Melander W R, Corradini D, Horváth C
J Chromatogr. 1984 Dec 28;317:67-85. doi: 10.1016/s0021-9673(01)91648-6.
Retention behavior in hydrophobic-interaction chromatography was examined within the framework of the solvophobic theory. The principal parameters which determine the effect of salt on the retention are salt molality and the molal surface tension increment of the salt. According to the theory, in the absence of special binding effects, increase in salt molality in the mobile phase or change of salt to one of greater molal surface tension increment will result in increased retention of proteins in hydrophobic chromatography. The theory is expanded to treat retention in gradient elution with linear decrease in salt concentration that is equivalent to linear increase in eluent strength. The results of the simple model lead to an expression with two parameters: the adjusted isocratic retention volume of the eluite with the gradient former and the slope of plot of logarithmic adjusted elution volume against salt molality, lambda. The latter parameter is linearly dependent on molal surface tension increment if no specific interactions between the eluite and the stationary phase and/or salt are present. In practice, deviations are to be expected from the predicted behavior due to such effects. The results of calculations are consistent with experimental results obtained with several proteins as the eluites and various salts in the eluent. Although unique values of the critical parameter lambda could not be obtained from the data, the trends showed that lambda is strongly correlated with the value of the molal surface tension increment. The prediction that increase in salt concentration in the initial eluent leads to increase in retention volume was found to be generally true, even when the isocratic retention volumes obtained with use of eluent having low salt concentration were small. Use of NaClO4 in the starting eluent led in some cases to decrease in retention volume with increase in the salt concentration at the beginning of the gradient elution. This effect may be due to specific binding effects.
在疏溶剂理论的框架内研究了疏水相互作用色谱中的保留行为。决定盐对保留作用影响的主要参数是盐的质量摩尔浓度和盐的摩尔表面张力增量。根据该理论，在没有特殊结合作用的情况下，流动相中盐质量摩尔浓度的增加或盐变为摩尔表面张力增量更大的盐之一将导致蛋白质在疏水色谱中的保留增加。该理论被扩展用于处理梯度洗脱中的保留，其中盐浓度呈线性下降，这等同于洗脱剂强度的线性增加。简单模型的结果得到了一个包含两个参数的表达式：用梯度形成剂时洗脱物的调整等度保留体积以及对数调整洗脱体积对盐质量摩尔浓度的图的斜率λ。如果洗脱物与固定相和/或盐之间不存在特定相互作用，则后一个参数与摩尔表面张力增量呈线性相关。在实际中，由于这些影响，预计会与预测行为存在偏差。计算结果与以几种蛋白质为洗脱物和洗脱剂中使用各种盐获得的实验结果一致。尽管无法从数据中获得关键参数λ的唯一值，但趋势表明λ与摩尔表面张力增量的值密切相关。发现初始洗脱剂中盐浓度的增加会导致保留体积增加这一预测通常是正确的，即使使用低盐浓度洗脱剂获得的等度保留体积较小。在起始洗脱剂中使用高氯酸钠在某些情况下会导致在梯度洗脱开始时随着盐浓度的增加保留体积减小。这种效应可能是由于特定的结合作用。
