Selectivity-column temperature relationship as a new strategy in predicting separation of structural analogues in HPLC by using different stationary phases

Abstract

The effect of changes in column temperature on the van't Hoff equation, as well as the relationship between separation efficiency and column temperature in high performance liquid chromatography (HPLC) by using different stationary phases, have been discussed and compared in this paper. For this purpose, six flavonoid glycosides were selected to establish van't Hoff equations on C₁₈, cholesterol, C₈ and porous polymer octadecyl bonded (ODP) stationary phases. The results indicated that, for all the columns, the changes in phase ratio arising from the varying column temperatures had no significant impact on the linearity of the van't Hoff equation, however, it indeed led to certain errors in the intercept (change in entropy, ΔS⁰) and slope (change in enthalpy, ΔH⁰) of the equation, which are considered to be important parameters in illuminating the chromatographic mechanism in HPLC. Thereby, a new protocol has been proposed in this paper to correct these errors, with the aim of offering solid data for ΔS⁰ and ΔH⁰. Furthermore, a relationship relating selectivity and column temperature was deduced in theory, and then validated by using the six flavonoid glycosides in this work. This relationship has been applied to predict the separation of six steroid hormones in HPLC with high consistency between experimental and predicted selectivity factors (average relative errors less than 2.2%, 1.0%, 6.1% and 5.1% for C₁₈, cholesterol, C₈ and ODP columns, respectively). The proposed new strategy in predicting selectivity greatly facilitates optimization processes for HPLC by avoiding tedious condition experiments, and furthermore, column temperature is proposed to be involved in the optimization processes as an important parameter, making separation of structural analogues effective. In this temperature-involved optimization method, a stationary phase sensitive to temperature change, i.e. cholesterol, is recommended.