Photodiode array spectral identification of modified amino acids in synthetic peptides following high resolution HPLC separation.

The products of peptide synthesis are routinely purified by reversed-phase HPLC with single wavelength detection at 214 nm. Additional information regarding the peptide product and any contaminating sequences can be obtained by high resolution HPLC combined with high sensitivity spectral analysis using photodiode array detection. Full spectral characterization can be used to ascertain the presence of aromatic amino acid residues as well as side chain modifications such as blocking groups for protection of labile side chains. Many of the protecting groups used in solid phase synthesis contain distinctive structural features that can be identified by their spectra. Although the synthesis protocol provides for hydrolysis of these groups, it is possible that the product mixture may include some residual blocked material. Since the presence of blocking groups will affect the properties of the synthesized peptide, detection and complete removal are essential. Spectral analysis using photodiode array detection in conjunction with high resolution HPLC separations can be used to identify protected amino acid residues in synthetic peptides.