Abstract
Analysis of peptides by reverse-phase high-pressure liquid chromatography would be simplified if retention times could be predicted by summing the contribution to retention of each of the peptide's amino acid side chains. This paper describes the derivation of values (“retention coefficients”) that represent the contribution to retention of each of the common amino acids and end groups. Peptide retention times were determined on a Bio-Rad “ODS” column at room temperature with a linear gradient from 0.1 M NaclO4, pH 7.4 or 2.1, at 0 min to 60% acetonitrile/0.1 M NaclO4 at 80 min. The NaclO4, a chaotropic agent, was added to improve peak shape and to minimize conformational effects. Retention coefficients for the amino acids were computed by using a Hewlett-Packard 9815A calculator programmed to change the retention coefficients for all amino acids sequentially to obtain a maximum correlation between actual and predicted retention times. Correlations of 0.999 at pH 7.4 and 0.997 at pH 2.1 were obtained for 25 peptides including glucagon, oxytocin, [Met]enkephalin, neurotensin, and somatostatin. This high degree of correlation suggests that, for peptides containing up to 20 residues, retention is primarily due to partition processes that involve all the residues. Although steric or conformational factors do have some effect on retention, the data suggest that under the above chromatographic conditions the retention of peptides containing up to 20 residues can be predicted solely on the basis of their amino acid composition. This possibility was tested by using data taken from the literature.
Keywords: lipophilicity, separation techniques
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