Investigation of the Influence of Hydrophobic Ions as Mobile Phase Additives on the Liquid Chromatographic Separation of Amino Acids and Peptides

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Abstract
Tetraalkylammonium, R4N+, and alkylsulfonate, RSO3 −, salts were evaluated as mobile phase additives for the separation of amino acids and peptides. The former were used in a basic mobile phase and the latter in an acidic one, conditions which convert the terminal carboxyl or amine groups in amino acids and peptides and the acidic or basic side chains if present into anionic or cationic forms, respectively. Because of the required strongly basic or acidic mobile phase pH, a polystyrene-divinylbenzene copolymer, PRP-1, was used as the reversed stationary phase. The retention is suggested to follow a dynamic interaction involving two major equilibria, namely retention of the hydrophobic ion and an ion exchange between the co-ion accompanying the hydrophobic ion and the amino acid or peptide ion of opposite charge. The effect of amino acid and peptide structure on retention is discussed. Key mobile phase variables are identified; a major one is the optimization of the hydrophobic ion concentration-mobile phase solvent composition to provide a sufficient number of charge sites on the stationary phase due to hydrophobic ion retention. Several separations are shown which focus on the advantages offered by using hydrophobic ions as mobile phase additives. In general, RSO3 − salts appear to be more versatile than R4N+ salts in improving selectivity and resolution in amino acid and peptide separations.