HPLC and mass spectrometry: basics of peptide analytics

HPLC: separate and measure

In high-performance liquid chromatography (HPLC), the dissolved sample is pumped through a column under pressure. The components travel at different speeds depending on their interaction with the column material and leave the column at different times – the retention time. A detector (for peptides usually UV at 210–220 nm) records a chromatogram: a signal over time with one peak per substance.

Column, solvent and gradient

Peptide work is usually done in reversed phase: the material in the column is non-polar, commonly C18-modified silica, while the mobile phase is a mixture of water and acetonitrile with an acid additive. The share of organic solvent is raised during the run, which is the gradient. More hydrophobic components are held back longer and elute later. Particle size, temperature, flow rate and the slope of the gradient decide how far two similar substances are pulled apart, which is why a retention time only means something together with the conditions under which it was recorded.

How this becomes a purity figure

The area under the main peak is set in relation to the total area of all peaks. The result is area percent – the number that appears as purity in the COA. Secondary peaks typically come from truncated sequences, oxidised variants or residual protecting groups from synthesis. Since the figure rests on integration, the integration threshold matters: what the software does not integrate does not enter the total area, and a peak hidden in the shoulder of the main one is not resolved at all.

Mass spectrometry: identity

Mass spectrometry (MS) ionises the sample and measures the mass-to-charge ratio (m/z). Peptides often carry several charges, so one molecule appears as a series of signals. From these the molecular mass can be calculated and compared with the theoretical value of the sequence. If the two match, identity is confirmed.

What agreement means in practice

Agreement is never exact. It falls within a tolerance set by the instrument, and the reference value has to be the right one: monoisotopic mass and average mass differ for a molecule of several thousand daltons, and comparing one against the other produces a mismatch that is only arithmetic. A deviation of about 18 daltons points to loss or gain of water, 16 to an added oxygen; a shift of a whole residue points to a different sequence rather than a measurement error.

Why both are needed

HPLC alone shows that one substance dominates – not which one. MS alone shows that the right molecule is present – not in what proportion. Only together do they answer the two questions a research reagent has to satisfy. Neither method covers everything: components without a suitable chromophore give a weak UV signal, and inorganic residues, endotoxins or solvent traces need tests of their own.

Terms from the certificate

• Retention time (RT): time until elution from the column; characteristic of the substance under the chosen conditions.
• Area percent: share of the main peak in the total area – the purity figure.
• m/z: mass-to-charge ratio of an ion in the mass spectrum.
• Theoretical mass: molecular mass calculated from the sequence; reference value for MS.
• Gradient: programmed change of solvent composition during a run.
• Baseline: detector signal with nothing eluting; drift makes small peaks harder to integrate.

Where the results appear

Which tests GPeptides regularly commissions is described in the Analytics and Quality Assurance section, and the reports themselves are published in the lab area under the product they refer to. Material without a published report is marked there as pending. The order of steps behind this is set out in the guide to quality control.

How to read such a report line by line is explained in the guide Reading a certificate of analysis; what the resulting percentage covers and what it leaves out is the subject of the purity guide.