METHODS

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Chiral Separations
Methods and Protocols
Second Edition

Edited by

Gerhard K.E. Scriba
Department of Pharmaceutical Chemistry, Friedrich Schiller University Jena,
Jena, Germany

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Editor
Gerhard K.E. Scriba
Department of Pharmaceutical Chemistry
Friedrich Schiller University Jena
Jena, Germany

ISSN 1064-3745
ISSN 1940-6029 (electronic)
ISBN 978-1-62703-262-9
ISBN 978-1-62703-263-6 (eBook)
DOI 10.1007/978-1-62703-263-6
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2012952732
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Dedication
To Beate, Sabrina, and Rebecca

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What can more resemble my hand or my ear, and be more equal in all points, than its image in a mirror?
And yet, I cannot put such a hand as is seen in the mirror in the place of its original.
Immanuel Kant
Prolegomena to Any Future Metaphysics That Will Be Able to Come Forward as Science (1783)

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Preface

The importance of the stereochemistry of compounds is well recognized in chemistry and
life sciences since Louis Pasteur discovered the phenomenon of chirality in 1848. The
enantiomers of chiral compounds often differ in their biological, pharmacological, toxicological, and/or pharmacokinetic profile. This has become evident specifically in pharmaceutical sciences, but it also affects chemistry, biology, food chemistry, forensics, etc., and is
reflected in the requirements for chiral compounds by regulatory authorities worldwide.
For example, the Food and Drug Administration (FDA) and the European Medicines
Agency (EMA) require the development of a single enantiomer of a drug candidate if the
enantiomers differ in their pharmacological action, toxicological profile, etc. As a consequence, seven drugs of the top ten drugs (not counting biotechnological drugs) according
to their sales in the USA in 2010 (www.drugs.com/top200.html, accessed February 21,
2012) are single enantiomer drugs, while two drugs are achiral compounds. One product
is a combination of a chiral and a racemic drug. In fact, the top three products are single
enantiomer drugs. However, the importance of chirality does not stop here but is important to any research in life sciences.
Generally, there is a great demand for analytical methods that are able to discriminate
between enantiomers in order to analyze the enantiomeric purity of compounds from natural or chemical sources not only in pharmaceutical sciences but in any field of bioactive
compounds including chemistry, biology, biochemistry, forensic and environmental sciences, and many others. Chromatographic techniques dominated the field of enantioseparations early on, but electrophoretic methods have gained increasing importance in recent
years. While some compounds may be analyzed only with one technique based on their
physicochemical properties, often the analyst can chose between two or more analytical
techniques for a given analyte. This requires knowledge of the strengths and weaknesses of
each technique in order to select the most appropriate method for the given problem.
The focus of Chiral Separations: Methods and Protocols, 2nd edition is clearly on analytical separation sciences by chromatographic and electrophoretic techniques although simulated moving bed chromatography has also been included, which is primarily used as a
preparative method. The book does not claim to comprehensively cover each possible chiral
separation mechanism but to give an overview and especially practically oriented applications of the most important analytical techniques in chiral separation sciences. Thus, the
book follows the well-established scheme of the Methods and Protocols series. Some review
chapters give an overview of the current state of art in the respective field. However, most
chapters are devoted to the description of the typical analytical procedures providing reliable and established procedures for the user. Critical points are highlighted so that the user
is enabled to transfer the described method to his/her actual separation problem.
Sixty-four authors from 34 research laboratories in 17 countries have contributed by
sharing their insight and expert knowledge of the techniques. I would like to take the
opportunity to thank all authors for their efforts and valuable contributions.

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Preface

Chiral Separations: Methods and Protocols, 2nd edition should be helpful for analytical
chemists working on stereochemical problems in fields of pharmacy, chemistry, biochemistry, food chemistry, molecular biology, forensics, environmental sciences, or cosmetics in
academia, government, or industry.
Jena, Germany

Gerhard K.E. Scriba

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Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix
xv

1 Chiral Recognition in Separation Science: An Overview. . . . . . . . . . . . . . . . . .
Gerhard K.E. Scriba
2 Enantioseparations by Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . .
Massimo Del Bubba, Leonardo Checchini, Alessandra Cincinelli,
and Luciano Lepri

3 Gas-Chromatographic Enantioseparation of Unfunctionalized
Chiral Hydrocarbons: An Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Volker Schurig and Diana Kreidler
4 HPLC Enantioseparation on Cyclodextrin-Based Chiral Stationary Phases. . . .
Yong Wang and Siu Choon Ng
5 Enantioseparations by High-Performance Liquid Chromatography
Using Polysaccharide-Based Chiral Stationary Phases: An Overview. . . . . . . . .
Bezhan Chankvetadze
6 Common Screening Approaches for Efficient Analytical Method
Development in LC and SFC on Columns Packed with Immobilized
Polysaccharide-Derived Chiral Stationary Phases . . . . . . . . . . . . . . . . . . . . . . .
Pilar Franco and Tong Zhang
7 Chiral Separations by HPLC on Immobilized Polysaccharide Chiral
Stationary Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Imran Ali, Zeid A. AL-Othman, and Hassan Y. Aboul-Enein
8 Enantioseparations by High-Performance Liquid Chromatography
Using Macrocyclic Glycopeptide-Based Chiral Stationary Phases:
An Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
István Ilisz, Anita Aranyi, Zoltán Pataj, and Antal Péter
9 Enantioseparations of Primary Amino Compounds by High-Performance
Liquid Chromatography Using Chiral Crown Ether-Based Chiral
Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Myung Ho Hyun
10 Screening of Pirkle-Type Chiral Stationary Phases for HPLC
Enantioseparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gregory K. Webster and Ted J. Szczerba
11 Enantioseparations by High-Performance Liquid Chromatography
Based on Chiral Ligand-Exchange. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Benedetto Natalini, Roccaldo Sardella, and Federica Ianni


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12 Enantioseparations by High-Performance Liquid Chromatography
Using Molecularly Imprinted Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
David A. Spivak
13 Chiral Mobile Phase Additives in HPLC Enantioseparations . . . . . . . . . . . . . .
Lushan Yu, Shengjia Wang, and Su Zeng
14 Chiral Benzofurazan-Derived Derivatization Reagents for Indirect
Enantioseparations by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Toshimasa Toyo’oka
15 Separation of Racemic 1-(9-Anthryl)-2,2,2-trifluoroethanol
by Sub-/Supercritical Fluid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . .
Xiqin Yang, Leo Hsu, and Gerald Terfloth
16 Chiral Separations by Simulated Moving Bed Method Using
Polysaccharide-Based Chiral Stationary Phases. . . . . . . . . . . . . . . . . . . . . . . . .
Toshiharu Minoda
17 Enantioseparations by Capillary Electrophoresis Using Cyclodextrins
as Chiral Selectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gerhard K.E. Scriba and Pavel Jáč
18 Application of Dual Cyclodextrin Systems in Capillary Electrophoresis
Enantioseparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anne-Catherine Servais and Marianne Fillet
19 Enantioseparations in Nonaqueous Capillary Electrophoresis
Using Charged Cyclodextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anne-Catherine Servais and Marianne Fillet
20 Use of Macrocyclic Antibiotics as the Chiral Selectors in Capillary
Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chengke Li and Jingwu Kang
21 Application of Polymeric Surfactants in Chiral Micellar
Electrokinetic Chromatography (CMEKC) and CMEKC Coupled
to Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jun He and Shahab A. Shamsi

22 Cyclodextrin-modified Micellar Electrokinetic Chromatography for
Enantioseparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Wan Aini Wan Ibrahim, Dadan Hermawan, and Mohd Marsin Sanagi
23 Cyclodextrin-Mediated Enantioseparation in Microemulsion
Electrokinetic Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Claudia Borst and Ulrike Holzgrabe
24 Chiral Separations by Capillary Electrophoresis Using Proteins
as Chiral Selectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jun Haginaka
25 Enantioseparation by Chiral Ligand-Exchange Capillary Electrophoresis . . . . .
Yi Chen and Lijuan Song
26 Experimental Design Methodologies in the Optimization
of Chiral CE or CEC Separations: An Overview . . . . . . . . . . . . . . . . . . . . . . .
Bieke Dejaegher, Debby Mangelings, and Yvan Vander Heyden

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233

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Contents

27 Chiral Capillary Electrophoresis–Mass Spectrometry . . . . . . . . . . . . . . . . . . . .
Elena Domínguez-Vega, Antonio L. Crego, and Maria Luisa Marina
28 Application of Chiral Ligand-Exchange Stationary Phases in Capillary
Electrochromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Martin G. Schmid
29 Polysaccharide-Derived Chiral Stationary Phases in Capillary
Electrochromatography Enantioseparations. . . . . . . . . . . . . . . . . . . . . . . . . . .
Zhenbin Zhang, Hanfa Zou, and Junjie Ou
30 Open Tubular Molecular Imprinted Phases in Chiral Capillary
Electrochromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Won Jo Cheong and Song Hee Yang
31 Enantioseparations in Capillary Electrochromatography Using Sulfated

Poly β-Cyclodextrin-Modified Silica-Based Monolith as Stationary Phase. . . . .
Ruijuan Yuan and Guosheng Ding
32 Cyclodextrin-Mediated Enantioseparations by Capillary
Electrochromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dorothee Wistuba and Volker Schurig
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors
HASSAN Y. ABOUL-ENEIN • National Pharmaceutical and Medicinal Chemistry

Department, Research Centre, Cairo, Egypt
IMRAN ALI • Department of Chemistry, Jamia Millia Islamia (Central University),
New Delhi, India
ZEID A. AL-OTHMAN • Department of Chemistry, King Saud University, Riyadh,
Kingdom of Saudi Arabia
ANITA ARANYI • Department of Inorganic and Analytical Chemistry, University of Szeged,
Szeged, Hungary
CLAUDIA BORST • Institute of Pharmacy and Food Chemistry, University of Würzburg,
Würzburg, Germany
MASSIMO DEL BUBBA • Department of Chemistry, University of Florence,
Sesto Fiorentino, Italy
BEZHAN CHANKVETADZE • Institute of Physical and Analytical Chemistry, Tbilisi State
University, Tbilisi, Georgia
LEONARDO CHECCHINI • Department of Chemistry, University of Florence,
Sesto Fiorentino, Italy
YI CHEN • Key Laboratory of Analytical Chemistry for Living Biosystems,
Chinese Academy of Sciences, Beijing, China
WON JO CHEONG • Department of Chemistry, Inha University, Incheon, South Korea
ALESSANDRA CINCINELLI • Department of Chemistry, University of Florence,
Sesto Fiorentino, Italy
ANTONIO L. CREGO • Department of Analytical Chemistry, University of Alcalá,
Alcalá de Henares, Spain
BIEKE DEJAEGHER • Department of Analytical Chemistry and Pharmaceutical Technology,
Vrije Universiteit Brussel, Brussels, Belgium
GUOSHENG DING • Analysis Center, Tianjin University, Tianjin, China
ELENA DOMÍNGUEZ-VEGA • Department of Analytical Chemistry, University of Alcalá,
Alcalá de Henares, Spain
MARIANNE FILLET • Department of Pharmaceutical Sciences, University of Liège, Liège,
Belgium
PILAR FRANCO • Chiral Technologies Europe, Illkirch, France

JUN HAGINAKA • School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s
University, Nishinomiya, Japan
JUN HE • Department of Chemistry, Center of Biotechnology and Drug Design,
Georgia State University, Atlanta, GA, USA
DADAN HERMAWAN • Department of Chemistry, Universiti Teknologi Malaysia, Johor,
Malaysia
YVAN VANDER HEYDEN • Department of Analytical Chemistry and Pharmaceutical
Technology, Vrije Universiteit Brussel, Brussels, Belgium
ULRIKE HOLZGRABE • Institute of Pharmacy and Food Chemistry, University of Würzburg,
Würzburg, Germany

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xvi

Contributors

LEO HSU • GlaxoSmithKline Research and Development, King of Prussia, PA, USA
MYUNG HO HYUN • Department of Chemistry and Chemistry, Pusan National University,
Busan, South Korea
FEDERICA IANNI • Dipartimento di Chimica e Tecnologia del Farmaco, Università degli
Studi di Perugia, Perugia, Italy
WAN AINI WAN IBRAHIM • Separation Science and Technology Group (SepSTec),
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia,
Johor, Malaysia
ISTVÁN ILISZ • Department of Inorganic and Analytical Chemistry, University of Szeged,
Szeged, Hungary

PAVEL JÁČ • Department of Pharmaceutical/Medicinal Chemistry, Friedrich Schiller
University Jena, Jena, Germany
JINGWU KANG • Chinese Academy of Sciences, Shanghai Institute of Organic Chemistry,
Shanghai, China
DIANA KREIDLER • Institute of Organic Chemistry, University of Tübingen, Tübingen,
Germany
LUCIANO LEPRI • Department of Chemistry, University of Florence, Sesto Fiorentino, Italy
CHENGKE LI • Chinese Academy of Sciences, Shanghai Institute of Organic Chemistry,
Shanghai, China
DEBBY MANGELINGS • Department of Analytical Chemistry and Pharmaceutical
Technology, Vrije Universiteit Brussel, Brussels, Belgium
MARIA LUISA MARINA • Department of Analytical Chemistry, University of Alcalá, Alcalá
de Henares, Spain
TOSHIHARU MINODA • Daicel Corporation, Niigata, Japan
BENEDETTO NATALINI • Dipartimento di Chimica e Tecnologia del Farmaco, Università
degli Studi di Perugia, Perugia, Italy
SIU CHOON NG • School of Chemical and Biomedical Engineering, Nanyang
Technological University, Singapore, Singapore
JUNJIE OU • National Chromatographic R&A Center, Dalian Institute of Chemical
Physics, Dalian, China
ZOLTÁN PATAJ • Department of Inorganic and Analytical Chemistry, University of Szeged,
Szeged, Hungary
ANTAL PÉTER • Department of Inorganic and Analytical Chemistry, University of Szeged,
Szeged, Hungary
MOHD MARSIN SANAGI • Department of Chemistry, Universiti Teknologi Malaysia, Johor,
Malaysia
ROCCALDO SARDELLA • Dipartimento di Chimica e Tecnologia del Farmaco, Università
degli Studi di Perugia, Perugia, Italy
MARTIN G. SCHMID • Institute of Pharmaceutical Sciences, Karl-Franzens-University,
Graz, Austria

VOLKER SCHURIG • Institute of Organic Chemistry, University of Tübingen, Tübingen,
Germany
GERHARD K.E. SCRIBA • Department of Pharmaceutical/Medicinal Chemistry, Friedrich
Schiller University Jena, Jena, Germany
ANNE-CATHERINE SERVAIS • Department of Pharmaceutical Sciences, University of Liège,
Liège, Belgium

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Contributors

xvii

SHAHAB A. SHAMSI • Department of Chemistry, Center of Biotechnology and Drug Design,
Georgia State University, Atlanta, GA, USA
LIJUAN SONG • Chinese Academy of Sciences, Key Laboratory of Analytical Chemistry
for Living Biosystems, Beijing, China
DAVID A. SPIVAK • Department of Chemistry, Louisiana State University, Baton Rouge,
LA, USA
TED J. SZCZERBA • Regis Technologies, Morton Grove, IL, USA
GERALD TERFLOTH • GlaxoSmithKline Research and Development, King of Prussia,
PA, USA
TOSHIMASA TOYO’OKA • Graduate School of Pharmaceutical Sciences, University of Shizuoka,
Shizuoka, Japan
SHENGJIA WANG • Department of Pharmaceutical Analysis and Drug Metabolism,
Zhejiang University, Hangzhou, China
YONG WANG • Department of Chemistry, School of Sciences, Tianjin University,
Tianjin, China
GREGORY K. WEBSTER • Abbott Laboratories, Abbott Park, IL, USA

DOROTHEE WISTUBA • Institute of Organic Chemistry, University of Tübingen,
Tübingen, Germany
SONG HEE YANG • Department of Chemistry, Inha University, Incheon, South Korea
XIQIN YANG • GlaxoSmithKline Research and Development, King of Prussia, PA, USA
LUSHAN YU • Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang
University, Hangzhou, China
RUIJUAN YUAN • School of Chinese Pharmacy, Beijing University of Chinese Medicine,
Beijing, China
SU ZENG • Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang
University, Hangzhou, China
TONG ZHANG • Chiral Technologies Europe, Illkirch, France
ZHENBIN ZHANG • National Chromatographic R&A Center, Dalian Institute of Chemical
Physics, Dalian, China
HANFA ZOU • National Chromatographic R&A Center, Dalian Institute of Chemical
Physics, Dalian, China

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Chapter 1
Chiral Recognition in Separation Science: An Overview
Gerhard K.E. Scriba
Abstract
Chiral recognition phenomena play an important role in nature as well as analytical separation sciences. In
separation sciences such as chromatography and capillary electrophoresis, enantiospecific interactions
between the enantiomers of an analyte and the chiral selector are required in order to observe enantioseparations. Due to the large structural variety of chiral selectors applied, different mechanisms and structural
features contribute to the chiral recognition process. This chapter briefly illustrates the current models of

the enantiospecific recognition on the structural basics of various chiral selectors.
Key words: Chiral separation, Chiral recognition mechanism, Chiral selector, Enantiodifferentiation

1. Introduction
The differentiation of enantiomers is a fundamental natural phenomenon as chiral bioactive compounds interact in a stereospecific
way with each other. Therefore, chiral molecules play an important
part in many aspects of life sciences, medical sciences, synthetic
chemistry, food chemistry, as well as many other fields. Consequently,
analytical techniques capable of differentiating stereoisomers,
specifically enantiomers, are required. With regard to analytical
enantioseparations, chromatography and electromigration techniques are the most important ones. Chromatographic techniques
include thin layer chromatography (TLC), gas chromatography
(GC), high-performance liquid chromatography (HPLC), as well as
super- and subcritical fluid chromatography (SFC). Capillary electromigration techniques which utilize electrophoretic phenomena
for the movement of the analytes toward the detector include capillary electrophoresis (CE), capillary electrokinetic chromatography
(EKC), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic chromatography (MEEKC), and capillary

Gerhard K.E. Scriba (ed.), Chiral Separations: Methods and Protocols, Methods in Molecular Biology, vol. 970,
DOI 10.1007/978-1-62703-263-6_1, © Springer Science+Business Media, LLC 2013

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G.K.E. Scriba

electrochromatography (CEC). Enantioseparations can be divided

into indirect and direct methods. In the indirect approach, the analyte enantiomers are reacted with an enantiopure reagent to form a
pair of diastereomers via covalent bonds. The diastereomers can be
subsequently separated under achiral conditions. Direct methods
refer to the separation of enantiomers in a chiral environment. This
requires the presence of a chiral selector either fixed to an immobile
support or as additive to the mobile phase or the background electrolyte. The separation is based on the formation of transient diastereomeric complexes in a thermodynamic equilibrium.
This introductory chapter of chiral separations will briefly
highlight the recognition mechanisms of the most frequently used
chiral selectors in stereoselective analysis, many of which are used
in the examples described in subsequent chapters. Considering all
selectors described in the literature, the present selection is far from
complete although some new developments such as aptamers or
chiral ionic liquids will also be discussed. No distinction will be
made between the individual basic techniques, i.e., between chromatography and electromigration methods. This is feasible because
there is no fundamental difference between the stereospecific interaction between enantiomers and a given chiral selector which is
bound to a stationary phase in chromatography or mobile in the
background electrolyte as in electrophoretic methods. The
stereospecific recognition is a chromatographic phenomenon independent of the mobility of a chiral selector (1). The fact that a
chiral selector is dissolved in the background electrolyte and mobile
in electrophoresis (a so-called pseudostationary phase) and not a
“true” stationary phase is not a conceptual difference. However,
one might argue that the stereoselectivity of a given selector may
be different whether it is fixed to a solid support compared to the
situation in solution so that the chiral recognition of a selector may
differ, whether it is fixed to a stationary phase, or whether it is
added to the liquid phase. For further reading on chiral recognition mechanisms in separation sciences, recent review papers (2–4)
and a monograph (5) are recommended.

2. Chiral
Recognition Model

In separation sciences, the reversible formation of diastereomers
between the enantiomers of a solute and the chiral selector is
the basis for chiral separations via direct methods. This equilibrium can be characterized by the equations:
KR

(R) − A + (R) − S ↔ [(R) − A
KS

(S ) − A + (R) − S ↔ [(S ) − A

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(R) − S]
(R) − S]


1

Chiral Recognition Mechanisms
C

D
A

C

A

D


B

B

B'

B'

C'

3

A'
R

C'

A'
R

Fig. 1. Scheme of the three-point interaction model.

Differences between the association constants, KR and KS, represent the physicochemical basis for the stereoselective retention of
the enantiomers by a chiral selector.
Early attempts to rationalize enantiospecific interactions at the
molecular level led to the Easson–Stedman “three-point attachment
model” (6) as a rigid geometric model (Fig. 1). One enantiomer
displays optimal fit forming three interactions with the selector,
while the other enantiomer is bound less tightly due to the formation of only two interactions. This simplistic model is only valid if
interactions of the chiral molecule with the selector can occur from

one side. Moreover, it does not reflect the nature of the interactions, i.e., attraction or repulsion. It has been noted that at least one
of the interactions has to be attractive to allow the formation of one
of the two possible diastereomeric complexes (7). Despite a lot of
criticism, the model may still be used for illustrative purposes considering that the chiral selector is not a plane but rather represented
by a three-dimensional structure. Furthermore, the criterion of
inequality of distance matrices of the diastereomeric complexes has
been introduced (8). This formalism allows the explanation of one-,
two-, and three-point mechanisms as the basis for chiral recognition. Moreover, interactions may rather be mediated via multiple
points instead of single points. For example, p–p and dipole–dipole
interactions are considered multipoint interactions. As a consequence, due to spatial requirements, one enantiomer of a selectand
exhibits an “ideal fit” with the chiral selector resulting in larger
binding constant compared to the other enantiomer possessing a
smaller binding constant due to its “nonideal fit.”
Complex formation is driven by several interactions, e.g., ionic
interactions, ion–dipole or dipole–dipole interactions, hydrogenbonds, van der Waals interactions, and p–p interactions. Ionic interactions are strong but may be primarily involved in the establishment
of the “first contact” due to their long-range nature. However, as
both enantiomers of an ionized solute are able to form these interac-

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G.K.E. Scriba

tions, they may not be stereoselective. In contrast, hydrogen bonds
and p–p interactions are short-range directional forces so that these
may be primarily responsible for stereoselective interactions, i.e., stereoselectivity (2). Furthermore, steric factors, i.e., fit or non-fit of
the solute in a cavity or cleft of the selector, contribute to the chiral
recognition. A conformational change of the selector during complex formation with the solute (induced fit) is also possible.


3. Recognition
Mechanisms of
Chiral Selectors

3.1. Polysaccharide
Derivatives

Several methods have been applied to the investigation of the chiral
recognition mechanisms of selectors (2, 5). Chromatographic and
electrophoretic studies have employed the variation of the structure
of the selectands or the selectors in order to establish “structure–
separation” relationships. Furthermore, the separation conditions
can be changed. Spectroscopic techniques include UV spectroscopy,
fluorimetry, Fourier transform and attenuated total reflectance IR
spectroscopy, NMR spectroscopy, as well as circular dichroism and
vibrational circular dichroism (VCD) spectroscopy. Especially NMR
techniques including nuclear Overhauser effect (NOE) and rotatingframe Overhauser enhancement (ROE) have the advantage of allowing conclusions about the spatial proximity of atoms or substituents
(9, 10). However, these methods can only by applied for soluble
selectors. Moreover, the selector–selectand interactions may vary
depending on the solvents so that the data have to be interpreted
with caution when solvents differ between NMR and separation
experiments. X-ray crystallography yields the structure of the selectand–selector complex in the solid state. It should be kept in mind
that the structure in solution may differ from the solid state. Finally,
chemoinformatics (11) and molecular modeling methods (12) have
been used to illustrate the selector–selectand interactions.
The suitability of natural polysaccharides for enantioseparations in
chromatography has been recognized in the early 1970s by Hesse
and Hagel using cellulose triacetate as stationary phase (13). The
modern polysaccharide-based chiral stationary phases have been

pioneered by Okamoto and coworkers (14, 15). These stationary
phases are based on the polysaccharides cellulose and amylose
which have been derivatized with aromatic substituents to yield a
large variety of derivatives with different selectivities and applications (16–19). To date they represent by far the most widely used
chiral stationary phases in HPLC due to their broad applicability
for a large structural diversity of compounds. Commercial products with a wide variety of substitutions and different immobilization chemistry are available from Chiral Technologies under the trade
names Chiralcel™ and Chiralpak™ or from Phenomenex as Lux

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Chiral Recognition Mechanisms

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Amylose™ and Lux Cellulose™ columns. It has been estimated that
the two most popular chiral stationary phases containing cellulose
tris(3,5-dimethylphenylcarbamate) (Chiralcel OD™, Chiralpak IB™,
Lux Cellulose-1™) and amylose tris(3,5-dimethylphenylcarbamate)
(Chiralpak AD™, Chiralpak IA™) account for about 2/3 of the chiral separations achieved with polysaccharide-derived selectors (16).
Cellulose tris(3,5-dimethylphenylcarbamate) and amylose
tris(3,5-dimethylphenylcarbamate) have been investigated in detail
by NMR, VCD, attenuated total reflectance IR spectroscopy, and
molecular modeling. The glucose units are arranged along the
helical axis with the substituents creating a helical groove. The carbamate groups are located inside, while the hydrophobic aromatic
moieties are located outside the polymer chain. In the case of amylose tris(3,5-dimethylphenylcarbamate), a left-handed 4/3 helix
has been derived from NMR and computational studies (20).
A left-handed helix was also concluded from VCD (21, 22). The

structure of cellulose tris(3,5-dimethylphenylcarbamate) appears
to be somewhat controversial as a left-handed helical structure has
been derived in molecular modeling studies (23). VCD measurements indicated a right-handed helix of the polymer as a film but a
left-handed helical structure in solution in dichloromethane (22).
The chiral groove of cellulose tris(3,5-dimethylphenylcarbamate)
appears to be slightly larger than the groove of amylose tris(3,5dimethylphenylcarbamate) (22, 24). The composition of the
mobile phase may cause changes in the structure of amylose
tris(3,5-dimethylphenylcarbamate) by affecting intramolecular
hydrogen bonds which seems to affect the chiral recognition of the
selector observed in HPLC enantioseparations using this stationary phase (22, 25–27).
When amylose tris(3,5-dimethylphenylcarbamate) encapsulates
rodlike poly(p-phenylenevinylene), a higher-ordered helical structure
compared to amylose tris(3,5-dimethylphenylcarbamate) without
the rodlike polymer in the interior cavity of amylase was concluded
from molecular modeling (28). This indicated a closer packing of the
phenylcarbamate residues in the poly(p-phenylenevinylene)-amylose
composite which would rationalize differences in the chiral recognition ability of the selectors in HPLC experiments.
In the case of polysaccharides, selector–selectand complex
formation may be mediated via hydrogen bonds to C=O or NH
of the carbamate groups as well as via p–p interactions with the
phenyl rings. The carbamate groups are located deeply inside the
cavities near the carbohydrate polymer backbone and are flanked
by the aromatic substituents which may affect the access to the
binding pocket via steric factors. The carbamate linkages allow
some flexibility for an adjustment of the aromatic moieties for
maximizing p–p interactions (induced fit). This binding mode
has been illustrated in several studies including techniques such
as NMR, attenuated total reflectance IR spectroscopy, and

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6

G.K.E. Scriba

Fig. 2. Energy-minimized structures of complexes of amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) with
(a) (1S,2R )-(+)-norephedrine (+PPA) and (b) (1R,2S )-(−)-norephedrine (−PPA). The dotted lines indicate hydrogen bonds,
p refers to p–p interactions (For the colored version of the figure, see the online version of the reference. Reproduced by
permission of Elsevier from ref. 30 © 2008).

molecular modeling (20, 29–31). Figure 2 shows the energyminimized structures of the complexes between amylose tris(3,5dimethylphenylcarbamate) and the enantiomers of norephedrine
(2-amino-1-phenyl-1-propanol, PPA) (30). The stronger retained
(1R,2S)-configured (−)-enantiomer displays three interactions,
two hydrogen bonds, i.e., (polymer)NH···OH(−PPA) and (polymer)C=O···H2N(−PPA), and one p–p interaction. In the case of
the weaker bound (1S,2R)-(+)-enantiomer, only two interactions,
one hydrogen bond and one p–p interaction, are observed.
Interestingly, the situation is reversed for cellulose tris(3,5dimethylphenylcarbamate). The stronger bound (+)-enantiomer
established one hydrogen bond and two p–p interactions with the

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Chiral Recognition Mechanisms

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selector, while the weaker complexed (−)-enantiomer forms only

one hydrogen bond and one p–p interaction. The modeling studies
are in accordance with the reversed elution order of the norephedrine enantiomers for the two chiral stationary phases (30).
It has also been shown that the selectand may change its conformation upon binding to the selector leading to a tight fit. For
example, in the protonated state, the stronger complexed (S)enantiomer of p-O-tert-butyltyrosine allyl ester folds when binding
to amylose tris(3,5-dimethylphenylcarbamate) in contrast to the
weaker bound (R)-enantiomer as evidenced from NMR and
molecular modeling studies (32).
A modified solvation parameter model has been developed in
order to rationalize the enantioselectivity of amylase tris(3,5-dimethylphenylcarbamate) and cellulose tris(3,5-dimethylphenylcarbamate) as chiral selectors in supercritical fluid chromatography
using a set of 135 structurally diverse solutes (33). Molecular properties including p and n electrons, hydrogen-bonding acceptor
and donor ability, molecular volume, flexibility, and globularity as
well as the respective interactions related to the solute descriptors
were selected. Factorial discriminant analysis was employed to identify significant factors. Steric fit associated to hydrogen-bonding
appeared to be the most important feature for enantiorecognition
by amylose tris(3,5-dimethylphenylcarbamate), while chiral recognition on cellulose tris(3,5-dimethylphenylcarbamate) requires
dipole–dipole and p–p interactions in addition to hydrogenbonding. The descriptors flexibility and globularity were highly relevant for the description of enantiorecognition in the model.
Furthermore, the study indicated that interactions providing the
principal contribution to retention on the stationary phase are not
necessarily the major contributors to enantioseparations which have
to be attributed to a combination of (stereo)selective interactions.
3.2. Cyclodextrins

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of a(1,
4)-linked D-glucose units produced by the digestion of starch by
cyclodextrin glycosyl transferase of various bacteria such as Bacillus
strains (34). The most important industrially produced CDs differ
in the number of glucose units, i.e., a-CD is composed of six glucose molecules, b-CD of seven molecules, and g-CD of eight molecules. The compounds are shaped like a hollow torus with a
lipophilic cavity and a hydrophilic outside. The narrower rim is
formed by the primary 6-hydroxyl groups, while the wider rim
contains the 2- and 3-hydroxyl groups of the glucose units. The

top and bottom diameters of the cavity of the CDs are 4.7 and
5.3Å for a-CD, 6.0 and 6.5Å for b-CD, and 7.5 and 8.3Å for gCD (35). The hydroxyl groups can be derivatized resulting in a
large variety of CD derivatives containing uncharged or charged
substituents. Due to their ability to form inclusion complexes, CDs
have found numerous applications. With regard to stereoisomer

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