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

Enantiomer & diastereomer

Characteristics

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

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

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)

Chiral separation technique(Direct)

Chiral separation technique(Direct)

Application of LC , GC , SEC , CCC , CPC

Chemical structure of (±)--cyclohexylmandelic acid.

(Lespedamine; Hexahydrobenzilic acid)

Oxybutynin(+)-enantiomer

Target

Drug for Urinary incotinence

Synthesis

Drug precursor

Experiment progress

Determine Distribution ratio Separation factor

Sample capacity Recovery of solutes

Chiral selector

Solvent systems

Chiral selector

Upper organic

Lower aqueousHydrophillic 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

Considerations

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

Determine Distribution ratio

D1 , α1 HP-β-CD 0.1M in aqD2, α2 (-)-2-Ethylhexyltartrate 0.3M in

org

Maximum ratio is 3:1 ( tartrate : HP-beta-CD )

pH effect

Ionic CHMA is formed with high pH in the aqueous phaseFinally pH 2.68 was selected for the CCC separation

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

Temperature & Thermodynamic effect

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

Theorogical considerations

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

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

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

HSCCC chromatogram (TBE – 20A]

N-hexane : MtBE : 0.1M phosphate salt buffer (pH=2.68)

3.5mg

7mg

12mg

22mg

HSCCC chromatogram (TBE – 300A]

Silica-gel column (remove CSs)

440mg

Recovery : 85-88%

99.5% , 186mg99.5% , 190mg

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.