Chiral Chromatography separates enantiomers using chiral stationary phases. It is the most effective method for resolving stereoisomers that have different biological activities. Various types of chiral stationary phases are used, including proteins, cyclodextrins, cellulose, amylose and macrocyclic glycopeptides bonded to silica. These phases interact differently with enantiomers through hydrogen bonding, hydrophobic interactions and other forces. Direct separation on chiral stationary phases or indirect separation after pre-column derivatization can resolve enantiomers that are important to analyze for pharmaceuticals.

1. Chiral Chromatography
Separation of enantiomers
Presented by
Vipul A. Sansare
Bombay College of
Pharmacy
Dept.: Pharmaceutics
2016-17

2.  Relationships of Stereoisomers
Isomers: Compounds with the
same molecular formula
Constitutional (or structural)
isomers
Stereoisomers
Same atom
connectivity
Different atom
connectivity
Interconvert through
rotation about a
single bond
Conformational
isomers or rotamers
Configurational
isomers
Not readily
Interconvertible
EnantiomersDiastereomers
Chiral
w/
chiral centers (optically active)
w/o
chiral centers (opt. inactive)
Geometric isomers
Achiral
Constitutional (structural)
isomers
mirror images at this carbon
Enantiomeric
Not mirror images
Diastereomers
Not mirror images at this carbon
Diastereomeric

3. Introduction
 Most successful method of separation
of enantiomers.
 Because of unique spatial orientation
of enantiomer it can interact with
enantiometric reagent and retain in
chromatographic system.
 Currently direct and most viable way
to achieve resolution of isomers.

4.  Separation of stereoisomers in drug is
very important analytical problem and
chiral chromatography has been
shown very effective solution for that.
 Enantiomer shave same
physicochemical properties but they
differ in the rate of reaction with
another chiral reagent.

5. Why??
 Albuterol (anti-asthmatic inhalant)
◦ D-albuterol may actually cause airway
constriction
◦ Levalbuterol (L-albuterol) avoids side
effects
 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

6. Continue..
 R isomer of thalidomide : sedative
S isomer of thalidomide : teratogenic
 (S,S) ethambutal : antituberculotic
(R,R) ethambutal : neuritis,blindness

7. Ways for separation of
enantiomers
Indirect liquid chromatographic
resolution via precolumn derivatization
Direct resolution of enantiomers on an
achiral SP.
Direct resolution of enantiomers using
selective chiral SP.

8. Precolumn derivatization
 In precolumn derivatization, enantiomers
are converted into diastereomers, before
they are fed to the column.
 Enantiomers react with a chiral substance
to get diastereomers.
 Because of difference in affinity of
diastereomers for the SP. diastereomers
can be easily separated by
chromatography.

9. Continue
 For eg. a mixture of S and R isomers of ibuprofen,
when react with R proline,produces two
diastereomers;
(S,R)IBP + (R)proline
(S)IBP (R)proline + (R)IBP (R)proline
(Diastereomer of (Diastereomer of
S- IBP) R- IBP)

10.  The chiral reagent should react
quantitatively with enantiomers for
accurate determination of
enantiomeric composition.
 Two diastereomers show different
affinity towards achiral SP. hence can
be separated on achiral SP. using an
achiral mobile phase.

11. Chiral stationary phase
 Different material employed as chiral
stationary phase.
 Material either bonded or coated on
the silica support and use as
stationary phase.
 Materials use as stationary phase:
-Natural proteins
-Cyclodextrine
-Polymers(cellulose, amylose)
-Macrocyclic glycopeptide

12. 1) Protein based stationary
phase
 It consist of protein bonded to the
silica support(5µm particle size)
 Protein contain no. of chiral centers
and interact strongly to many small
chiral analyte through
Hydrogen bonding
Hydrophobic interaction
Electrostatic interaction

13.  Available columns
- Chiral AGP from ChromeTech.
- Chiral HAS from ChromeTech.
- Chiral BSA from Regis Technologies.
- Chiral RSA from Chrome Tech.
- Chiral DSA from Chrome Tech
- Chiral MSA from Chrome Tech

14.  Chiral AGP
-Extremely broad applicability.
Separate all kinds of compounds:
-amines(10,20,30, and quaternary)
-acids(strong and weak)
- amides, esters, alcohols)
• Chiral HAS
-Separate preferentially acids and
amides and esters.

15. Acid glycoprotein CSP Human Serum Albumin CSP
Naproxen separation using
protein based SP.

16. Polymer based stationary
phase
 Currently available as cellulose/amylose
coated on silica support as oppose to the
bonded on the silica support.
 Cellulose and amylose unit contain five
chiral centers.
 Two basic form;
- Derived from cellulose
- Derived from amylose
 Polymer contain large no. of chiral
centers.

17.  Cellulose derivatise using following;
3,5 dimethyl phenyl carbamate 4 methyl benzoate
 Both structure contain aromatic nuclei
offer strong polar interaction with strong
polar group of solute.

18.  Amylose derivatise using following;
α methyl benzyl carbamate 3,5 dimethyl phenyl carbamate
 Both structure contain aromatic nuclei
offer strong polar interaction with polar
group.
 Eg: Chiralpak-AD-H , AD-RH, RS.

19. Macrocyclic glycopeptide
 Macrocyclic glycopeptide covalently
bound to the silica support.
 Eg. Vancomycin, avoparcin,teicoplanin
 Vancomycin contain 18 chiral centers.
 Very stable SP. has high sample capacity
and it can use with mobile phase with
high water content.
Chirobiotic A (Avoparcin),
Chirobiotic V ( Vancomycin)
Chirobiotic T(Teicoplanin)

20. Cyclodextrine based SP.
 Cyclodextrine/ their derivatives
bonded to the silica support which are
then pack into the column.

21. Suggested applications of
CSP
ChirobioticPeptides
PirkleOxazolindones
Pirkle, cellulose, proteinNSAIDS
Cellulose, PirkleDihydropyridines
Cellulose, PirkleAlkaloids
Pirkle, celluloseCyclic ketones
Cellulose, PirkleLactones
Protein, PirkleAromatic drugs
ProteinCyclic drugs
Cyclodextrins
Polycyclic aromatic
hydrocarbons
Pirkle, celluloseβ-Lactams
PirkleBinaphtols
Pirkle, ChirobioticHydantoins
PirkleSuccinamides
Pirkle, ChirobioticAmino alcohols
PirkleThiols
CyclodextrinsMetallocenes
CyclodextrinsCrown ethers
PirkleUreas
PirkleCarbamates
Pirkle, cellulose, protein, ChirobioticSulfoxides
Pirkle, cellulose, ChirobioticEsters
Protein, cellulose/amylose, Pirkle, cyclodextrins,
ChirobioticAmides
Protein, cellulose/amylose, Pirkle, cyclodextrinsAlcohols
Protein, cellulose/amylose, Pirkle, cyclodextrins,
ChirobioticAmines
Cyclodextrins, protein, ChirobioticAmino Acids
Protein, cellulose/amylose, Pirkle, ChirobioticAcids
Type(s) of CSPClass of Compound
ChirobioticPeptides
PirkleOxazolindones
Pirkle, cellulose, proteinNSAIDS
Cellulose, PirkleDihydropyridines
Cellulose, PirkleAlkaloids
Pirkle, celluloseCyclic ketones
Cellulose, PirkleLactones
Protein, PirkleAromatic drugs
ProteinCyclic drugs
Cyclodextrins
Polycyclic aromatic
hydrocarbons
Pirkle, celluloseβ-Lactams
PirkleBinaphtols
Pirkle, ChirobioticHydantoins
PirkleSuccinamides
Pirkle, ChirobioticAmino alcohols
PirkleThiols
CyclodextrinsMetallocenes
CyclodextrinsCrown ethers
PirkleUreas
PirkleCarbamates
Pirkle, cellulose, protein, ChirobioticSulfoxides
Pirkle, cellulose, ChirobioticEsters
Protein, cellulose/amylose, Pirkle, cyclodextrins,
ChirobioticAmides
Protein, cellulose/amylose, Pirkle, cyclodextrinsAlcohols
Protein, cellulose/amylose, Pirkle, cyclodextrins,
ChirobioticAmines
Cyclodextrins, protein, ChirobioticAmino Acids
Protein, cellulose/amylose, Pirkle, ChirobioticAcids
Type(s) of CSPClass of Compound

22.  Interaction with polar group of analyte
take place at polar hydroxyl group.
Non polar, lipophilic group of analyte
fit into hydrophobic cavity.
 One enantiomer will able to fit into
cavity than other.
 Analyte must have lipophilic group to
fit into cyclodextrine cavity.
 Derivative provide specific type of
interaction with analyte and increase
selectivity.

23. Thank you