Haidacher D, Vailaya A, Horváth C
Biotechnology Department, Sandoz Pharma, Basel, Switzerland.
Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2290-5. doi: 10.1073/pnas.93.6.2290.
The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.
通过高效液相色谱法在三种固定相上研究了5℃至50℃的温度对氨基酸丹磺酰衍生物在疏水相互作用色谱（HIC）中保留率的影响。在很宽的温度范围内，对数保留因子与倒数温度的关系图是非线性的，这表明与保留相关的热容量变化很大且为负值。通过使用基尔霍夫关系，分别根据热容量的不变性和对温度的平方反比依赖性，将数据拟合到对数方程和二次方程，从不同温度下的对数保留因子评估焓、熵和热容量的变化。在实验温度区间内，发现热容量变化随温度升高而增加，并且可以用算术平均值来近似。对于一组丹磺酰氨基酸的HIC保留，正如文献中针对基于疏水效应的其他过程所描述的那样，焓变和熵变在低温时均为正值，但在高温时为负值。这里介绍的方法表明，色谱测量不仅可以作为量热法的有用辅助手段，而且是评估热力学参数的另一种方法。
