Chiral separation of (+/-)-dinitrobenzoyl(DNB) amino acids was successfully performed in both analytical and preparative scales by high-speed CCC, using a chiral selector (CS), N-dodecanoyl-L-proline- 3,5-dimethylanilide. The separations were carried out with a two-phase solvent system composed of hexane/ethyl acetate/ methanol/10mM HCl (8:2:5:5) by adding a proper amount of the CS in the organic stationary phase. The sample size ranging up to 2g was separated in 2-7 hours. Various parameters involved in the chiral resolution were investigated using a set of DNB-amino acid racemates. The results indicated that increasing the concentration of the CS in the stationary phase improves both separation factor (alpha) and peak resolution (Rs). Increasing the hydrophobicity of the solvent system also increased the alpha of the racemate while it affected the peak resolution differently according to the partition coefficient of the racemate. Our results indicated that the best separation will be achieved by applying a high CS concentration and adjusting the hydrophobicity of the solvent system so that the partition coefficient of (K) the racemates falls between 0.6 and 0.8. A simple mathematical analysis on the chiral separation shows good agreement with the experimental data.
