Analytical Testing
Analytical data is what separates a peptide from a bottle of powder. This hub explains the methods we use at release, what each one measures, how to read the chromatogram and the mass spectrum, and which additional tests are worth requesting for a particular application.
Reverse-phase HPLC and the purity value
Analytical reverse-phase HPLC separates the peptide from deletion sequences, truncated fragments and oxidised variants on a C18 column with a water-acetonitrile gradient. The purity value is the area of the target peak as a percentage of total integrated area at the detection wavelength, usually 214 or 220 nm. It is a method-dependent number: a different gradient, column or wavelength can change the result, which is why the method is printed on the certificate.
LC-MS and mass confirmation
Electrospray ionisation LC-MS confirms that the dominant component has the expected molecular mass. The observed multiply charged envelope is deconvoluted to a neutral mass and compared with the theoretical value; a difference outside the method tolerance indicates a missing residue, an incomplete deprotection or an unintended modification. Mass confirmation proves identity, not purity, so it is always reported alongside the chromatogram.
MS/MS sequencing and fragment analysis
Tandem mass spectrometry fragments the peptide along the backbone and reads the b- and y-ion series. It is used when a sequence must be verified beyond nominal mass, when isobaric residues are present, or when a customer needs independent evidence for a novel peptide. MS/MS is more demanding than a single mass measurement and is quoted separately.
Net peptide content, water and counter-ions
A lyophilised peptide is not 100 percent peptide. Residual water, trifluoroacetate or acetate counter-ions and salts reduce the net peptide content, sometimes to 70 or 80 percent of the vial weight. Amino-acid analysis, Karl Fischer titration and ion chromatography quantify these contributions. For assays where the molar concentration matters, net peptide content should be measured rather than assumed.
Stability, solubility and forced degradation
Stability work answers a different question from release testing: how the peptide behaves over time under defined temperature, humidity and light. Forced degradation can identify the weak points in a sequence, such as methionine oxidation or asparagine deamidation. Solubility screening in water, buffer and co-solvent systems prevents a costly failure at the bench.
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Frequently asked questions
What does 95 percent HPLC purity mean?
It means the target peak accounts for 95 percent of the integrated peak area in the specified HPLC method. It does not identify the remaining 5 percent, and it does not correct for water, salts or counter-ions, which is why net peptide content is reported separately.
Why does the measured mass differ from the theoretical mass?
Small differences are expected from the charge state and calibration of the instrument. A larger difference usually indicates a missing residue, an incomplete deprotection, oxidation or a salt form. The certificate states the observed and theoretical masses so the difference can be judged directly.
Do I need MS/MS or is a single mass enough?
A single mass confirms the molecular formula. MS/MS is needed when sequence order must be proven, when isobaric residues are present, or when a regulator or reviewer asks for fragment-level evidence. For routine research peptides, mass confirmation with HPLC is normally sufficient.
Can you test a peptide supplied by another manufacturer?
Yes. Independent testing can cover HPLC purity, mass identity, water content, net peptide content and, where requested, MS/MS. The sample is logged with its own identifier and the results are reported against the methods stated in the quotation.
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