Peptide Analytical Methods Compared: HPLC, LC-MS, Amino Acid Analysis, NMR and Karl Fischer

Reversed-phase HPLC with UV detection: purity

A peptide is injected onto a reversed-phase column and eluted with a gradient of increasing organic solvent. The detector, usually set at 214 nm, records the absorbance of the peptide bond. The area of the main peak divided by the total area gives the HPLC purity. The result depends on the column, the gradient, the wavelength and the integration, so a report should give the method.

It cannot see compounds that do not absorb at 214 nm and does not show the amount of peptide in the vial.

LC-MS: identity and impurity assignment

Coupling the HPLC to a mass spectrometer assigns a mass to each peak. The main peak should have the expected mass, and minor peaks can be explained with known mass shifts such as oxidation or deamidation. High-resolution instruments allow the exact mass to be compared with the calculated one.

LC-MS is the single most informative test for peptide identity, but it is not a content assay and does not distinguish D- from L-amino acids.

Amino acid analysis: content

In amino acid analysis the peptide is hydrolysed to free amino acids, which are separated and quantified against standards. The amounts of the amino acids, compared with the known composition, give the amount of peptide present. It is the traditional method for net peptide content and also verifies the amino acid composition.

Some residues, such as tryptophan and cysteine, are partly destroyed during hydrolysis and need special treatment.

Quantitative NMR

Quantitative NMR compares the signals of the peptide with those of an internal standard of known purity and can measure peptide content, residual solvents and counter-ions such as trifluoroacetate in a single experiment. It needs more material and a specialised laboratory than HPLC, and it works best for peptides that give resolved signals.

Karl Fischer titration and loss on drying: water

Water is part of the powder but not of the peptide. Karl Fischer titration measures it selectively and precisely, and loss on drying measures all volatile components. Water content is needed to calculate net peptide content and changes when the material takes up moisture from the air.

Counter-ions, stereochemistry and sterility

Ion chromatography and 19F NMR quantify trifluoroacetate, and ion chromatography also measures acetate and chloride. Enantiomeric purity is determined by hydrolysing the peptide and analysing the amino acids with a chiral column or a derivatising agent, or by direct chiral chromatography of the intact peptide.

Sterility and bacterial endotoxin are determined by microbiological tests and are relevant for products that are labelled sterile.

Putting it together

A COA that reports only HPLC purity answers one of at least four questions. For a given use, decide which properties matter: identity is almost always essential, purity matters for most work, content matters when the concentration of a solution has to be known, and counter-ion and water matter when comparing the amounts of different batches.

The compound pages on this site list the expected mass of each compound so that the identity question can be checked quickly.