Ketoprofen is a non-steroid anti-inflammatory drug (NSAID) that has analgesic, anti-inflammatory and antipyretic properties, of which pharmacological activity has been contributed mainly by the S-ketoprofen enantiomer. To gain highly purified S-ketoprofen enantiomer from ketoprofen racemate, suitable stationary phase was investigated by comparing R-type naphthylglycine and 3,5-dinitrobenzoic acid of Chirex^® 3005 and O,O'-bis(4-tertbutyl-benzoyl)-N and N'-diallyl-L-tartardiamide of Kromasil^® CHI-II. S-ketoprofen enantiomer was successfully isolated by using the Kromasil column as well as the mobile phase of hexane/tert-butyl methyl ether/acetic acid (v/v)=60/40/0.1. Equilibrium constants of R- and S-ketoprofen were obtained by pulsed injection method (PIM) at various concentrations of ketoprofen racemate and loading sample amounts, which serve the basic information on overloading sample volume and amounts. A volume of 100 μl containing 3.0 mg of ketoprofen racemate to Kromasil column (250 mm×4.6 mm) resulted in 85% recovery of injected sample under an high concentration (30 mg/ml) feed condition.