Forecasting of Solute Retention Time in Reversed Phase High Performance Liquid Chromatography

Abstract

Solutes are separated in a reversed phase HPLC (RP-HPLC) according to their partitions betweenstationary and mobile phases. The separation principle is very similar to that of gas liquid chromatography(GLC). The difference is the mobile phases, where solvent (s) or liquid is used as the mobile phase in HPLCwhile an inert gas is used as the mobile phase in GLC. Therefore, it is speculated that the equation proposedby Krisnangkura et al (Ink = a+bz+c/T+dz/T) for predicting retention in GLC should be able to predict thesolutes retention times (or volumes) in RP-HPLC at different temperatures and flow rates. For n-alkanes(C6 – C12) eluted from Nuclosil ODS column between 20 -40 °C, the highest difference between theexperimental and predicted values was 0.01 min. or 0.19%. For n-alkyl benzenes eluted by methanol-water(4:1 and 9:1 w/w) from Hypersil C18 column between 30 – 51 °C, the highest differences in k values were0.242 or -2.03% and 0.131 or 2.85 %, respectively.

HPLC is more versatile than GLC in term of the selectivity of the mobile phases and they can betailored for the best separation of a mixture, but it is considered as a draw back in term of the prediction ofthe solute retention time because the 4 column constants are changed as the mobile phase (or mobile phasecomposition) is changed.

Keywords : Reversed Phase High Performance Liquid Chromatography / Retention Time