase HPLC with UV detection | How pure is it? | Main-peak area % at 210–220 nm | Co-elution; unequal UV response; water and salts invisible |
| Mass spectrometry (ESI or MALDI) | Is it the right molecule? | Observed mass or m/z versus theoretical | Isomers share a mass; signal size is not a purity measure |
| LC-MS | What mass sits under each peak? | Mass spectrum for each chromatographic peak | TFA in the mobile phase suppresses the ESI signal |
| Tandem MS (MS/MS) | Is the sequence in the right order? | Fragment-ion ladder | Requires expert interpretation |
| Amino acid analysis | What is the composition, and how much peptide? | Amino acid ratios and quantity | No sequence information |
| Karl Fischer; ion chromatography | How much water and counter-ion? | % water; % TFA or acetate | Says nothing about identity or purity |
In reverse-phase HPLC (RP-HPLC), the sample is pumped through a column packed with a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent. More hydrophobic molecules are retained longer, so a peptide and its near-relatives leave the column at different times. Silica-based packings remain the most widely used for peptide HPLC, and in the experience of Mant, Hodges and colleagues the best approach to most analytical peptide separations is a linear gradient from aqueous trifluoroacetic acid (TFA) to TFA in acetonitrile at about pH 2 and room temperature, with resolution tuned by the steepness of the gradient (generally 0.5–2.0% acetonitrile per minute). TFA also acts as an ion-pairing agent, which helps separate complex mixtures.2
The peptide bond itself absorbs in the far ultraviolet, which is why peptide purity is read at 210–220 nm.1 At 280 nm, by contrast, only peptides containing tryptophan or tyrosine absorb strongly. Kuipers and Gruppen measured the molar extinction coefficient of the peptide bond at 214 nm as 923 M⁻¹ cm⁻¹, and found that tryptophan absorbs about 30 times more strongly, while phenylalanine, tyrosine and histidine absorb about six times more.3
A chromatogram plots detector response against time. Purity is calculated as the area of the main peak relative to the total area of all peaks.1 On a good trace, look for a single dominant peak, small and well-separated impurity peaks, a flat baseline and stated integration settings, because the choice of which peaks to integrate changes the answer.
Two practices close some of these gaps. The first is co-elution with a reference sample: if the test material and an authentic standard of the same peptide give one peak when injected together, that supports identity, and Bachem lists it among its identity tests for peptides.1 The second is an orthogonal separation. Size-exclusion and ion-exchange HPLC separate peptides by size and charge rather than hydrophobicity, and Mant and colleagues describe a mixed-mode hydrophilic interaction and cation-exchange approach as a useful complement to reverse-phase HPLC.2 An impurity that hides under the main peak in one system often separates in another.
A mass spectrometer ionises molecules and measures their mass-to-charge ratio (m/z). Two soft ionisation methods dominate peptide work:
PubChem lists BPC-157 (GEPPPGKPADDAGLV) as C62H98N16O22, with a molar mass of 1419.5 g/mol and a monoisotopic mass of 1418.70 Da.6 Adding protons (1.0073 Da each) and dividing by the charge gives the ions an ESI instrument would look for:
| Ion | Charge | Expected m/z |
|---|---|---|
| [M+H]+ | 1+ | 1419.71 |
| [M+2H]2+ | 2+ | 710.36 |
| [M+3H]3+ | 3+ | 473.91 |
An observed mass that matches within the instrument's stated accuracy supports identity; a mass that matches a different peptide means the material, or the paperwork, is not what it claims. Masses that differ from the expected value by a characteristic amount point to specific impurities, as the table further down shows. The compound itself is covered in our BPC-157 research overview, and research material is listed on the BPC-157 product page.
Each peak in a peptide mass spectrum is really a cluster. Carbon-13 and other heavy isotopes produce a series of peaks one mass unit apart, and on the m/z scale that spacing shrinks to 1/z: about 1.0 for a singly charged ion, 0.5 for a doubly charged ion and 0.33 for a triply charged one. Reading the spacing is how an analyst, or the software, assigns the charge state before calculating the mass. The first peak of the cluster corresponds to the monoisotopic mass; the average (molar) mass reflects the whole isotope distribution, which is why BPC-157 can be described as 1418.7 Da or 1419.5 g/mol and both figures are right. What matters is that an analytical report says which one it is quoting, and that the instrument resolved the cluster well enough to support it.
Coupling HPLC directly to the mass spectrometer (LC-MS) assigns a mass to each chromatographic peak, which catches co-eluting or unexpected components that UV alone would miss. There is a trade-off in the mobile phase. In a systematic comparison, TFA produced slightly narrower peaks than formic acid but significantly more suppression of the electrospray signal7, an effect attributed to ion pairing and to changes in surface tension.8 LC-MS methods therefore commonly use formic acid while UV purity methods use TFA, so the two chromatograms for one sample may not look identical.
Tandem mass spectrometry (MS/MS) isolates one ion, fragments it along the peptide backbone and reads the sequence from the ladder of fragment masses.9 It can confirm the order of residues, which an intact mass cannot: two peptides containing the same amino acids in a different order have exactly the same intact mass.
Most research peptides are made by solid-phase peptide synthesis (SPPS), which leaves characteristic by-products. A review of impurities in peptide medicines groups them into synthesis-related impurities (deletions and insertions of amino acids, racemisation, incompletely removed protecting groups, side-chain reactions, oxidation, dimers and oligomers, counter-ions and contamination by unrelated peptides) and degradation products (β-elimination, diketopiperazine, pyroglutamate and succinimide formation).10
| Impurity | Typical origin | Mass difference from target | Best detected by |
|---|---|---|---|
| Deletion sequence | Inefficient Fmoc deprotection during SPPS | Minus one residue (for example −57.02 Da for glycine) | LC-MS |
| Insertion sequence | Excess amino acid reagent during SPPS | Plus one residue | LC-MS |
| Diastereomer | Racemisation of a residue | 0 Da | HPLC separation; mass alone cannot tell |
| Oxidation (for example methionine sulfoxide) | Air, light or storage | +15.99 Da | LC-MS |
| Deamidation (Asn or Gln) | Moisture, pH and temperature | +0.98 Da | High-resolution LC-MS |
| Dimer | Oxidation or aggregation | About twice the mass | LC-MS; size-exclusion HPLC |
Origins follow the review cited above10; mass differences are calculated from monoisotopic atomic masses. For how storage conditions drive oxidation and deamidation, see how to store peptides.
A complete analysis also accounts for the mass that is not peptide. Bachem lists water by Karl Fischer titration, residual solvents by gas chromatography, acetate by HPLC or ion chromatography, residual TFA by ion chromatography, and nitrogen content from elemental analysis as a measure of peptide content. Amino acid analysis breaks the peptide down with strong acid and quantifies the released amino acids, confirming composition but not sequence.1 Endotoxin is measured with the bacterial endotoxins test in USP chapter <85>, which the FDA's March 2026 guidance describes as covering gel-clot, photometric and kinetic methods.11 How these results should appear on paper is covered in how to read a peptide certificate of analysis.
Whatever the answers, an HPLC percentage and a matching mass together show that one main component of the expected mass dominates the UV trace. On their own they do not prove the sequence, the stereochemistry or the peptide content. For how synthesis and lyophilisation produce the material being tested, see what research peptides are.
HPLC peptide testing separates a peptide sample on a reverse-phase column using a water-to-acetonitrile gradient, usually containing trifluoroacetic acid, and detects the components by ultraviolet absorbance at about 210–220 nm. Purity is reported as the main peak's area as a percentage of all peak areas. It shows how dominant the main component is, not its identity or how much of the powder is peptide.
Mass spectrometry measures the mass-to-charge ratio of ionised molecules. Electrospray ionisation gives a series of multiply charged ions that software converts into one molecular mass, while MALDI mostly gives singly charged ions. Identity is supported when the observed mass matches the theoretical mass of the sequence, and tandem MS fragments the peptide to confirm the order of its residues.
Because the peptide bond absorbs ultraviolet light in that region, so every peptide gives a signal there, whereas at 280 nm only peptides containing tryptophan or tyrosine absorb strongly. Absorbance at 214 nm still varies with composition: tryptophan absorbs about 30 times more than a peptide bond, and phenylalanine, tyrosine and histidine about six times more.
No. Impurities that co-elute with the main peak, or absorb weakly at the detection wavelength, can be missed or under-counted, and water and counter-ions are not part of the purity figure at all. Diastereomers have exactly the same mass, so mass spectrometry cannot distinguish them either; only chromatographic separation can. That is why purity, identity and content are measured separately.
Purity, measured by HPLC, is the share of UV-absorbing material that is the target peptide. Net peptide content is the share of the whole powder that is peptide, as opposed to non-peptide material such as water and counter-ions like trifluoroacetate. A peptide can be highly pure yet have a much lower content, so both matter when you calculate concentrations.
A section-by-section guide to reading a peptide certificate of analysis: identity, HPLC purity, net peptide content, counter-ions, water, endotoxin, and the red flags that mean a COA does not check out.
A plain-English primer on research peptides: what a peptide is, how peptides are built one amino acid at a time on a resin bead, why they arrive as a freeze-dried powder, and what "research use only" does and does not mean.
A laboratory guide to storing lyophilised and reconstituted peptides: freezer temperatures, moisture, oxidation-prone residues, light, freeze–thaw cycles and adsorption to containers.
