Does phase ratio in reversed phase high performance liquid chromatography vary with temperature?

Area of Science:

• Analytical Chemistry
• Chromatography
• Physical Chemistry

Background:

• The phase ratio (Φ) in HPLC, defined as the ratio of stationary phase volume (Vs) to void volume (Vm), is crucial for retention factor (k) calculations (k = KΦ).
• Accurate determination of Φ is essential, yet debated, as it can vary with mobile phase composition and temperature.
• Existing methods for Φ evaluation often rely on measuring retention factors (k) for known compounds.

Purpose of the Study:

• To evaluate the phase ratio (Φ) for commercially available C18 columns using varying mobile phase compositions (water/acetonitrile).
• To investigate the effect of temperature on the phase ratio (Φ) within the range of 20 °C to 50 °C.
• To assess the implications of temperature-dependent phase ratio on thermodynamic calculations in reversed-phase HPLC.

Main Methods:

• Utilized high-performance liquid chromatography (HPLC) with three distinct C18 columns.
• Employed water/acetonitrile mobile phase compositions at temperatures ranging from 20 °C to 50 °C.
• Determined phase ratio (Φ) by measuring retention factors (k) of hydrocarbons with known octanol/water partition coefficients (log Kow).

Main Results:

• The effective phase ratio (Φ) was found to decrease as the column temperature increased.
• This temperature-dependent behavior of Φ aligns with the observed decrease in retention times at higher temperatures in reversed-phase HPLC.
• The study highlights that the changing phase ratio with temperature complicates accurate calculation of thermodynamic functions from van't Hoff plots.

Conclusions:

• Phase ratio (Φ) in HPLC is not constant but varies with temperature, decreasing as temperature rises.
• This temperature dependence of Φ has significant implications for the precise calculation of thermodynamic parameters like enthalpy and entropy.
• Accurate thermodynamic analysis in reversed-phase HPLC requires accounting for the temperature-dependent effective phase ratio.