色氨酸在TOYOPEARL HW-40S 凝胶填料上非理想排阻行为的研究

Deviation of Size Exclusion Chromatographic Behavior of Tryptophan on TOYOPEARL HW 40S Column

在发现１８种氨基酸特别是色氨酸在ＴＯＹＯＰＥＡＲＬＨＷ－４０Ｓ树脂所填色谱柱上表现出非理想排阻行为的基础上，以色氨酸在ＨＷ柱上的保留时间为指标，探讨不同的流动相改良剂对ＨＷ凝胶与色氨酸之间相互作用的影响，从而阐明这种反常行为是疏水、离子交换以及氢键三者综合作用的结果，而疏水作用是最主要的影响因素。实验结果说明流动相组成对ＨＷ凝胶色谱行为有明显的影响作用，这不但有助于确立ＨＷ凝胶理想的排阻色谱条件，而且为利用其所具有的吸附性能分离分析肽类和氨基酸等小分子物质奠定了基础。

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.