Separation of small cationic bioactive peptides by strong ion-exchange chromatography

The high resolving power of a Hyper D cation-exchange column was evaluated to achieve the separation of small cationic bioactive peptides derived from tryptic digest of K-casein: MAIPPK (pI=9.9), MAIPPKK (pI=10.5), KNQDK (pI=9.6) and NQDK (pI=6.35). The influence of pH (1.5-6), gradient slope (2-10 mM sodium chloride/min) and elution of the mixture under isocratic conditions was investigated. Although their physico-chemical properties are very similar, these four peptides were readily resolved with an excellent selectivity and recovery. The selectivity of the exchanger was also expressed toward peptides of the same net positive charge; the most hydrophilic peptide always eluted last. It was also shown that the elution order of these molecules depends on pH. From the observed retention limes and the elution order, we have established a simple approach to linearization of peptide retention behaviour on the S-Hyper D support.