Methods for determining the retention of peptides in reverse phase chromatography using linear solvent strength theory

A method for separating or isolating a peptide with a known amino acid sequence from a sample, the method comprising:

• (a) providing a sample comprising the peptide;
• (b) determining for the peptide a peptide charge, a peptide length, a hydrophobicity of the peptide, a distribution of hydrophobic amino acids within the peptide and a number of occurrences of each amino acid in the peptide;
• (c) using the function:
• S=C1*ZC2+C3*NC4+C5*HIC6+C7/Z+C8/N+C9/HI+C10*ZN+C11*ZHI+C12*NHI+C13*Z*N*HIC14+B+Saa+SSSF;
• to determine a slope S for the peptide, wherein the slope S represents a change in retention of the peptide in a reverse phase chromatography column with respect to a change in a mobile phase gradient slope during reverse-phase chromatographic separation and where N is the peptide length, Z is the peptide charge, HI is the hydrophobicity of the peptide, Saa is calculated based on the number of occurrences of each amino acid in the peptide, SSSF is calculated based on the sequence of the peptide and where constant B and coefficients C1 to C14 are empirically determined;
• (d) determining a retention time for the peptide in a reverse phase chromatography column based on the slope S;
• (e) introducing the sample into the reverse phase chromatography column with a mobile phase;
• and
• (f) collecting an eluent out of the column at the retention time determined in (c), wherein the eluent contains the peptide.