1. 2010 4 29 Minseok Kang Separation of α-cyclohexylmandelic acid enantiomers using biphasic chiral recognition high-speed counter-current chromatography Shengqiang Tonga,b, Jizhong Yanb, ∗, Yi-Xin Guana, ∗∗, Yaner Fub,c, Yoichiro Ito Journal of Chromatography A

2. Enantiomer & diastereomer

3. Characteristics In nature, they usually exist in only one of the one of the two possible enantiomeric forms.

4. Characteristics Oftentimes, Only a single enantiomer of a chiral molecule is desired. Because some enantiomers show completely different biological activities than their optical isomers. (S)-citalopram, ( aka Lexapro ) (R)-citalopram (aka Celexa ) DOSAGE 2 1

5. Enatiomeric separation by chromatography Using Auxiliary chiral reagent Convert to diastereomer Adding chiral selectors to mobile phase Using chiral Stantionary phase Achiral separation technique (Indirect) Achiral separation technique (Indirect) Chiral separation technique (Direct) Chiral separation technique (Direct) Chiral separation technique (Direct) Chiral separation technique (Direct) Application of LC, GC, SEC, CCC, CPC

6. Chemical structure of (±)--cyclohexylmandelic acid. (Lespedamine; Hexahydrobenzilic acid) Oxybutynin (+)-enantiomer Target Drug for Urinary incotinence Synthesis Drug precursor

7. Experiment progress Determine Distribution ratio Separation factor Sample capacity Recovery of solutes Chiral selector Solvent systems

8. Chiral selector Upper organic Lower aqueous Hydrophillic chiral selector ( hydroxypropyl-beta-cyclodextrin) Highly selective in the liquid phase Combination of solvent does not destroy selectivity and retains the capacity to elute chiral isomers of interest Lipophillic chiral selector ( (- )-isobuthyl tartrate Lipophillic chiral selector ( (- )-isobuthyl tartrate Considerations

9. 1. Chiral selector should be soluble in only one phase 2. Racemic mixtures should be easily soluble in both phases 3. For a perfect separation, Distribution ratio is about 1 Solvent system Adjust Lipophillic chiral selector Chiral selector

10. Determine Distribution ratio D1, α1HP-β-CD 0.1M in aq D2, α2(-)-2-Ethylhexyltartrate 0.3M in org Maximum ratio is 3:1 ( tartrate : HP-beta-CD )

11. pH effect Ionic CHMA is formed with high pH in the aqueous phase Finally pH 2.68 was selected for the CCC separation

12. Temperature & Thermodynamic effect Apply “ Van’t Hoff equation “ Chemical thermodynamics relates the change in Temperature to the change in the equilibrium constant given the standard enthalpy change for the progress

13. Temperature & Thermodynamic effect If the enthalpy change of reaction is assumed to be constant with temperature, a plot gives a straight line.

14. Theorogical considerations Schematic diagram of chemodynamic equilibrium between the racemates (A±) and chiral selector (CS) in the separation column based on biphasic recognition.

15. Sample capacity We may derive the langmuir isotherm by treating the adsorption process as we would any other equilibrium process the number of filled surface sites (SP) is proportional to θ, the number of unfilled sites (S * ) is proportional to 1-θ

16. Langmuirian isotherms and estimation of the operating conditions in chiral CCC separation of-CHMA. Parameters for Langmuirian isotherms: a+ = 1.594; b+=−0.0322; a−= 3.215; b−= 0.197 Sample capacity (-)-enantiomer (+)-enantiomer Loading limits : molar ratio CS/analyte = 1:1 Sample volume : less than 5% of total column volume

17. HSCCC chromatogram (TBE – 20A] N-hexane : MtBE : 0.1M phosphate salt buffer (pH=2.68) 3.5mg 7mg 12mg 22mg

18. HSCCC chromatogram (TBE – 300A] Silica-gel column (remove CSs) 440mg Recovery : 85-88% 99.5%, 186mg 99.5%, 190mg

19. Conclusion 1. A chiral separation method for complete resolution of the racemic mixture on a preparative scale was established 2. But Further purification step is needed to recover enantiomers in isloated form. 3. The success of a CCC enantioseparation is highly dependent on the choice of the solvent system.