ALL INFORMATION ABOUT CHIRAL CHROMATOGRAPHY

1. Chiral Chromatography
Subject :- :- Methods in Pharmaceuticals Research (MPR)
DEPARTMENT OF PHARMACEUTICAL SCIENCES
Dr. HARISINGH GOUR VISHWAVIDYALAYA
Sagar ( M.P)- 470003, India
(A Central University )
SUBMITTED BY :-
Suruchi Dahiya
( M Pharm Sem I )
Roll No;- Y22254028
SUBMITTED TO :-
PROF. ASHMITA GAJBHIYE
(PROFESSOR, DOPS)
PROF. S.K. KASHAW
(PROFESSOR, DOPS)

2. CONTENTS
❖ Introduction
❖ Types of Isomers
❖ Method of chiral separation
❖ Chiral detectors
❖ Advantages
❖ Disadvantages
❖ References

3. INTRODUCTION
Chiral chromatography is the separation of enantiomeric compounds.
Most successful method of separation of enantiomers.
Because of unique spatial orientation of enantiomer it can interact
with enantiomeric reagent and retain in chromatographic system.
Currently direct and most viable way to achieve resolution of isomers.
Separation of stereoisomers in drug is very important analytical
problem and chiral chromatography has been shown very effective
solution for that.
Enantiomer have same physicochemical properties but differ in the
rate of reaction with another chiral reagent.

4. Chiral chromatography is typically performed in classical elution
mode.
A mobile phase is continuously passed through a column containing a
stationary phase .
The mobile phase can be gaseous (gas chromatography, GC), liquid
(LC), or a supercritical fluid (SFC).
Chiral molecule:- Is defined as a molecule that is not superimposable
on its mirror image.
The two mirror image forms of a chiral molecule are..........
▪ Enantiomers,
▪ Diastereomers
A chiral molecule has one or more stereogenic centers which are
typically carbons. These stereogenic carbon atom are attached to four
different substitute.
• Example -Chlorobromomethanol.

5. TYPES OF ISOMERS

6. Isomerism, the existence of molecules that have the same numbers of
the same kinds of atoms (and hence the same formula) but differ in
chemical and physical properties.
There are two general types of isomers:-
❑ Constitutional isomers
Isomers that differ in connectivity are called constitutional (sometimes
structural) isomers. They have the same parts, but those parts are
attached to each other differently.
The simplest hydrocarbons—methane (CH4), ethane (CH3CH3), and
propane (CH3CH2CH3)—have no constitutional isomers, as there is
no other way to connect the carbons and hydrogens of these molecules
consistent with the tetravalency of carbon and the univalency of
hydrogen.

7. ❑ Stereoisomers
stereoisomers are isomers that have the same composition (that is, the
same parts) but that differ in the orientation of those parts in space.
There are two kinds of stereoisomers:
• enantiomers and diastereomers.
❖ Enantiomers
• Enantiomers are a pair of molecules that exist in two forms that are
mirror images of one another but cannot be superimposed one upon
the other.
• Have identical physical properties except for the ability to rotate
plane-polarized light
• Present in pairs
• Similar molecular shape

8. ❑ Diastereomers
Diastereomers are defined as compounds with the same molecular
formula and sequence of bonded elements but are non-superimposable
non-mirror images.
Distinct physical properties
There can be several molecules
Different molecular shape

9. METHODS OF CHIRAL SEPARATION
Chromatographic Techniques Non-chromatographic Techniques
Thin-layer chromatography(TLC) Polarimetry
High-performance liquid chromatography (HPLC). Enzyme Techniques
Supercritical fluid chromatography (SFC). Calorimetry
Capillary electrophoresis (CE). Nuclear Magnetic Resonance
Gas chromatography(GC).. Isotopic dilution

10. Five general types of CSPs used in chromatograpy:
• Polymer-based carbohydrates
• Pirkle or brush-type phases
• Cyclodextrins.
• Chirobiotic phases
• Protein-based

11. ❑ 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
❑ Pirkle or Brush-type Phases:
• (Donor-Acceptor)Small chiral molecules bonded to silica
More specific applications; strong 3-point interactions through 3 classes:
• alpa-donor phases
• alpha-acceptor phases
• Mixed donor-acceptor phases

12. • Binding sites are x-basic or -acidic aromatic rings (n-n 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
❑ Cyclodextrin CSPs
• Alpha, beta and gamma-cyclodextrins bond to silica and form chiral
cavities
• 3-point interactions by:
o Opening of cyclodextrin cavity contains hydroxyls for H-bonding
with polar groups of analyte
o Hydrophobic portion of analyte fits into non-polar cavity (inclusion
complexes)

13. • 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
❑ 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:
o л-л complexes, H-bonding, ionic interactions
o Inclusion complexation, steric interactions
• Capable of running in RP-HPLC, normal phase, polar organic, and
polar ionic modes

14. CHIRAL DETECTORS
▪ Chiral detectors are used for detection of optically active compounds
such as amino acids, sugars, terpenes and other compounds containing
an asymmetric carbon.
▪ There are two chiral detection techniques, polarimetry or optical
rotary dispersion (ORD) and circular dichorism (CD).
▪ ORD detectors are based on differences in refractive index and CD
detectors differentiate enantiomers by measuring differences between
the absorption of light and left-handed circularly polarized light due to
existence of a chiral chromophores.

15. ADVANTAGES OF CHIRAL CHROMATOGRAPHY
The majorities of inorganic molecules are chiral and are analyzed by
this method.
Board ranges of the chiral column are available.
This chromatographic method provides fast and accurate results.
You can get different selectivity’s from the chiral phases.
Good chromophoric or fluorophoric properties of the reagent
(enhanced sensitivity can be achieved)
Low cost of achiral columns
Method development is simple
Selectivity can be increased (better separation is often achieved than
with a direct method)
No sample derivatization is required (except for very polar analytes)&
High efficiency at high speed & Sensitive &
Temperature-programming tools

16. DISADVANTAGES OF CHIRAL CHROMATOGRAPHY
It is used only to separate chiral compounds. This is the major
disadvantage of chiral chromatography.
we need to be an expert to handle the system.
The major disadvantage of chiral chromatography is the need for pure
chiral substances and their relative slowness.
Several chiral additives are expensive, and must often be synthesized.
Volatility
Thermal stability
Stereochemical integrity (not prone to racemization)
Derivatization is required, if compound is polar
Optically pure chiral reagent (ideally 99% optically pure)

17. REFERENCES
Churig, V., Separation Of Enantiomers By Gas Chromatography. J.
Chromatogr. A 2001, 906 (1-2), 275-299.2.
Schurig, V., Chiral Separations Using Gas Chromatography. Trac,
Trends Anal. Chem. 2002, 2/ (9/10), 647-661.3.
Morrison, C. In Chromatographic Separations And Analysis: Chiral
Gas Chromatography, Elsevier B.V.: 2012; Pp 333-353.
Pirkle W. H. And Pochapsky T.C., "Advances In Chromatography"
Giddings G.C, Grushka E & Brown P. R.; Marcel Dekker Inc. NY
Volume 27, 73-127 [ 1987.
D. R Taylor & K. Maher, "Chiral Separation By High Performance
Liquid Chromatography," J. Chromatography Sci., 30, 67-85,[1992].