SUPELCO. Chiral GC Using Cyclodextrin-Based Capillary Columns. Jamie Desorcie Gas Separations R&D. Supelco, Supelco Park, Bellefonte, PA T499136

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1 Chiral GC Using Cyclodextrin-Based Capillary Columns Jamie Desorcie Gas Separations R&D Supelco, Supelco Park, Bellefonte, PA T499136

2 utline Introduction Chirality and Its Implications Chiral Separation Methods Chiral Gas Chromatography Stationary Phases DEX Column Selection and Method Development Applications

3 Definitions Chirality The property of nonsuperimposability of an object on its mirror image. Chiral Molecule Not superimposable on its mirror image. Achiral Molecule Superimposible on its mirror image.

4 Mirror Image Relationships Adapted from Chirotechnology: Industrial Synthesis of ptically Active Compounds R.A. Sheldon, Marcel Dekker, Inc., New York, Used with permission

5 Lactic Acid Enantiomers 3 C C C C 3

6 Enantiomers Differ in Structure nly in the Left- and Right-andedness of their Spatial rientations. Rotate the Plane of Polarized Light in the pposite Directions in Equal Amounts. React at Different Rates with ther Chiral Compounds.

7 Enantioselectivity L R Adapted from Chirotechnology: Industrial Synthesis of ptically Active Compounds R.A. Sheldon, Marcel Dekker, Inc., New York, Used with permission

8 (S)/(R)-Limonene C 3 C 3 (S)-Limonene Lemon dor (R)-Limonene range dor

9 Taste Selectivity N 2 2 C N C 2 C 3 (S,S) Sweet (Aspartame) C 2 Ph N 2 3 C 2 C N C 2 (R,R) Bitter C 2 Ph

10 (S/R)-Dopa C 2 2 C (S)-Dopa anti-parkinson N 2 2 N (R)-Dopa serious side effects

11 (R/S)-Thalidomide N N N N (R)-Thalidomide Good Sedative No Teratogenic Effect (S)-Thalidomide No Sedative Effect Potent Teratogen

12 Chiral Separation Methods Crystallization Conversion to Diastereomers Enzymatic Transformations Differential Adsorption (Chromatography)

13 Crystallization C C C C C C CN 4 CN 4 Pasteur, 1848

14 Conversion to Diastereomers C C (S) (R) C 3 3 C Ph Ph 2 N C 3 Ph (S) C 3 (S,S) C 2 N Ph C 3 Ph Ph + 3 C C 3 (R,S) C 2 N Ph

15 Enzymatic Transformations C C R N 2 2 N R Esteraze / R 1 C 2 R' Very Slow Reaction R N 2

16 Chromatographic Separation

17 Chiral GC Stationary Phases Amino Acid Derivatives/Analogues Metal Complexes Native and Derivatized Cyclodextrins

18 Amino Acid Derivatives/Analogues F 3 C N C N-Trifluoroacetyl-(L)-isoleucine lauryl ester (Gil-Av, 1966) 23 C 11 N N C 4 9 -t N-Lauroyl-(L)-valine tert-butylamide (SP-300)

19 Metal Complexes F 3 C M M = Mn, Co, Ni CF 3 Schurig, 1988

20 Cyclodextrins α-cyclodextrin β-cyclodextrin γ-cyclodextrin ,0576,

21 Possible rientations of Enantiomers Tail Complex ead Complex

22 Per--methyl-α-cyclodextrin C 3 C 3 C 3 C3 C3 C 3 C 3 C3 C 3 C 3 C 3 C 3 C 2 C 3 C 2 C

23 Chiral Stationary Phases DEX 225 R = C 3 C R R R R R R R R R R R R DEX 325 R = C 3 C 2 -SiMe 2 Bu-t C 2 -SiMe 2 Bu-t

24 DEX Columns Chiral Stationary Phase: Cyclodextrin derivative in polysiloxane SPB -20 or SPB-35 Cyclodextrin Derivative: Series 100: per--methyl-cd Series 200: 2,3-di--acetyl-6-tert-butyldimethylsilyl-CD Series 300: 2,3-di--methyl-6-tert-butyldimethylsilyl-CD Example: β-dex 120 β-cd 20% per--methyl

25 Enantioseparations on DEX Columns α-dex TM β-dex γ-dex Menthols Largest number Carvone Substituted benzenes of analytes α -Ionone and phenols Menthone Epoxides Methamphetamine Small molecules Large molecules

26 Enantioselectivity: Chiral Selector Percentage 3,3-Dimethyl-2-butanol α-pinene α 3-Methyl-2-heptanone % Per--methyl-β-cyclodextrin

27 Peak Resolution (Rs) can be expressed via chromatographic factors of retention (k'), efficiency (N), and enantioselectivity (α) t 2 t 1 t 0 W 1 W 2 t 2 - t 1 N 2 α-1 k' 2 R s = 2 = W 1 + W 2 4 α 1 + k' 2 where: N 2 = 16(t 2 / W 2 ) 2 α = (t 2 - t 0 ) / (t 1 - t 0 ) k' 2 = (t 2 - t 0 ) / (t 0 )

28 Effect of Column ID on Efficiency and Resolution Column: β-dex 120, 30m Column ID N α Rs 100µm µm µm µm

29 Column Selection: Chiral Column Kit II 30m, 0.25mm ID, 0.25um Film 20% Per-- methyl-β-cyclodextrin 25% 2,3--Diacetyl-6--TBDMS-βcyclodextrin 25% 2,3--Diacetyl-6--TBDMS-γcyclodextrin 25% 2,3--Dimethyl-6--TBDMS-βcyclodextrin

30 Column Selection: verall Approach Prepare Sample and Set Initial Instrument Parameters Temperature-Programmed Analysis Isothermal Analysis

31 Sample & Instrument Parameters Sample Concentration: 10mg/mL in Dichloromethane or Methanol Injection Volume: 1uL, Split (100:1) Injector: 230 C Detector: FID, 250 C Carrier Gas: elium (25cm/sec at 180 C) or ydrogen (50cm/sec at 180 C)

32 Temperature Programmed Analysis Initial Temperature: 50 C Program Rate: 10 C/minute Final Temperature: 230 C old Time: 60 minutes

33 Reduce Injector Temperature or Use n-column Injection Temperature Programmed Analysis Suspected Compound Decomposition Broad Peak or No Peak Chromatogram Compound Not Suitable for GC Narrow Peak Isothermal Analysis

34 Reduce ven Temperature Isothermal Analysis ven Temperature Too igh Poor Separation Short Retention Chromatogram Poor Separation Long Retention Evaluate Another Column Satisfactory Separation Column Selected

35 Temperature Programmed Analysis β-dex 120 Column * 3,4-Dihydroxyphenyl Glycol Min G000771

36 Temperature Programmed Analysis β-dex 120 Column C 3 * Benzoin Methyl Ether Min G000772

37 Isothermal Analysis β-dex 120 Column C 3 * Benzoin Methyl Ether Min G000773

38 Isothermal Analysis (180 C) γ-dex 225 Column C 3 * Benzoin Methyl Ether Min G000774

39 Isothermal Analysis (150 C) γ-dex 225 Column C 3 * Benzoin Methyl Ether Min G000775

40 Effect of Temperature on Enantioselectivity Column: β-dex 120 α Me-2-heptanone 1.06 alpha-pinene ,3-diMe-2-butanol Temperature ( C)

41 Temperature-Dependent Enantioreversal T ( C) k R k S k R / k S R S Analyte: Ethyl mandelate Column: β-dex 120, 30m x 0.25mm x 0.25µm film

42 Isoenantioselective Temperature RT ln α = δ( ) + Tδ( S) where: δ( ) = difference in enthalpy of sorption of the enantiomers δ( S) = difference in entropy of sorption of the enantiomers T isoen = δ( ) / δ( S) T isoen : enantioseparation is impossible

43 Enantioseparation of Terpinen-4-ol at 100 C Columns: 30m x 0.25mm ID x 0.25µm film β-dex TM 225 (+) ( ) (+) β-dex ( ) min

44 Enantiomeric Excess Determination: β-dex 120 (C 2 5 ) 2 Zn R Asymmetric Catalyst R Jun, Guo, and Zhang* J. rg. Chem.

45 Enantiomeric Excess Determination: β-dex 120 * * * R R R = C 3, C 2 5, C 4 9 -n R = C 3, C 6 5 R R =, C 3, C 3, Cl Ph * * R * * 3 C R =, C 3

46 Enantiomeric Excess Determination: β-dex 120 R 1 Asymmetric + + Catalyst R 2 R 1 R 2 Jiang, Jiang, Zhu, and Zhang* Tetrahedron Letters 1997, 38, 6565

47 Enantiomeric Excess Determination: R 2 β-dex 120 * * * R 1 R 1 =, R 2 = C 3 R 1 =, R 2 = C 2 5 R 1 = R 2 = C 3 * * * R R = C 3, Br

48 Enantiomeric Excess Determination: γ-dex 225 1) IpcB 2 2) 2 2 Zhu, Cao, Jiang, and Zhang* J. Am. Chem. Soc. 1997, 119, 1799

49 Enantiomeric Excess Determination: β-dex 225 N + 2 Asymmetric Catalyst N * Zhu and Zhang* Tetrahedron: Asymmetry 1998, 9, 2415

50 Carvone (Enantioreversal) 3 C C = C 2 α-dex TM 120 S R * β-dex 120 Columns: 30m x 0.25mm ID x 0.25µm film ven: 90 C R,S γ-dex 120 S R

51 Linalool Enantiomers in Tea Sample: Gao-sun tea (Taiwan) SPME Fiber: 65µm polydimethylsiloxane / divinylbenzene Column: β-dex 325, 30m x 0.25mm ID x 0.25µm film ven: 100 C 9.52 (R)-Linalool (S)-Linalool

52 Enantiomeric Ratio of Linalool in Teas can be used to identify / control quality of tea Type of Tea (Source) R/S Ratio Gao-sun (Taiwan) 9:91 Assam (Yamamoto, rient, Los Angeles) 34:66 Spring arvest, Lichee's Black (China) 36:64 Arati (Tea Packs Specialty Ltd., Calcutta) 40:60 Darjeeling (Jacksons of Picadilly) 44:56 Green Label (Lipton) 47:53 oji-cha (Yamamotoyama, Japan) 50:50 Yunnan Tuo (Xiaguan Tea Factory, China) 56:44 Red Label (Brooke Bond Lipton India, Calcutta) 58:42 Lapsang Souchong (China) 65:

53 Environmental Phenols Column: α-dex 120, 30m x 0.25mm ID x 0.25µm film ven: 130 C to 220 C at 3 C/min min Chlorophenol 2. Phenol 3. o-cresol 4. p-cresol 5. m-cresol 6. 2,4-Dimethylphenol 7. 2,4-Dichlorophenol 8. 2,6-Dichlorophenol 9. 4-Chloro-3-methylphenol 10. 2,3,5-Trichlorophenol 11. 2,4,6-Trichlorophenol 12. 2,3,4-Trichlorophenol 13. 2,4,5-Trichlorophenol Nitrophenol 15. 2,3,5,6-Tetrachlorophenol 16. 2,3,4,6-Tetrachlorophenol 17. 2,3,4,5-Tetrachlorophenol 18. 4,6-Dinitrophenol 19. 2,4-Dinitrophenol 20. Pentachlorophenol

54 Conclusions Derivatized cyclodextrins are the most useful chiral selectors for enantioselective capillary GC A broad range of volatile and semi-volatile enantiomers can be resolved using cyclodextrin-based capillary columns Selecting the appropriate DEX TM column is an empirical process