Comparison of retention behavior of oligolysine and oligoarginine in ion-pairing chromatography using heptafluorobutyric acid

This paper describes the retention behavior of oligolysine and oligoarginine peptides of different lengths as a function of heptafluorobutyric acid (HFBA) concentration in ion-pairing reversed-phase chromatography in isocratic elution. A mixture of oligolysine and a mixture of oligoarginine with number of amino acid residues (dp) from two to eight were conveniently prepared by one-pot protease-catalyzed synthesis. Analysis of the logarithm of the retention factor k as a function of [HFBA] for each oligopeptide component, using a closed pairing model, provided values for (1) number (n) of paired HFBA anions per peptide molecule, (2) equilibrium constant (K _ip,m) for ion pairing between oligopeptides and HFBA anions, and (3) product of the phase ratio and the distribution constant of the paired oligopeptide between the mobile and stationary phases (βK _d,ip). We found that βK _d,ip of oligoarginine is larger compared with oligolysine having the same dp. A linear relationship was obtained for ln βK _d,ip as a function of n + g·dp. By optimizing constant g separately for oligolysine and oligoarginine, we determined that g is larger for oligoarginine, in agreement with the higher hydrophobicity of arginine residues. Plotting the fraction of paired oligoarginine and oligolysine as a function of [HFBA] shows that the cooperative effect in forming ion pairs is greater for oligoarginine than oligolysine. [Figure not available: see fulltext.]