Peptide separations using fluorescence detection

The formation of fluorophores by the action of o-phthalaldehyde with amino acids and peptides has provided a highly sensitive assay for these compounds. A relatively simple system for the analysis and separation of peptides, in the range 5 nmole to 10 micromole, normally derived from enzymic digestion of proteins, is described. The system comprises a gradient-generating device feeding volatile pyridine buffers via a pump to a column of cation-exchange resin. Eluate from the column is fed through a proportioning pump to a fluorocolorimeter, output from which is displayed on a recorder. For analytical runs the eluate is mixed with o-phthalaldehyde in borate buffer containing Brij 35 and 2-mercaptoethanol prior to its passage into the detector. For preparative work the eluate stream is split, one reacting with 0-phthalaldehyde, the other for collection. Results on the analysis and preparation of tryptic peptides derived from cytochrome c and Salmonella histidinol dehydrogenase are discussed.