Chiral enantioseparation of racemic drugs

CHIRAL SEPARATION OF RACEMIC DRUGS





POON YIN FUN
(B.Sc.(Hons.). NUS)





A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF CHEMISTY
NATIONAL UNIVERSITY OF SINGAPORE
2005

i
ACKNOWLEDGEMENTS

I would like to express my heartfelt gratitude to my supervisor, A/P Ng Siu
Choon for his advice, useful discussions and guidance throughout the whole project. I
am also grateful to Prof Chan Sze On, Hardy for the follow-up supervision done on
my pH.D candidature. My sincere thanks also go to Dr I Wayan Muderawan for his
kind advice in organic synthesis and his help in proofreading this thesis. I like to

thank Dr Chen Lei for sharing her ideas in the analytical aspects involved in this
project and being ever so helpful to clear my doubts on the project.

I would like to thank the technical and laboratory staff in the Faculty of
Science of the University for the immense help. In particular, I am indebted to Mdm
Frances Lim Guek Choo who has been so helpful to clarify my doubts concerning
chromatographic separations and the use of the HPLC instrument; Mdm Han Yanhui
and Miss Peggy Ler for helping with the Nuclear Magnetic Resonance analyses; Mdm
Toh Soh Lian and Miss Serene Lim for providing me with precious knowledge and
life-long skills which will be useful for me as a chemist.

Lastly, I will also remember all my friends and seniors in the Functional
Polymer Laboratory and Collaborative Research Laboratory with whom I have
worked and have helped me in one way or another. I am also grateful for the
understanding and support rendered by my family and friends throughout my work.

ii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS i
TABLE OF CONTENT ii
SUMMARY vii
LIST OF SYMBOLS AND ABBREVIATIONS xi
LIST OF PUBLICATIONS xii

Chapter 1: INTRODUCTION 1
1.1. The History of Enantiomeric Resolution 3
1.2. Chiral Analysis 5
1.3 Major Racemate Separation Technologies 11
1.4 Liquid Chromatography – Direct and Indirect Methods 13
1.5 Mechanism of Separation 15

1.6 Classification of Chiral Stationary Phases 17
1.6.1

Dinitrobenzoyl phases (Pirkle CSP) 18
1.6.2

Protein bonded phases 20
1.6.3

Chiral phases with inclusion effects

1.6.3.1 Cyclodextrin bonded phases 20

1.6.3.2 Crown-ether phases 24
1.6.4

Polymeric phases 25
1.6.5

Chiral ligand exchange phases (CLEC) 26
1.7 Chiral Simulated Moving-Bed Chromatography (SMB) 28
1.8 Time Frame of Cyclodextrin Discovery 29
1.9 Physical Properties of Cyclodextrins 30
1.10 Application of Cyclodextrins in Chromatography 31

iii
1.11 Work Done by our Research Group 33
1.12 Racemic Drugs and Compounds 36
1.13 Scope of Work 37

Chapter 2 PREPARATION OF CSPs 41
2.1 Synthetic Methodologies 42
2.2 Purpose of Immobilization at C2-OH position 42
2.3 Modifications involving reaction of C6 Primary Hydroxy Group 44
2.4 Modifications involving reaction of C2 Secondary Hydroxy
Group
44
2.5 Selective Tosylation of Cyclodextrin 46
2.6 Differentiation between C2-tosylated-CD and C6-tosylated-CD 48
2.7 Differentiation between C2-tosylated-CD and C3-tosylated-CD 50
2.8 Azidolysis 52
2.9 Carbamoylation and Staudinger Reaction 56
2.10 The Hydrosilylation Route 58
2.11 List of CSPs 61
2.12 Column Efficiency and Surface Coverages 63

Chapter 3 STUDY OF THE EFFECTS OF IMMOBILIZATION POSITION
OF CYCLODEXTRIN AND SUBSTITUENTS ON
ENANTIOSELECTIVITY

67
3.1 Introduction 68
3.2 Characteristics of CSPs 72
3.3 Basic Concepts for Chromatographic Separation 73
3.4 Chromatographic Properties of Perphenylcarbamoylated CD CSPs 75

iv

3.4.1

Comparisons between CSP-AM-2-PC and CSP-AM-6-PC under
Normal Phase and Reversed-Phase

75
3.5 Chromatographic Properties for P
eracetylated CD CSPs
90
3.6 Comparison between Phenylcarbamate CD CSPs and Acetyl CD
CSPs using Flavanone Compounds

94
3.7 Permethylated series and Perbenzoylated series 97
3.7.1

Chromatographic Properties of Permethylated CD CSPs 98
3.7.2

Chromatographic Properties of Perbenzoylated CD CSPs 101
3.8 Conclusion 103

Chapter 4 STUDY OF THE EFFECTS OF SUBSTITUENTS ON PHENYL
GROUPS OF PHENYLCARBAMOYLATED CD ON
ENANTIOSELECTIVTY


107
4.1 Introduction 108
4.2 Characteristic of the CSPs 111
4.3 Discussion on the π-basic columns 112
4.4 Comparison between π-basic and π-acidic phases 117

4.5 Influence of the Substituents present on Phenyl Groups on
Carbamate Moiety

121
4.6 Separation and Comparisons using Racemic drugs 122
4.7 Conclusion 124

Chapter 5 COMPARISON BETWEEN TWO IMMOBILIZATION
PROCEDURES AND PROPOSAL OF NEW SYNTHETIC
METHODOLOGIES


126
5.1 Introduction 127
5.2 Comparisons between Immobilization Procedures using

v

Perphenylcarbamoylated CD CSPs 128
5.3 Comparisons between Immobilization Procedures using
Permethylated CD CSPs

132
5.4 Chromatographic Properties of the CSP made from a New
Procedure

134
5.5 Effect of Phenyl Isocyanate Capping on Enantioselectivity 137
5.6 Conclusion 141

Chapter 6 CHROMATOGRAPHIC PROPERTIES AND CONDITIONS
INVESTIGATION FOR A CSP BASED ON C2-
ALLYLCARBAMIDO-PERMETHYLATED CD


143
6.1 Introduction 144
6.2 Comparison of
CSP-2-ME
with other CSPs (CD immobilized at
primary rim)

145
6.3 Investigation of HPLC separation conditions using Permethylated
CD CSP

148
6.3.1

Influence of pH on Chromatographic Properties 149
6.3.2

Influence of Composition of Organic Modifier on
Chromatographic Properties

155
6.4 Chiral Amino Acid Separations 157
6.5 Conclusion 160

Chapter 7 EXPERIMENTAL 163

7.1 Reagents and Apparatus 164
7.2 HPLC Conditions 165
7.3 Carbamoylation 166

vi

7.4 Methylation 172
7.5 Acetylation 174
7.6 Benzoylation 175
7.7 Preparation of mono-(6
A
-derivatised)- β-CD 176
7.8 Immobilization Reactions 183
7.9 Preparation of HPLC column 186

Chapter 8 CONCLUSIONS 187
Future Developments 194

REFERENCES 196

vii

SUMMARY


Enantiomers are molecules which cannot be superimposed on their mirror
images. The separation of enantiomers is important in many industries and scientific
disciplines. The pharmaceutical industry in particular is concerned with the separation
of enantiomers since many of these isomers are known to have different physiological
activities. For example, in the case of the β-blocker propranolol, only the l-isomer is

beneficial in treating angina pectoris, while d-propranolol mediates the anti-
arrhythmic and antihypertensive activity of the racemic mixtures. Since Louis Pasteur
first discovered the spontaneous resolution of racemic ammonium sodium tartrate
(1848), many physical and chemical separative methods have evolved with the
fundamental aim to afford drugs safe for consumption.

Separation of drug enantiomers through the use of chiral stationary phases
(CSPs) via chromatographic means offers a convenient, rapid and reliable technique
to separate enantiomers. A search of the literatures concerning separation science
reveals that there is an abundance of CSPs on the market that can achieve
enantioseparation to racemates. Over the past two decades, there has been a dynamic
growth in CSP research; in 1987, a classification system was drawn out for these
CSPs according to the type of separating materials (namely cellulose, cyclodextrin,
crown ether, proteins, Pirkle-type molecules and metal complexes) with their unique
mechanisms. Even though there is an abundance of CSPs in the market, there is, till
today, no one single CSP that can universally separate all types of racemates (acids,
bases and bulky solutes), and the exact separation mechanism between CSP and
solute still remains obscure.

viii
This thesis reports the application of several CSPs based upon β-cyclodextrin
(β-CD) as chiral selector in chromatographic separations of several classes of
racemates. β-CD, consisting of an optically active hydrophobic cavity, was chosen
due to its excellent selectivity in chiral separations when bonded to silica supports. It
is also relatively inexpensive and easily available. It is hoped that this work can help
to gain a better understanding of the mechanism behind chiral separation using β-CD
chiral stationary phases.

Our research group has done several studies on the development of novel CD-
based CSPs using different synthetic approaches with successful outcomes. Many

enantioseparations have been achieved using these columns and they are feasible for
use in the analytical as well as the preparative scale. Drawing on this success, this
work seeks to expand the application of the existing CD columns by developing chiral
separating phases based on several modified CDs.

In this dissertation, fifteen modified CD CSPs, based on a stable single urea
linkage to the silica support, were prepared and their chromatographic properties were
evaluated using High performance liquid chromatography (HPLC) and compared
using a broad range of racemic drugs and compounds. Research in this project
focused on several areas, the effects of different CD immobilization position (via C2-
OH and C6-OH) to the silica support, different substituent groups (phenylcarbamate,
acetyl, methyl, benzoyl) on the CD and different immobilization approaches on
chromatographic properties. The influence of changing pH of the buffer and mobile
phase composition on enantioseparation of several racemic drugs and compounds

ix
were also examined using a permethylated CD CSP based on the chiral selector,
mono-(2
A
-allylcarbamido-2
A
-deoxy)-permethylated β-CD.

HPLC results have shown that all fifteen modified CD CSPs can give
effective separations. In brief, the perphenylcarbamoylated CD CSP gave separations
to all 31 racemates tested in this thesis under normal as well as reversed-phase
conditions and was found to be particularly useful in the separations of a diverse
range of racemates compared to peracetylated CD CSP, permethylated CD CSP and
perbenzoylated CD CSP. A series of CSPs, where CD derivatives were
regioselectively bonded at the C2-OH position to the silica gel, were prepared, and

when evaluated for chiral separations, showed different separation behaviours as
compared to those bonded at the C6-OH position. Results indicated that inclusion
complexation to the CD cavity with bulky CD substituents was sterically hindered on
the phase where the wider opening (secondary face) of the CD torus was bonded to
the silica support. Their synthetic approaches and characterisations were described in
chapter 2. Three other phenylcarbamate CD CSPs, i.e. per(p-
chloro)phenylcarbamoylated CD CSP, per(3,5-dimethy)phenylcarbamoylated CD
CSP and per(p-methoxy)phenylcarbamoylated CD CSP, were also synthesized and
compared with the unsubstituted perphenylcarbamoylated CD CSP. Among the four,
the unsubstituted perphenylcarbamoylated CD CSP was demonstrated to give the best
separations to a range of racemates, and π-π interactions were demonstrated to be
important forces in the separation mechanisms. Chromatographic properties can be
affected by different CD immobilization procedures and this had been demonstrated
by the use of four synthetic methods to generate different types of CD CSPs. HPLC
results showed that achiral, non-selective interactions with surface amino and silanol

x
groups could have adverse effects on the retention times as well as
enantioselectivities. The last chapter showed that a newly developed permethylated
CSP based on mono-(2
A
-allylcarbamido-2
A
-deoxy)-permethylated β-CD could be
used to “condition” separations by the adjustments of pH of the buffer and polarity of
the mobile phase, and that these HPLC conditions in turn depended greatly on the
characteristics of the analytes.


xi

Lists of Symbols and Abbreviations

α Selectivity
AM Amino
AC Peracetylated
BZ Perbenzoylated
ClPC Per(p-chloro)phenylcarbamoylated
CSP Chiral stationary phase
δ NMR chemical shift
DEPT Distortionless enhancement by polarization transfer
DMPC

Per(3,5-dimethy)phenylcarbamoylated
ME Permethylated
GC Gas chromatography
k’ Retention factor
LC Liquid chromatography
OMPC

Per(p-methoxy)phenylcarbamoylated
PC Perphenylcarbamoylated
Rs Resolution
t
1
, t
2
Retention times for enantiomer 1 and enantiomer 2

xii
List of Publications

1. Poon Y.F., Muderawan I.W., Ng S.C.; J. Chromatogr. A; 2006, 1101, 1-2, 185

2. Poon Y.F., Muderawan I.W., Ng S.C.; Preparation and Chromatographic
Properties of a Chiral Stationary Phase based on Mono-2-allylcarbamido-2-deoxy-
permethylated β-cyclodextrin; In preparation

3. Poon Y.F., Muderawan I.W., Chen L., Ng S.C.; Synthesis and application of
mono-2
A
-azido-2
A
-deoxyperphenylcarbamoylated β-cyclodextrin and mono-2
A
-
azido-2
A
-deoxyperacetylated β-cyclodextrin as chiral stationary phases for high-
performance liquid chromatography; paper presented in Symposium : Recent
Advances in Separation & Purification Techniques for Biological &
Pharmaceutical Products Development; Nanyang Technological University,
Singapore, 21-23 Feb. 2005





1

CHAPTER 1:
INTRODUCTION

2

Within the past decade, there has been a rapid increase in separations of
enantiomeric compounds (optical or chiral isomers). Enantiomers are isomeric
molecules that have one or more chiral (asymmetric) centers in the molecule – usually
a carbon atom substituent with four different groups. Interest in these compounds
stems from their importance to pharmaceutical, agricultural and other related fields. In
general, the biological activity of two chiral isomers can be different, for example, in
the case of the drug penicillamine, the D-isomer possesses therapeutic properties,
while the L-isomer is highly toxic. Methamphetamine is also a chiral molecule where
L-methamphetamine is a decongestant with no stimulant activity while the D-isomer
is a banned stimulant. Very often, one of the enantiomers may represent the more
active isomer for a given action (eutomer), while the other (distomer) might be even
active in a different way, contributing to side effects, displaying toxicity, or acting as
antagonist. Therefore it is extremely crucial to carry out enantiomeric analysis and
separation.
1-3
Figure 1.1 shows two examples of drugs that can cause different effects
due to the contribution by two different isomers.

In 2002, the sales of chiral compounds as drugs (individual isomers as well as
racemic mixtures) had reached $159 billion, of which approximately 30% of all drug
sales were single enantiomers. Global sales of single-enantiomer compounds are
expected to grow annually by 11.4%.
2
This increasing demand of chiral compounds in
the pharmaceutical industry has also stimulated the development of new and

specialized companies in asymmetric synthesis giving enantiomerically pure
substances.
1-3



3


Figure 1.1 Examples of chiral drugs

1.1 The History of Enantiomeric Resolution

The resolution of enantiomers can be said to be started by Pasteur
4
who first
discovered the spontaneous resolution of racemic ammonium sodium tartrate, which
yielded two enantiomorphic crystals. Hand-picked crystals gave an optical rotation of
polarized light in solution. He proposed that the two sets of crystals are mirror images
of each other.

Later, Schenk
5
introduced urea for resolution of enantiomers, which relied on
the formation of inclusion compounds. The urea molecules are arranged in a left-
handed or right-handed manner forming two enantiomorphous lattice. Each lattice has

4
a different affinity to include one of the enantiomers. Cyclodextrin, deoxycholic acid,
Cram’s crown ethers and other compounds also form, similar to urea, chiral inclusion

compounds.

In 1951, Kotake
6
studied the influence of a chiral mobile phase on the
resolution of several amino acids on paper chromatography and found that the
separation could be ascribed to the chirality of the support (cellulose). Dalgliesh
7
later
suggested a necessary three-point simultaneous attachment between the resolvable
amino acids and the cellulose surface.

This was followed by Prelog’s
8
usage of starch to separate Troger’s base and
Klemn and Reed’s
9
nitroaromatic compounds which relied on the formation of
molecular complex with other aromatic hydrocarbon via π-π interaction for
separation.

In 1966, Gil-Av, Feibush and Charles-Sigler
10
noted the special relationship
between the solvent and solute in order to establish a separation (difference) between
the enantiomers in the GC columns. He had proposed a model, highlighting the
importance of strong interactions such as π-π interactions, hydrogen bonds and
minimal non-contributing associative forces, to yield transient diastereomers.

In 1968, Rogozhin and Davankov

11
showed that ligand exchange
chromatography (LEC) can be used for chiral separations. When a metal is introduced
into a system capable of reversibly coordinating with an asymmetric stationary phase

5
and the individual enantiomer, complex diastereomeric species are formed. Such a
ligand exchange process, involving a chiral-bonded ligand, led to chiral resolution.

Cram and his group
12
discovered a different kind of inclusion complex in
crown ether, which was later chemically bonded to silica gel to resolve the
enantiomers by the HPLC technique. Since then, an increased number of chiral phases
are available for separating various classes of racemates.
13-16


1.2 Chiral Analysis

The analysis of enantiomers can be divided into two categories: separation-
based and non-separation based.
17-18

• Separation-based methods for chiral analysis include gas, liquid, thin-layer
and affinity chromatography, and capillary electrophoresis.

• Non-separation methods include polarimetry, nuclear magnetic resonance
(NMR), isotopic dilution, calorimetry and enzymatic assay. Main features of
these techniques are summarized in Table 1.1.

The separation methods are primarily used to separate the isomers to obtain
pure enantiomers while the non-separation methods simply detect the isomers.

Separation-based methods for chiral analysis can be classified according to
four main methods, chiral liquid chromatography (LC), chiral gas chromatography

6
(GC), supercritical fluid chiral chromatography (SFC) and chiral capillary
electrophoresis (CE). Table 1.2 compares the brief features of these techniques.

The general principle of any chromatography is based on the partitioning of a
substance between a stationary phase and a mobile phase. Compounds in the mobile
phase partition themselves on the stationary phase in a manner that depends on the
compound’s preference for one phase or the other. There is an associative force
between the chemical compound and either the stationary or the mobile phase, and
this associative force depends on the type and number of chemical functional groups
of the subject compound.

Chiral chromatography, however, does not rely on the nature of the functional
groups in the analysed compound, but with their spatial arrangement, i.e. their
stereoisomerism. The isomers in a racemate have identical chemical functional
groups, but differ in how they are oriented. Therefore, the basis of chiral
chromatography lies in the selector material in the system.

Chiral LC can be done by providing the mobile liquid phase with an
enantiomeric compound as a chiral mobile phase additive (CMA). However, this
approach has substantially been replaced with HPLC or LC which incorporates a
chiral stationary phase or CSP.
20

Compared with the great interest in the application of chiral LC, chiral GC
21

seems less popular nowadays. Chiral GC which is a well-established technique before
LC is nonetheless still widely used. The chiral selector in gas chromatography can be


7
Table 1.1 Features for non-separation methods for chiral analyses
17
Methods Features
Polarimetry
Makes use of the unique property of a chiral compound to rotate the plane
of polarization of plane-polarised light. The asymmetry of the chiral
carbon atom, with its four dissimilar substituent groups, is the basis for
optical isomerism and the ability to rotate a plane of polarized light.
Individual enantiomers rotate the plane of polarized light, but a racemic
mixture will not. The specific rotation [α] can be defined in an equation
and is often solvent and concentration dependent.
Nuclear
Magnetic
Resonance
(NMR)

In NMR, enantiomers first have to be complexed with chemical-shift
reagents (such as optically active lanthanide-shift reagents, (R)-(-)-2,2,2-
trifluoro-1-phenylethanol, chiral selectors such as cyclodextrins, etc),
forming diastereomers that NMR can then differentiate. The peak splitting
resulting from the solvent-induced chemical shift difference is a

consequence of the preferential interaction of one of the enantiomers with
the chiral solvent. Results obtained from NMR methods by peak
integration give the concentration ratio (r) of the enantiomers, and the
enantiomeric purity or enantiomeric excess.
Isotope dilution
This method requires the determination of two variables, the specific
rotation and the isotope content. This technique is based on a procedure
that first dilutes an isotope of known starting concentration with the
isotope present in an unknown sample, and then measures the diluted
concentration. With these measurements, isotope concentration in the
unknown can be inferred. It requires a polarimeter for determining
specific optical rotation, and an instrument for determining isotope
concentration.
Calorimetry
Uses a differential scanning calorimeter. It can detect the temperature
point at which a solid compound melts after its temperature has been
increased by a measured amount. In principle, the energy absorbed or
evolved by a sample is determined as a function of the temperature. For
racemate compounds, DSC traces for the sample of unknown optical
purity as well as for the racemates recorded, giving the necessary data for
a calculation of the enantiomeric composition.




8
Methods Features
Enzymes
techniques
Enzymes have an intrinsic chiral selectivity due to their nature and the

process of evolution in natural systems. Presented with an enantiomeric
pair, an enzyme operating on a chiral compound usually will promote the
transformation of one and not the other isomer, or will do so at a faster
rate on one, more than the other isomer.
X-Ray
crystallography
Discovered by Bijvoet
19
in 1949, X-ray crystallography reveals the
absolute or true orientations of atoms within the molecule and reveal true
enantiomeric spatial configuration. This technique relies on the
interpretation of an anomalous dispersion caused by heavy atoms present
in the crystal lattice. The Bijvoet technique has now been highly refined
by the use of modern computerized diffractometers; it is no longer
necessary to introduce heavy atoms as anomalous scatterers in the crystal
lattice and it is possible to use atoms as light as oxygen as anomalous X-
ray scatterers with present-day technology.


Table 1.2 Features of four main techniques for chiral analysis
HPLC GC CE SFC
Solvent 500-1000 ml Uses high purity
gases
Less than 10 ml Uses mainly carbon
dioxide
CSP with
column
Chiral column Chiral capillary
column
Chiral selector and

glass capillary
Chiral column
Time Time required
to condition the
column
No need to
condition
column
No need to condition
column
Shorter conditioning
time than HPLC
Operating
Cost
High Medium Low Medium
Scale of
Operation
Analytical /
Preparative
Mainly
analytical
Analytical Analytical /
Preparative





9
added in the mobile gas phase. This means adding volatile, enantiomerically pure

chiral substances into the gas mixture with regular, achiral sweep gas such as helium.
However, most chiral gas chromatography today incorporates the chiral selector in the
stationary phase. Cyclodextrin CSPs are now very popular in chiral GC, and with the
benefits of the high efficiency and resolving power of capillary GC it is possible to
resolve large numbers of components of complex mixtures into their enantiomer pairs
in a single run. Chiral GC is frequently used in chiral environmental chemistry to
measure the enantiomeric distribution of chiral organochlorine compounds, e.g.
polychlorinated biphenyl congeners, pesticides and pesticide components in soil and
water.
22


SFC involves the use of a supercritical fluid, which is a material at its critical
point, where it no longer behaves like a normal liquid or gas. The most popular
supercritical fluid used has been carbon dioxide, which displays similar polarity as the
solvents in chiral LC. In principle, dipolar interactions involved in solvation by
carbon dioxide could lead to better enantioselectivity than in LC. The lower viscosity
and higher diffusivity of supercritical fluids often lead to reduced analysis times and
improved efficiency when compared to LC. Improved peak resolution is also provided
by using SFC. Additionally, method development time is reduced because column
equilibration generally occurs more rapidly in SFC than in LC. The ease of mobile
phase removal in SFC also facilitates preparative scale separations. In SFC, separation
can be performed without the need for preparation of aqueous buffers.
23
Rapid

chiral
SFC analysis together with a robotic system was recently employed in an automated
screening method for enzymatic transformations.
24

Additionally chiral semi-

10
preparative SFC to obtain enantiomers of high purity and quantities have been
reported.
24

CE is a separation technique based on the mobility of ions in an electric field.
Like all charged particles, positively-charged ions migrate towards a negative
electrode and negatively-charged ions migrate towards a positive electrode. Ions
migrate at different rates depending on their total charge, size and shape, and can be
separated on the basis of these parameters. They can be used for chiral separation with
the use of a chiral selector, e.g. cyclodextrin. It is well-known for its high efficiency
and high resolving power. However, there may be some loss in efficiency arising
from slow mass transfer, because formation and breakup of the transient
diastereomeric complexes in the running buffer is generally slower than the normal
adsorption and desorption processes occurring in chromatographic techniques.
Advantages also include its speed and ease with which method development can be
performed. With one capillary, the chiral selector can be changed from run to run and
very small amounts of often expensive chiral selectors are consumed, thereby
diminishing the cost associated with the use of chiral CE. This technique is, however,
currently limited to analytical applications.
17-18

However, the mode of separation using CE is less straightforward compared to
HPLC. In CE, the migration time and the resolution of the enantiomers depend on the
pH (which affects electroosmotic flow as well as electrophoretic mobility of the
analyte), the direction of the electric field and the concentration of the chiral selectors.
On the other hand, in HPLC, a fixed amount of the chiral selector is covalently

11
attached to the stationary phase material and the mobility of the analyte in the bulk
liquid is not influenced by the pH of the eluent as in CE.
25


Enantiomeric CE separations of various compounds, e.g., pharmaceuticals,
drug candidates,
26
amino acids,
27
di- and tri peptides,
28
and diastereomeric
flavanones,
29
have been performed using different chiral selectors. Native and
derivatized cyclodextrins are the most widely used chiral selectors while other chiral
selectors such as natural and synthetic chiral micelles, crown ethers, chiral ligands,
proteins, oligo- and polysaccharides, and macrocyclic antibiotics have also been
applied to chiral CE separations.
30

1.3 Major Racemate Separation Technologies

Chromatographic methodologies are the most utilized techniques at the
analytical as well as preparative scale. However, to obtain single enantiomers on a
large scale, crystallization
31
and kinetic resolution procedures

32
are also commonly
used.

Technologies for chiral manufacturing separations divide into two major
categories: physical separation methods and kinetic separation methods. Physical
separation technologies rely on differences in the physical properties of enantiomeric
pairs. These include crystallisation, chromatography and solvent extraction. Kinetic
separation technologies rely on differences in chemical reactivity between the
enantiomers of a pair. Recently, a series of racemic aryl β-hydroxyalkyl sulfones have
been successfully transformed into the corresponding optically active O-acetyl

12
Table 1.3 Features of crystallisation, solvent extraction and kinetic resolution
Techniques Features
1. Crystallisation Is the manipulation of a solution of a material in solvent in such a way
that one material in a mixture crystallises. Then it can be separated
from the solution by filtration, centrifuge or other means. If the desired
enantiomer is crystallised, it can be directly removed. If the undesired
enantiomer or other unwanted product is crystallised, then the desired
product remains in solution and then can be further treated to purify
and reclaim it. Two types of crystallisation technology have been used:
preferential and diastereoisomer.
a. Preferential
crystallisation
Involves seeding a supersaturated solution of racemate with pure
crystals of one of the enantiomers; this seeding causes only one isomer
to crystallise out of solution.
b. Diastereomeric
crystallisation

A chiral precipitating agent (or resolving agent) is added to a racemic
solution to precipitate one enantiomer from the solution. This agent
forms a diastereomeric complex with both isomers of the racemate, but
because of solubility differences, one of the isomer complexes
crystallises out more readily than the other.
2. Solvent
extraction
Involves prudent choice of system and solvent because the two isomers
often have very similar properties, including their solubility in common
solvents. Thus, innovative techniques are required in chiral solvent
extraction. For instance, a chiral selector of opposite character may be
incorporated into each of the liquids and counter-current flow of the
two liquid streams may be employed to effect separation.
3. Kinetic
resolution
Uses enzymes to catalyse a reaction that converts one enantiomer faster
or more completely than the other. Separation is achieved on the basis
of differences in chemical reactivity of the different isomers. For
instance, hydrolysis of one enantiomeric ester which generates an acid
and an alcohol. The ionic acid can then be separated from the neutral,
unreacted enantiomeric ester by ion-exchange chromatography. To
increase the efficiency of the process, the unreacted isomer can be
recycled into the process through a deliberate racemisation.