The chiral recognition mechanism for a series of d,l phenoxypropionic acid herbicides (PPAs) on a teicoplanin stationary phase was investigated in reversed phase liquid chromatography (RPLC) over a wide range of mobile phase pH and column temperature. The effect of methanol on the enantiomeric separation was studied by varying its fraction (v/v) in the mobile phase. The thermodynamic data indicated that the chiral recognition was controlled by the interaction between the anionic form of the solute and the teicoplanin phase while those with the neutral form played a minor role. In addition, it was demonstrated that the enhancement of the separation factor observed as the methanol fraction increased in the mobile phase was enthalpically controlled owing to stereoselective binding interactions. Such behavior was used to optimize the chromatographic conditions for separation of PPAs herbicides on teicoplanin.