[Deviation of size-exclusion chromatographic behavior of tryptophan on TOYOPEARL HW-40S column]

A deviation of size-exclusion chromatographic behavior of amino acids on TOYOPEARL HW-40S column is reported. On the column 18 amino acids were not eluted in the same fraction in the total volume, but separated into 5 fractions with tryptophan as the last one far behind the total volume. Interaction between tryptophan and the matrices was investigated under different conditions in order to elucidate the chemical nature of the deviation. It was found that addition of trifluoroacetic acid and sodium chloride of high concentration in mobile phase, dramatically increased the retention time of trypophan, while addition of acetonitrile, sodium chloride of lower concentration and the hydrogen bond breaker urea reduced the interaction. It is concluded that adsorption of tryptophan on TOYOPEARL HW-40S is basically due to the hydrophobic nature with limited influence of hydrogen bond and ionic interaction. The former factor can be eliminated by keeping acentonitrile concentration over 50% (V/V), while the latter two can be reduced to the minimum by adding 0.17mol/L sodium chloride and 3.51mol/L urea. The results not only lead to better understanding of size-exclusion chromatography of this matrices, but also help to apply hydrophobic chromatography of this matrices to the isolation of amino acids and peptides.