Synthetic peptide purification by application of linear solvent strength gradient theory

Abstract The purification of crude synthetic peptide by isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) requires time to determine suitable chromatographic conditions, which limits the use of isocratic RP-HPLC as a purification procedure for the newer, multiple-peptide synthetic techniques. An empirical or semi-empirical procedure to determine the appropriate chromatographic conditions based on a knowledge of the peptide's sequence or molecular weight would allow direct purification of the crude synthetic peptide without the initial exploratory work. Using linear solvent strength gradient theory, a formula was derived which predicts the optimum gradient time from the number of residues in the sought-after peptide and certain chromatographic parameters. Using a variety of crude synthetic peptides of approximately twenty residues and this gradient time, the mass capacity of various commercially available RP-HPLC columns was determined. Typically, the described procedure was capable of isolating