Peptide Purification

Purpose

Cleavage from the resin delivers a mixture. Purification separates the target sequence from the process-related impurities described in synthesis — deletion and truncated sequences, residual protecting groups, and side-reaction products — so that subsequent analytical work reflects the intended molecule.

Reversed-phase separation

The dominant method is reversed-phase high-performance liquid chromatography. The stationary phase is nonpolar (typically C18-bonded silica); the mobile phase starts rich in water and becomes progressively richer in organic solvent. Peptides bind according to hydrophobicity and elute in order, with more hydrophobic species retained longer. Gradients are tuned per peptide, which is why purification is developed rather than applied blindly.

Detection

Peptide bonds absorb in the far UV, so detection is commonly performed around 214 nm, where most peptides respond regardless of composition. Peptides containing tryptophan or tyrosine also absorb near 280 nm. Because absorbance depends on composition and on wavelength, purity figures are only comparable when the method is comparable.

Fraction collection

Eluting material is collected as a series of fractions, each analysed, and those meeting the chosen criterion are pooled. In practice the target is the main peak, with judgement applied to closely eluting species that may be difficult to separate.

Desalting and lyophilisation

Pooled fractions typically contain organic solvent and buffer components. These are removed by desalting or buffer exchange before the peptide is freeze-dried. The result is a stable lyophilised powder, which is the form normally shipped and stored.

Documented per batch

Purification is part of the process record for each batch, and its outcome is reflected in that batch's certificate of analysis rather than assumed from a previous run.