1. Chiral Separations: A Tutorial
Christine Aurigemma
Pfizer Global Research & Development,
La Jolla, CA
July 24, 2006
July 24-27, 2006, San Diego, CA

2. Outline Stereochemistry Refresher Chiral Separations
Relationships of Stereoisomers
Terminology
Chiral Separations
Why do we need chiral separations?
Different approaches to enantiopure products
Chromatographic Chiral Separations
What is Chiral Recognition? 3-point rule
SFC vs. HPLC
Types of CSP’s
Screening option
Problem solving
Absolute Stereochemistry (Oliver McConnell)
July 24-27, 2006, San Diego, CA

3. Relationships of Stereoisomers
Isomers: Compounds with the
same molecular formula
Constitutional (or structural)
isomers
Stereoisomers
Same atom
connectivity
Different atom
Interconvert through rotation about a
single bond
Conformational
isomers or rotamers
Configurational
isomers
Not readily Interconvertible
Constitutional (structural)
isomers
Configurational isomers
Enantiomers
Diastereomers
Chiral w/
chiral centers (optically active)
w/o
chiral centers (opt. inactive)
Geometric isomers
Achiral
Conformational isomers
mirror images at this carbon
Enantiomeric
Not mirror images
Diastereomers
Not mirror images at this carbon
Diastereomeric
Cis, Trans
(E,Z) isomers
cis and trans isomers
mirror images
Enantiomers
Courtesy of Brown/Foote, Organic Chemistry, 3/e, Figure 1
Harcourt, Inc. items and derived items copyright by Harcourt, Inc. and
July 24-27, 2006, San Diego, CA

4. Chiral vs Achiral Compounds
Chiral Molecule:
Has one stereogenic center (typically C, but can be N, P, etc.), which is attached to 4 different substituents  asymmetric
one that is not superimposable on its mirror image (the two are not identical)
i.e. hands, keys, shoes
the two mirror image forms are called enantiomers
Optically active
Achiral Molecule:
Has no stereogenic center; the carbon atom has less than 4 non-equivalent substituents attached
has a plane of symmetry
one that is superimposable on its mirror image (the two are identical)
i.e. nail, ball, a baseball bat
Not optically active
July 24-27, 2006, San Diego, CA

5. Determination of Optical Activity
Each enantiomer has an equal but opposite optical rotation; can be measured using optical rotation polarimeter
One enantiomer rotates polarized light in a clockwise direction and is then designed as (+), or dextrorotatory
The other enantiomer rotates polarized light in counter-clockwise direction and is the (-) enantiomer, or levorotatory
Racemates (1:1 mixture of enantiomers) have no observable optical rotation; they cancel each other out
Specific Rotation = []D = 
l * c
where  = observed rotation, l = cell length in dm,
c = concentration in g/mL, and D is the 589nm light from a sodium lamp
©1999 William Reusch, All rights reserved   (most recent revision 7/14/2006)
July 24-27, 2006, San Diego, CA

6. Stereochemistry Terms
Isomers: Compounds with the different chemical structures and the same molecular formula
Stereoisomers: compounds made up of the same atoms but have different arrangement of atoms in space
Enantiomers are the 2 mirror image forms of a chiral molecule
can contain any number of chiral centers, as long as each center is the exact mirror image of the corresponding center in the other molecule
Identical physical and chemical properties, but may have different biological profiles. Need chiral recognition to be separated.
Different optical rotations (One enantiomer is (+) or dextrorotatory (clockwise), while the other is (-) or levorotatory (counter clockwise))
Racemate: a 1:1 mixture of enantiomers.
Separation of enantiomers occurs when mixture is reacted with a chiral stationary phase to form 2 diastereomeric complexes that can be separated by chromatographic techniques
Diastereomers: stereoisomers that are not enantiomers
Have different chemical and physical characteristics, and can be separated by non-chiral methods.
Has at least 2 chiral centers; the number of potential diastereomers for each chiral center is determined by the equation 2n, where n=the number of chiral centers
July 24-27, 2006, San Diego, CA

7. Outline Stereochemistry Refresher Chiral Separations
Relationships of Stereoisomers
Terminology
Chiral Separations
Why do we need chiral separations?
Different approaches to enantiopure products
Chromatographic Chiral Separations
What is Chiral Recognition? 3-point rule
HPLC vs. SFC
Types of CSP’s
Screening option
Problem solving
July 24-27, 2006, San Diego, CA

8. Racemate vs. Single Enantiomer
C&EN, May 5, 2003, pg. 56
July 24-27, 2006, San Diego, CA

9. Chiral Blockbuster Drugs
Nine of the top 10 drugs have chiral active ingredients
Note: Sales figures from IMS Health
Courtesy of C&EN, September 5, 2005, Volume 83, Number 36, pp
July 24-27, 2006, San Diego, CA

10. Examples Albuterol (anti-asthmatic inhalant)
D-albuterol may actually cause airway constriction
Levalbuterol (L-albuterol) avoids side effects
Allegra (allergy medication)
Single enantiomer of Seldane that avoids life-threatening heart disorders of Seldane
Fluoxetine (generic name for Prozac, depression medication)
R-Fluoxetine – improved efficacy; minimizes side effects, i.e. anxiety and sexual dysfunction. Other indications (eating disorders)
S-Fluoxetine – use for treatment of migraines
July 24-27, 2006, San Diego, CA

11. Approaches to Pure Enantiomers
Few Samples
Large Scale
Many Samples
Small Scale
Courtesy of Christina Kraml, Wyeth
July 24-27, 2006, San Diego, CA

12. Outline Stereochemistry Refresher Chiral Separations
Relationships of Stereoisomers
Terminology
Chiral Separations
Why do we need chiral separations?
Different approaches to enantiopure products
Chromatographic Chiral Separations
What is Chiral Recognition? 3-point rule
HPLC vs. SFC
Types of CSP’s
Screening option
Problem solving
July 24-27, 2006, San Diego, CA

13. Chiral Chromatography
Chiral Recognition: Ability of chiral stationary phase, CSP, to interact differently with each enantiomer to form transient-diastereomeric complexes; requires a minimum of 3 interactions through:
H-bonding
π-π interactions
Dipole stacking
Inclusion complexing
Steric bulk
Five general types of CSPs used in chromatography:
Polymer-based carbohydrates
Pirkle or brush-type phases
Cyclodextrins
Chirobiotic phases
Protein-based
CSP
Biphenyl derivative
July 24-27, 2006, San Diego, CA

14. Classification of Chiral Stationary Phases (CSP)
Polymer-based Carbohydrates
Chiral polysaccharide derivatives, i.e. amylose and cellulose, coated on a silica support
Enantiomers form H-bonds with carbamate links between side chains and polysaccharide backbone
Steric restrictions at polysaccharide backbone may prevent access of one of enantiomers to H-bonding site
Can be used with normal phase HPLC, SFC, RP-HPLC
Limitations: Not compatible with a wide range of solvents other than alcohols
Available columns:
i.e. Chiralpak AD, AD-RH, AS, AS-RH, and Chiralcel OD, OD-RH, OJ, OJ-RH, etc. from Chiral Technologies, Inc.
Chiralpak IA and IB…same chiral selectors as AD and OD, respectively, but these are immobilized on the silica; more robust and has much greater solvent compatibilities
Courtesy of Chiral Technologies, Inc.
July 24-27, 2006, San Diego, CA

15. Naproxen examples using polymer-based CSPs
Conditions:
Chiralpak AD-H
Hexane/IPA/TFA, 80:20:0.1
Flow: 1.0 mL/min
Conditions:
Chiralpak AS-RH
aq. H3PO4 (pH2)/ACN, 60:40
Flow: 0.7mL/min
Conditions:
Chiralpak AD-H, 100x4.6mm
CO2/MeOH, 80:20
Flow: 5.0 mL/min
Conditions:
Chiralpak AD-H, 100x4.6mm
CO2/MeOH, 90/10
Flow: 2.0 mL/min
Courtesy of Chiral Technologies, Inc.
July 24-27, 2006, San Diego, CA

16. Classification of Chiral Stationary Phases (CSP)
Pirkle or Brush-type Phases: (Donor-Acceptor)
Small chiral molecules bonded to silica
More specific applications; strong 3-point interactions through 3 classes:
π-donor phases
π-acceptor phases
Mixed donor-acceptor phases
Binding sites are π-basic or π-acidic aromatic rings (π-π interactions), acidic and basic sites (H-bonding), and steric interaction
Separation occurs through preferential binding of one enantiomer to CSP
Mostly used with normal phase HPLC, SFC. May get less resolution with RP-HPLC; compatible with a broad range of solvents
Limitations: only works with aromatic compounds
Available columns:
Whelk-O 1, Whelk-O 2, ULMO, DACH-DNB (mixed phases), -Burke 2, β-Gem 1 (π-acceptor phases), Naphthylleucine (π-donor phases), from Regis Technologies, Inc.
Phenomenex Chirex phases
Courtesy of Regis Technologies, Inc.
July 24-27, 2006, San Diego, CA

17. Naproxen examples using Pirkle-type CSP
(Reversed phase)
(Normal phase)
Courtesy of Regis Technologies, Inc.
July 24-27, 2006, San Diego, CA

18. Classification of Chiral Stationary Phases (CSP)
Cyclodextrin CSPs
Alpha, beta and gamma-cyclodextrins bond to silica and form chiral cavities
3-point interactions by:
Opening of cyclodextrin cavity contains hydroxyls for H-bonding with polar groups of analyte
Hydrophobic portion of analyte fits into non-polar cavity (inclusion complexes)
One enantiomer will be able to better fit in the cavity than the other
Used in RP-HPLC and polar organic mode
Limitations: analyte must have hydrophobic or aromatic group to “fit” into cavity
Available columns:
Cyclobond (-, -, and -cyclodextrins) from Astec, Inc.
ORpak CDA (), ORpak CDB (), ORpak CDC () from JM Sciences
July 24-27, 2006, San Diego, CA

19. Chlorpheniramine example using Cyclodextrin-type CSP
Conditions Results
Column: CYCLOBOND I 2000
Dimensions (mm): 250x4.6mm
Catalog Number: 20024
Mobile Phase: 10/90: CH3CN/1% TEAA, pH 4.1
Flow Rate (mL/min): 1.0 mL/min.
Temp (oC): 23°C
Chart Speed (cm/min): 0.4cm/min.
Detection (nm): 254nm
Injection Volume (µL): 2.0µL
Sample Concentration (mg/mL): 5.0mg/mL
Peak Peak2 18.1
July 24-27, 2006, San Diego, CA

20. Classification of Chiral Stationary Phases (CSP)
Chirobiotic Phases
Macrocyclic glycopeptides linked to silica
Contain a large number of chiral centers together with cavities for analytes to enter and interact
Potential interactions:
π-π complexes, H-bonding, ionic interactions
Inclusion complexation, steric interactions
Capable of running in RP-HPLC, normal phase, polar organic, and polar ionic modes
Available columns:
Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2 (Teicoplanin), Chirobiotic R (Ristocetin A) from Astec
July 24-27, 2006, San Diego, CA

21. Naproxen example using Chirobiotic-type CSP
Conditions Results
Column: CHIROBIOTIC V
Dimensions (mm): 250x4.6
Catalog Number: 11024
Mobile Phase: 10/90:THF/0.1% TEAA, pH7
Flow Rate (mL/min): 1.0 mL/min.
Temp (oC): 25°C
Chart Speed (cm/min): 0.5
Detection (nm): 254
Injection Volume (µL): 2
Sample Concentration (mg/mL): 5
Naproxen
Peak Peak
July 24-27, 2006, San Diego, CA

22. Classification of Chiral Stationary Phases (CSP)
Protein-based CSPs
Natural proteins bonded to a silica matrix
Proteins contain large numbers of chiral centers and interact strongly with small chiral analytes through:
Hydrophobic and electrostatic interactions, H-bonding
Limitations:
Requires aqueous based conditions in RP-HPLC
Analyte must have ionizable groups such as amine or acid.
Not suited for preparative applications due to low sample capacity
Available columns:
Chiral AGP (-glycoprotein) from ChromTech
HSA (human serum albumin) from ChromTech
BSA (bovine serum albumin) from Regis Technologies
July 24-27, 2006, San Diego, CA

23. Naproxen examples using Protein-based type CSP
Human Serum Albumin CSP
Acid glycoprotein CSP
July 24-27, 2006, San Diego, CA

24. Selecting a CSP General use column with no solubility issues
Polymer-based phases
Specific applications; solubility issues
Pirkle-type
Chirobiotic phases
SFC only
Polymer-based, Pirkle-type, Chirobiotic
Biological Samples
Protein-based phases
July 24-27, 2006, San Diego, CA

25. Suggested Applications of CSPs
Compiled from Snyder, et. al, “Practical HPLC Method Development”, 2nd ed., John Wiley and Sons, Inc. 1997, p. 549
July 24-27, 2006, San Diego, CA

26. Chiral SFC vs. HPLC Advantages Disadvantages Reduced solvent
Amounts (CO2 reduces liquid waste)
Reduced toxicity
Solvent types (alkanes, chlorinated, etc)
CO2 has a net zero environmental impact
Safety
Reduce flammability
Separation speed/efficiency
Disadvantages
Equipment costs
Maintenance/robustness
Solubility
July 24-27, 2006, San Diego, CA

27. Flurbiprofen examples using HPLC and SFC
HPLC (normal phase)
SFC (normal phase)
 = 1.76
Run time = 20.5 minutes
Flow rate = 1.5 mL/min
 = 1.35
Run time = 10 minutes
Flow rate = 0.4 mL/min
July 24-27, 2006, San Diego, CA

28. Chiral Screen Mobile phases: CO2 + methanol or isopropanol Columns:
Detector
SFC
Solvent selector
valve
Column selector
Solvents
Mobile phases: CO2 + methanol or isopropanol
Columns:
Chiralpak AD-H, AS-H
Chiralcel OD-H, OJ-H
Chiralpak IA (immobilized AD)
July 24-27, 2006, San Diego, CA

29. Changing Stationary Phase
Daicel Chiralcel OD-H
25% MeOH, 140 bar
Daicel Chiralpak AD-H
30% MeOH, 140 bar
> LOADABILITY
July 24-27, 2006, San Diego, CA

30. Problem Solving Approaches
Derivatization of final products and intermediates
Use of protecting groups such as t-BOC and CBZ (carbobenzyloxy)
CBZ derivatization of chiral primary and secondary amines (common intermediates or final products of enantioselective synthesis)
Adding CBZ can improve compound solubility, enables high efficiency purifications through repetitive, stacked injections
Enhances chiral recognition and improves 3-point interactions; improves baseline separation ability by either HPLC or SFC
CBZ protecting group easily attached and removed during synthetic processes
Acylation of amine with benzyl chloroformate
Amine is regenerated by catalytic hydrogenolysis using palladium on carbon
Product is isolated by simple filtration and evaporation of the solvent
Pd/C H2
PhCH2OCOCl
iPr2NEt
+ CO2 + toluene
Kraml, Christina et. al ,“Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromato
Graphy”, J. of Chrom A, 1100 (2005)
July 24-27, 2006, San Diego, CA

31. CBZ-Derivatization underivatized Purify: ~2g/hr 47.5 g per day
Isolated 70 g
35.4 hrs. purification time
Aurigemma, C., BSAT 2005, Boston, MA
Low S/N ratio
Poor separation
underivatized
Purify: ~2g/hr
Aurigemma, C., BSAT 2005, Boston, MA
July 24-27, 2006, San Diego, CA

32. Addition of strong acid additives to mobile phase
Especially useful for separation of chiral amines
0.1% ethanesulfonic acid (ESA) added to ethanol, or 0.1% methanesulfonic acid (MSA) added to methanol will cause formation of ion pairs with the amine to increase chances of successful enantioseparation
Courtesy of Roger Stringham, Chiral Technologies, Inc
July 24-27, 2006, San Diego, CA

33. Use of Basic Additives to Mobile Phase and Sample solvent*
(S,S) Whelk-O 1, 250x4.6mm, 10u i.d (Regis Technologies, Inc.) % IPA w/ 0.1% IPAm bar
Analytical SFC
Isopropylamine in
Mobile phase only
IPAm in sample solvent
No additive in sample solvent
Preparative
Result: better peak shapes, allowing for high throughput purifications
through stacked injections and yielding pure enantiomers
*Trans()-2-Phenylcyclopropanamine•HCl, CAS No
Aurigemma, C., BSAT 2005, Boston, MA
July 24-27, 2006, San Diego, CA

34. NO residual base in collected sample
Use of Basic Additives to Sample Solvent only
No IPAm in sample solvent
IPAm added to sample solvent
NO residual base in collected sample
IPAm
Aurigemma, C., BSAT 2005, Boston, MA
July 24-27, 2006, San Diego, CA

35. Summary Direct separations of enantiomers achieved by changing CSP’s
Solubility issues can be resolved by adding CBZ or another protecting group
Poor peak shapes can be overcome by addition of additives to MP, MP + sample solvent, or sample solvent only
July 24-27, 2006, San Diego, CA

36. Questions??
July 24-27, 2006, San Diego, CA