1. Chiral chromatography refers to the separation of enantiomers using a chiral stationary phase in HPLC. Approximately 60% of pharmaceutical drugs are chiral. 2. There are several types of chiral stationary phases used for separation, including polymer-based carbohydrates, Pirkle phases, cyclodextrins, chirobiotic phases, and protein-based phases. Each type interacts differently with enantiomers through mechanisms like hydrogen bonding and pi-pi interactions. 3. Being able to separate enantiomers is important for drug development since individual enantiomers may have different biological effects and safety profiles. Chromatographic techniques allow for the analysis and purification of single enantiomers.

1. BY
Yogesh K. Chaudhari
M Pharm 1st
year (Pharmacology
University of Mumbai

2. Chiral: A molecule is chiral if it is not superimposable
on its mirror image.
Most chiral molecules can be identified by their lack of a
plane of symmetry or center of symmetry.
Enantiomers or Optical isomers: Two mirror images of
chiral molecules.
Chiral chromatography refers to the separation of
enantiomers using a chiral HPLC column, an HPLC
column packed with chiral stationary phases.
Approximately 60% of Pharmaceutical Drugs are
chiral.

3. 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)
Geometric isomers
Achiral
Configurational isomers
Constitutional (structural)
isomers
mirror images
Enantiomers

4. Chiral MoleculeChiral Molecule::
• Has one stereogenic centerHas one stereogenic center
(typically C, but can be N, P, etc.),(typically C, but can be N, P, etc.),
which is attached to 4 differentwhich is attached to 4 different
substituentssubstituents ⇒⇒ asymmetricasymmetric
• one that isone that is notnot superisuperi
mposable on its mirror image (themposable on its mirror image (the
two are not identical)two are not identical)
– i.e. hands, keys, shoesi.e. hands, keys, shoes
• the two mirror image forms arethe two mirror image forms are
calledcalled enantiomersenantiomers
• Optically activeOptically active
Achiral MoleculeAchiral Molecule::
• Has no stereogenic center; theHas no stereogenic center; the
carbon atom has less than 4 non-carbon atom has less than 4 non-
equivalent substituents attachedequivalent substituents attached
• has a plane of symmetryhas a plane of symmetry
• one thatone that isis superimposable on itssuperimposable on its
mirror image (the two are identical)mirror image (the two are identical)
– i.e. nail, ball, a baseball bati.e. nail, ball, a baseball bat
• Not optically activeNot optically active

5. • 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 = [α]D
α
l * c
where α = observed rotation, l = cell length in dm,
c = concentration in g/mL, and D is the 589nm light from a
sodium lamp

6.  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

7. • 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 (i.e.
increased toxicity)
• Increased pressures by regulatory authorities to
switch from racemic to single enantiomer APIs
• 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

8.  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

9.  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:
1. Polymer-based carbohydrates
2. Pirkle or brush-type phases
3. Cyclodextrins
4. Chirobiotic phases
5. Protein-based
CSP Biphenyl derivative

10. 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 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

11. CH3
O
OH
MeO
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

12. 2) 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), α-Burke 2,
β-Gem 1 (π-acceptor phases), Naphthylleucine (π-donor phases), from
Regis Technologies, Inc.
• Phenomenex Chirex phases

13. (Normal phase)(Reversed phase)

14. 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 (α-, β-, and γ-cyclodextrins) from Astec, Inc.
– ORpak CDA (α), ORpak CDB (β), ORpak CDC (γ) from JM
Sciences

15. 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 (o
C): 23°C
Chart Speed (cm/min): 0.4cm/min.
Detection (nm): 254nm
Injection Volume (µL): 2.0µL
Sample Concentration (mg/mL): 5.0mg/mL
Peak1 16.1
Peak2 18.1
Chlorpheniramine example
using
Cyclodextrin-type CSP
chlorpheniramine

16. 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:
 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

17. Peak1 8.78
Peak2 10.48
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 (o
C): 25°C
Chart Speed (cm/min): 0.5
Detection (nm): 254
Injection Volume (µL): 2
Sample Concentration (mg/mL): 5
Naproxen example using
Chirobiotic-type CSP
Naproxen

18. 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, 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 (α-glycoprotein) from ChromTech
– HSA (human serum albumin) from ChromTech
– BSA (bovine serum albumin) from Regis Technologies

19. Naproxen examples using
Protein-based type CSP
Human Serum Albumin CSP Acid glycoprotein CSP

20. 1.0 It is possible to effect an enantiomeric separation using conventional
HPLC stationary phases by adding a chiral selector to the mobile phase.
2.0 Chiral selector additives generally interact via ion pair ligand exchange
or inclusion interactions with enatiomeric analytes, forming diastermeric
complexes that free separable to conventional normal phase or reversed
phase columns.
3.0 When free cyclodexrins are added to the mobile phase, inclusion
complexes are formed and separation can approach those obtained on
cyclodextrins based chiral stationary phases.
Chiral separation of Ketoprofen on an achiral C8 column by HPLC using
norvancomycin as chiral mobile phase additives.
S (+) Analgesic and antiinflammatory
R (-) slows periodontal bone loss

21. Chiral derivatising agent (chiral resolving agent) react with enantiomers to
gives diasteromers. Since diasteromers have different physical properties,
that they further analyzed by HPLC and NMR spectroscopy.
Two compounds that are enatiomers have same NMR spectral properties.
e.g. Analysis of enantiomers of chiral phenylethylamine i.e. Amphetamine
vy capillary GC/MS/FID and precolumn chiral derivatisation from biological
fluids.
Following derivatising agent widely used
N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA),
2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-
nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-
phthalaldehyde/isobutyryl-L-cysteine (OPA-IBLC),

22. One enantiomers exhibits desired biological activity and other enantiomers
may exhibit undesired sideffects thereby making chiral purity an important
part.
Circular Dichiorism (CD) detector: Optically active compounds with
chromophore close to a chiral center may be absorb circularly polarized
light , which can be detected with excellent sensitivity and selectivity.
When chiral compounds are measured using UV-visible detectors , d and l
enantiomers cannot distinguished eventhough separated by chiral column.
Chiral detectors measures the angle of rotation of plane polarised light
caused by optically active isomers and useful for the chiral compounds
with no absorption.

23. ULTRA PERFORMANCE LIQUID
CHROMATOGRAPHY (UPLC)
 UPLC brings dramatic improvements in sensitivity, resolution and speed
of analysis can be calculated. It has instrumentation that operates at
high pressure than that used in HPLC & in this system uses fine
particles (less than 2.5 µm) and mobile phases at high linear velocities
decreases the length of column, reduces solvent consumption & saves
time. Therefore, by using smaller particles, speed and peak capacity
(number of peaks resolved per unit time in gradient separations) can be
extended to new limits, termed ultra performance liquid
chromatography.
 UPLC system allows shortening analysis time up to nine times
comparing to the conventional system.

24. ADVANCES IN HPLC SYSTEM
High temperature liquid chromatography
Increased temperature of liquid mobile phase is correspondingly lowers
mobile phase viscosity, which allows increased mobile phase flow rate
through liquid chromatography system while maintaining desired
chromatographic analysis attributes. Zironica a packing material for
stationary phase, is thermally stable and provides relatively stable
analytical separations at temperature even in excess of 200 °C. In fact,
recent tests have demonstrated that packing materials utilizing Zironica as
the substrate material are chemically and thermally stable at temperature
approaching the critical point of water (375 °C). Water increasingly
resembles an organic solvent as temperature increasingly towards critical
temperature of water. In fact, recent tests and calculations indicate that at
250 °C, water exhibits solvent properties approaching those of the pure
organic solvent, such as methanol and acetonitrile. Thus, in reversed
phase applications, the use of only water as a mobile phase is
environmentally and economically highly desirable.

25. ADVANCES IN HPLC SYSTEM
Monolithic reversed phase silica column
A monolithic HPLC column is a special type of column used in HPLC with
porous channels rather than beads. In these, tiny beads of an inert
substance, typically a modified silica, are packed tightly into a tube.
Monolithic columns possess a different structure from traditional columns.
Their construction is more akin to a rod with lots of random channeling and
outcroppings. Monoliths support high flow rates without sacrificing
resolution as they exhibit no void volume and can withstand flow rates up
to 9.0 mL/minute.

26. ADVANCES IN HPLC SYSTEM
Microchip HPLC system:
Recent microchip HPLC focused on control of pumping pressure and
sample injections in polymer and glass microsystems. Rapid microchip RP-
HPLC of peptides and proteins at pressure gradients of 180 psi/cm has
been performed using a microdevice that integrates on-chip injection,
separation, and detection with a miniaturized LIF detector. Separation was
achieved via definition of a C18 side-chain porous polymer monolith using
contact lithography, and injection was achieved via definition of a pressure
switchable fluoropolymer valve using projection lithography. Preliminary
separations of peptide standards and protein mixtures were performed in
40-200 s, and switching between samples with no detectible sample
carryover has been performed at 72 injections/h. Sample volumes ranging
from 220 to 800 pL could be linearly metered by controlling the pressure
injection pulse duration with conventional timing and valving.

27. ADVANCES IN HPLC SYSTEM
 Development of new high-capacity, high-selective and high efficient
stationary phases for separation of chiral molecules, i.e. molecules that
exist as different mirror image forms.
 The stationary phase particles become even smaller to increase
performance further; at present particles with 1.7 μm in diameter are
commercially available. LC columns packed with such materials require
ultra-high pressure pumps to provide sufficient flow rates this technique
also called ultra performance liquid chromatography (UPLC).
 Column miniaturization to decrease sample and mobile phase
consumption.
 The development of monolithic stationary phases. Instead of packing
the column with spherical particles, a single-piece stationary phase is
synthesized by in situ polymerization.
 Operation at very high temperatures to decrease mobile phase
viscosity, increase solute solubility and to enable the use of nontoxic
eluents such as water. Stationary phases are developed to withstand
extreme conditions, such as very high or very low pH.