The ratio between the volume of the stationary phase and the void volume of column in HPLC is known as the phase ratio (denoted by Φ). In almost all the thermodynamic studies based on van’t Hoff plots for estimating the values of standard enthalpy and standard entropy, the phase ratio of HPLC column is assumed to be temperature invariant. The validity of this assumption is examined in the present work by studying the variation of Φ with temperature (T) in the range of 20–50 °C for three commercially available C18 columns and two mobile phase compositions methanol/water. A procedure based on proportionality of retention factors k on octanol/water partition coefficients log K_ow for several aromatic hydrocarbon homologues was used for the measurement of Φ. Variation of Φ with temperature was previously reported for RP-HPLC separations in an acetonitrile/water mobile phase. However, while for acetonitrile/water mobile phase the effective value of Φ is decreasing as the temperature increases, in the case of methanol/water mobile phase the variation is more complicated. In some cases Φ is decreasing similar to the case of acetonitrile/water mobile phase, but in other cases it decreases up to a point and then shows a slight increase. The study proves that the findings regarding variability of Φ with temperature for the case of acetonitrile/water mobile phase is not an unique effect. In addition, present study evaluates how much the calculation of standard enthalpy and entropy from van’t Hoff plots differ when the variation of Φ with temperature is taken into consideration as compared to the classic approach when Φ is considered a constant.

HPLC中固定相的体积与色谱柱空隙体积之间的比称为相比（以Φ表示）。在几乎所有基于van't Hoff图来估算标准焓和标准熵值的热力学研究中，HPLC色谱柱的相比均假定为温度不变的。通过研究三个商用C18色谱柱和两个甲醇/水流动相组成的Φ在20–50°C范围内随温度（T）的变化，检验了该假设的有效性。基于保留因子k对辛醇/水分配系数log K _ow的比例的程序几种芳香族烃的同系物用于Φ的测量。先前报道了乙腈/水流动相中RP-HPLC分离过程中Φ随温度的变化。但是，对于乙腈/水流动相，Φ的有效值会随着温度的升高而降低，而在甲醇/水流动相的情况下，变化更为复杂。在某些情况下，与乙腈/水流动相类似，Φ下降，但在其他情况下，Φ下降到一个点，然后略有上升。该研究证明，对于乙腈/水流动相而言，有关Φ随温度变化的发现并不是唯一的影响。此外，