Introduction:

Chiral chromatography has recently become a preferred method for rapidly separation of enantiopure compounds in the pharmaceutical industry. Chiral chromatographic enantioseparation has been practiced for a decade by researchers. 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

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

where = observed rotation, l = cell length in dm, c = concentration in g/mL, and D is the 589nm light from a sodium lamp

WHY DO WE NEED CHIRAL SEPARATIONS.:

RACEMATE Vs SINGLE ENANTIOMER
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Single enantiomers of chiral active pharmaceutical ingredients (APIs) may have different: Pharmacokinetic properties in animal models Absorption, distribution, metabolism and excretion Pharmacological or toxicological effects Biologically active isomer may have desirable effects Biologically inactive isomer may have undesirable side effects (increased toxicity) Increased pressure by regulatory authorities to switch from racemic to single enantiomer. Development of chiral APIs raises issues regarding: Acceptable manufacturing control of synthesis and impurities Pharmacological and toxicological assessment of both enantiomers Proper assessment of metabolism and distribution Proper clinical evaluation of these drugs.

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 lifethreatening heart disorders of Seldane
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 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

Approaches to Pure Enantiomers:

Chiral synthetic approach Steroselective or asymmetric synthesis Biotransformation or enzymatic resolution Catalytic enantioselective processes Racemic approach Crystallization Chiral salt resolution CE(capillary electrophoresis) SMB(simulated moving bed technology) Chromatography

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 - interactions Dipole stacking Inclusion complexing Steric bulk Five general types of CSPs used in chromatography: 1. Polymer-based carbohydrates 2. Pirkle or brush-type phases 3. Cyclodextrins 4. Chirobiotic phases 5. Protein-based

Classification of Chiral Stationary Phases (CSP):

1) 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 Hbonding site Can be used with normal phase HPLC, SFC, RPHPLC

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 IBsame chiral selectors as AD and OD, respectively, but these are immobilized on the silica; more robust and has much greater solvent compatibilities

2.Pirkle or Brush-type Phases: (Donor-Acceptor) Small chiral molecules bonded to silica More specific applications; strong 3-point interactions through 3 classes: -donor phases -acceptor phases Mixed donor-acceptor phases Binding sites are -basic or -acidic aromatic rings (- 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:
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Whelk-O 1, Whelk-O 2, ULMO, DACH-DNB (mixed phases), - Burke 2, -Gem 1 (-acceptor phases), Naphthylleucine (-donor phases), from Regis Technologies, Inc. Phenomenex Chirex phases 3. 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 . 4)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: - 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 5.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, Hbonding Limitations: Requires aqueous based conditions in RPHPLC 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
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 HSA (human serum albumin) from ChromTech BSA (bovine serum albumin) from Regis Technologies

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

Suggested applications of CSP:

Cs la

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