(Methods in molecular biology 970) gerhard k e scriba (auth ), gerhard k e scriba (eds ) chiral separations methods and protocols humana press (2013)

69 Yong Wang and Siu Choon Ng 5 Enantioseparations by High-Performance Liquid Chromatography Using Polysaccharide-Based Chiral Stationary Phases: An Overview.. 137 István Ilisz, Anita

Series Editor

John M Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651

Chiral Separations

Methods and Protocols

Second Edition

Edited by

Gerhard K.E Scriba

Department of Pharmaceutical Chemistry, Friedrich Schiller University Jena,

Jena, Germany

Department of Pharmaceutical Chemistry

Friedrich Schiller University Jena

Jena, Germany

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

© Springer Science+Business Media, LLC 2013

This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, speci fi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction

on micro fi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied speci fi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law.

The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a speci fi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

Humana Press is a brand of Springer

Springer is part of Springer Science+Business Media (www.springer.com)

To Beate, Sabrina, and Rebecca

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)

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, toxico-logical, and/or pharmacokinetic pro fi le This has become evident speci fi cally in pharma-ceutical sciences, but it also affects chemistry, biology, food chemistry, forensics, etc., and is

re fl ected 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 pro fi le, etc As a conse-quence, 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 impor-tant 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 natu-ral or chemical sources not only in pharmaceutical sciences but in any fi eld of bioactive compounds including chemistry, biology, biochemistry, forensic and environmental sci-ences, and many others Chromatographic techniques dominated the fi eld of enantiosepa-rations 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

analyti-cal separation sciences by chromatographic and electrophoretic techniques although lated 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 applica-tions of the most important analytical techniques in chiral separation sciences Thus, the

simu-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 fi eld However, most chapters are devoted to the description of the typical analytical procedures providing reli-able 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

Chiral Separations: Methods and Protocols, 2nd edition should be helpful for analytical

chemists working on stereochemical problems in fi elds of pharmacy, chemistry, try, food chemistry, molecular biology, forensics, environmental sciences, or cosmetics in academia, government, or industry

1 Chiral Recognition in Separation Science: An Overview 1

Gerhard K.E Scriba

2 Enantioseparations by Thin-Layer Chromatography 29

Massimo Del Bubba, Leonardo Checchini, Alessandra Cincinelli,

and Luciano Lepri

3 Gas-Chromatographic Enantioseparation of Unfunctionalized

Chiral Hydrocarbons: An Overview 45

Volker Schurig and Diana Kreidler

4 HPLC Enantioseparation on Cyclodextrin-Based Chiral Stationary Phases 69

Yong Wang and Siu Choon Ng

5 Enantioseparations by High-Performance Liquid Chromatography

Using Polysaccharide-Based Chiral Stationary Phases: An Overview 81

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 113

Pilar Franco and Tong Zhang

7 Chiral Separations by HPLC on Immobilized Polysaccharide Chiral

Stationary Phases 127

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 137

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 165

Myung Ho Hyun

10 Screening of Pirkle-Type Chiral Stationary Phases for HPLC

Enantioseparations 177

Gregory K Webster and Ted J Szczerba

11 Enantioseparations by High-Performance Liquid Chromatography

Based on Chiral Ligand-Exchange 191

Benedetto Natalini, Roccaldo Sardella, and Federica Ianni

12 Enantioseparations by High-Performance Liquid Chromatography

Using Molecularly Imprinted Polymers 209

David A Spivak

13 Chiral Mobile Phase Additives in HPLC Enantioseparations 221

Lushan Yu, Shengjia Wang, and Su Zeng

14 Chiral Benzofurazan-Derived Derivatization Reagents for Indirect

Enantioseparations by HPLC 233

Toshimasa Toyo’oka

15 Separation of Racemic 1-(9-Anthryl)-2,2,2-trifluoroethanol

by Sub-/Supercritical Fluid Chromatography 249

Xiqin Yang, Leo Hsu, and Gerald Terfloth

16 Chiral Separations by Simulated Moving Bed Method Using

Polysaccharide-Based Chiral Stationary Phases 257

Toshiharu Minoda

17 Enantioseparations by Capillary Electrophoresis Using Cyclodextrins

as Chiral Selectors 271

Gerhard K.E Scriba and Pavel Jáč

18 Application of Dual Cyclodextrin Systems in Capillary Electrophoresis

Enantioseparations 289

Anne-Catherine Servais and Marianne Fillet

19 Enantioseparations in Nonaqueous Capillary Electrophoresis

Using Charged Cyclodextrins 297

Anne-Catherine Servais and Marianne Fillet

20 Use of Macrocyclic Antibiotics as the Chiral Selectors in Capillary

Electrophoresis 307

Chengke Li and Jingwu Kang

21 Application of Polymeric Surfactants in Chiral Micellar

Electrokinetic Chromatography (CMEKC) and CMEKC Coupled

to Mass Spectrometry 319

Jun He and Shahab A Shamsi

22 Cyclodextrin-modified Micellar Electrokinetic Chromatography for

Enantioseparations 349

Wan Aini Wan Ibrahim, Dadan Hermawan, and Mohd Marsin Sanagi

23 Cyclodextrin-Mediated Enantioseparation in Microemulsion

Electrokinetic Chromatography 363

Claudia Borst and Ulrike Holzgrabe

24 Chiral Separations by Capillary Electrophoresis Using Proteins

as Chiral Selectors 377

Jun Haginaka

25 Enantioseparation by Chiral Ligand-Exchange Capillary Electrophoresis 393

Yi Chen and Lijuan Song

26 Experimental Design Methodologies in the Optimization

of Chiral CE or CEC Separations: An Overview 409

Bieke Dejaegher, Debby Mangelings, and Yvan Vander Heyden

27 Chiral Capillary Electrophoresis–Mass Spectrometry 429

Elena Domínguez-Vega, Antonio L Crego, and Maria Luisa Marina

28 Application of Chiral Ligand-Exchange Stationary Phases in Capillary

Electrochromatography 443

Martin G Schmid

29 Polysaccharide-Derived Chiral Stationary Phases in Capillary

Electrochromatography Enantioseparations 457

Zhenbin Zhang, Hanfa Zou, and Junjie Ou

30 Open Tubular Molecular Imprinted Phases in Chiral Capillary

Electrochromatography 469

Won Jo Cheong and Song Hee Yang

31 Enantioseparations in Capillary Electrochromatography Using Sulfated

Poly β-Cyclodextrin-Modified Silica-Based Monolith as Stationary Phase 489

Ruijuan Yuan and Guosheng Ding

32 Cyclodextrin-Mediated Enantioseparations by Capillary

Electrochromatography 505

Dorothee Wistuba and Volker Schurig

Index 525

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

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 ,

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

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 TER FL OTH • 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

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

The differentiation of enantiomers is a fundamental natural nomenon as chiral bioactive compounds interact in a stereospeci fi c 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 fi elds Consequently, analytical techniques capable of differentiating stereoisomers, speci fi cally enantiomers, are required With regard to analytical enantioseparations, chromatography and electromigration tech-niques 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 fl uid chromatography (SFC) Capillary elec-tromigration techniques which utilize electrophoretic phenomena for the movement of the analytes toward the detector include capil-lary electrophoresis (CE), capillary electrokinetic chromatography (EKC), micellar electrokinetic chromatography (MEKC), microe-mulsion electrokinetic chromatography (MEEKC), and capillary

1 Introduction

electrochromatography (CEC) Enantioseparations can be divided into indirect and direct methods In the indirect approach, the ana-lyte 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 fi xed to an immobile support or as additive to the mobile phase or the background elec-trolyte The separation is based on the formation of transient diaste-reomeric complexes in a thermodynamic equilibrium

This introductory chapter of chiral separations will brie fl y 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 chro-matography and electromigration methods This is feasible because there is no fundamental difference between the stereospeci fi c inter-action 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 stereospeci fi c recognition is a chromatographic phenomenon inde-pendent 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 fi xed to a solid support compared to the situation in solution so that the chiral recognition of a selector may differ, whether it is fi xed to a stationary phase, or whether it is added to the liquid phase For further reading on chiral recogni-tion mechanisms in separation sciences, recent review papers ( 2– 4 ) and a monograph ( 5 ) are recommended

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 equilib-rium can be characterized by the equations:

Differences between the association constants, K R and K S , resent the physicochemical basis for the stereoselective retention of the enantiomers by a chiral selector

Early attempts to rationalize enantiospeci fi c 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 fi t forming three interactions with the selector, while the other enantiomer is bound less tightly due to the forma-tion 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 re fl ect the nature of the interac-tions, 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 con-sidering 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 recogni-tion 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 conse-quence, due to spatial requirements, one enantiomer of a selectand exhibits an “ideal fi t” with the chiral selector resulting in larger binding constant compared to the other enantiomer possessing a smaller binding constant due to its “nonideal fi t.”

Complex formation is driven by several interactions, e.g., ionic interactions, ion–dipole or dipole–dipole interactions, hydrogen-bonds, van der Waals interactions, and p – p interactions Ionic inter-actions are strong but may be primarily involved in the establishment

of the “ fi rst contact” due to their long-range nature However, as both enantiomers of an ionized solute are able to form these interac-

B

C

R A' C'

Fig 1 Scheme of the three-point interaction model

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., ste-reoselectivity ( 2 ) Furthermore, steric factors, i.e., fi t or non- fi t of the solute in a cavity or cleft of the selector, contribute to the chiral recognition A conformational change of the selector during com-plex formation with the solute (induced fi t) is also possible

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,

fl uorimetry, Fourier transform and attenuated total re fl ectance IR spectroscopy, NMR spectroscopy, as well as circular dichroism and vibrational circular dichroism (VCD) spectroscopy Especially NMR techniques including nuclear Overhauser effect (NOE) and rotating-frame Overhauser enhancement (ROE) have the advantage of allow-ing 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 select-and–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 applica-tions ( 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 prod-ucts with a wide variety of substitutions and different immobiliza-tion chemistry are available from Chiral Technologies under the trade names Chiralcel™ and Chiralpak™ or from Phenomenex as Lux

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 chi-ral 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 re fl ectance IR spectroscopy, and molecular modeling The glucose units are arranged along the helical axis with the substituents creating a helical groove The car-bamate groups are located inside, while the hydrophobic aromatic moieties are located outside the polymer chain In the case of amy-lose 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 measure-ments indicated a right-handed helix of the polymer as a fi lm 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(3,5-dimethylphenylcarbamate) ( 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 station-ary 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 tion 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 fl anked

by the aromatic substituents which may affect the access to the binding pocket via steric factors The carbamate linkages allow some fl exibility for an adjustment of the aromatic moieties for maximizing p – p interactions (induced fi t) This binding mode has been illustrated in several studies including techniques such

as NMR, attenuated total re fl ectance IR spectroscopy, and

molecular modeling ( 20, 29– 31 ) Figure 2 shows the minimized structures of the complexes between amylose tris(3,5-dimethylphenylcarbamate) and the enantiomers of norephedrine (2-amino-1-phenyl-1-propanol, PPA) ( 30 ) The stronger retained

energy-(1 R ,2 S )-con fi gured (−)-enantiomer displays three interactions,

two hydrogen bonds, i.e., (polymer)NH···OH(−PPA) and mer)C=O···H 2 N(−PPA), and one p – p interaction In the case of

(poly-the weaker bound (1 S ,2 R )-(+)-enantiomer, only two inter actions,

one hydrogen bond and one p – p interaction, are observed Interestingly, the situation is reversed for cellulose tris(3,5-dimethylphenylcarbamate) The stronger bound (+)-enantiomer established one hydrogen bond and two p – p interactions with the

Fig 2 Energy-minimized structures of complexes of amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) with

( a ) (1 S ,2 R )-(+)-norephedrine (+PPA) and ( b ) (1 R ,2 S )-(−)-norephedrine (−PPA) The dotted lines indicate hydrogen bonds,

p refers to p – p interactions (For the colored version of the fi gure, see the online version of the reference Reproduced by permission of Elsevier from ref 30 © 2008)

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 norephed-rine enantiomers for the two chiral stationary phases ( 30 )

It has also been shown that the selectand may change its formation upon binding to the selector leading to a tight fi t For example, in the protonated state, the stronger complexed ( S )-

con-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 modi fi ed solvation parameter model has been developed in order to rationalize the enantioselectivity of amylase tris(3,5-dime-thylphenylcarbamate) and cellulose tris(3,5-dimethylphenylcar-bamate) as chiral selectors in supercritical fl uid chromatography using a set of 135 structurally diverse solutes ( 33 ) Molecular prop-erties including p and n electrons, hydrogen-bonding acceptor and donor ability, molecular volume, fl exibility, and globularity as well as the respective interactions related to the solute descriptors were selected Factorial discriminant analysis was employed to iden-tify signi fi cant factors Steric fi t associated to hydrogen-bonding appeared to be the most important feature for enantiorecognition

by amylose tris(3,5-dimethylphenylcarbamate), while chiral nition on cellulose tris(3,5-dimethylphenylcarbamate) requires dipole–dipole and p – p interactions in addition to hydrogen- bonding The descriptors fl exibility and globularity were highly rel-evant 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

recog-to be attributed recog-to a combination of (stereo)selective interactions 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 cose molecules, b -CD of seven molecules, and g -CD of eight mol-ecules 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 g -

glu-CD ( 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

3.2 Cyclodextrins

separations, they have been used as chiral stationary phases in GC ( 36, 37 ) and HPLC ( 38– 40 ) Commercial columns for GC include DEX™ columns (Supelco), Lipodex™ columns (Macherey-Nagel),

or Chirasil-DEX™ columns (Agilent Technologies) In HPLC Cyclobond™ columns from Astec, ChiraDex™ columns from E Merck, or Ultron ES-CD™ columns from Shinwa have been applied Furthermore, native CDs and CD derivatives are by far the most frequently used chiral selectors in CE, MEKC, and MEEKC ( 41– 43 ) CDs can be obtained from numerous compa-nies including Sigma-Aldrich, Fluka, CDT Inc., PAC L.P., or CyDex Inc Probably the most complete selection of CDs including variations in the degree of substitution and isomeric purity is sup-plied by Cyclolab Beyond separation sciences, CDs are used for a wide variety of further applications in the pharmaceutical, cosmetic, food, textile, chemical, and agrochemical industries ( 44, 45 ) The complexation between CDs and guest molecules has been studied extensively due to the widespread technological applica-tions besides separation sciences A comprehensive overview of CD complexes can be found in a recent monograph ( 46 ) A web-based database for CD–ligand complexes has been established ( 47 ) Numerous techniques including thermodynamics, NMR spectros-copy, mass spectrometry, X-ray crystallography, molecular model-ing, as well as chromatographic and CE investigations have contributed to their characterization In most cases, 1:1 complexes are formed, but guest–host complexes with other ratios such as 2:1, 2:2, or higher-order equilibria also exist Complexation involves the insertion of lipophilic moieties of the guest molecules into the cavity of the CD displacing solvent molecules (typically water) from inside the cavity ( 35 ) Van der Waals and hydrophobic interactions are believed to be primarily involved, but hydrogen- bonding with the hydroxyl groups and steric effects also play a role For derivatized CDs, additional interactions such as ionic interactions in the case of CDs containing charged substituents or

p – p interactions in the case of CDs containing aromatic ents have to be considered as well Depending on the solute and the CD, inclusion can occur from the narrower or wider side of the

substitu-CE In fact, it has been shown that inclusion complexation is not a prerequisite for CD-mediated enantioseparations in CE ( 48, 49 ) Especially NMR studies have contributed to the understand-ing of the structures of the CD–guest molecule complexes as reviewed in ( 9, 50, 51 ) For example, opposite migration order in

CE was observed for the enantiomers of the drug ide when using b -CD or g -CD as chiral selectors in the background electrolyte Rotating-frame nuclear Overhauser effect spectroscopy

aminoglutethim-(ROESY) suggested the inclusion of the p -aminophenyl moiety of

the drug into the cavity of b -CD from the wider secondary side,

while the complex with g CD is formed by inclusion of the p

-aminophenyl ring from the narrower primary side ( 52 ) Another

example is the interaction between propranolol and b -CD or

heptakis(6- O -sulfo)- b -CD where inclusion of the naphthyl ring

occurs from the secondary side in the case of b -CD and from the narrow primary side in the case of heptakis(6- O -sulfo)- b -CD

( 53 ) Furthermore, different moieties of a molecule may interact with different CDs as shown for clenbuterol and b -CD as well as

heptakis(2,3-di- O -acetyl)- b -CD, respectively ( 54 ) The phenyl

ring of the drug enters b -CD, while the tert -butyl moiety of the

compound is included into the cavity of heptakis(2,3-di- O

-acetyl)- b -CD A recent study has shown that the structure of the complex can also depend on the background electrolyte used in

CE enantio separations ( 49 ) Thus, as shown in Fig 3 , the

com-plex between ( R )-propranolol and heptakis (2,3-di- O

O -sulfo)- b -CD is formed by inclusion of the side chain of the

drug into the cavity of the CD from the wider secondary side in methanolic solution In contrast, the naphthyl moiety enters hep-takis (2,3-di- O -methyl-6- O -sulfo)- b -CD from the narrow pri-

mary side in an aqueous background electrolyte The same group observed enantioselective nuclear Overhauser effects for the com-

plexes between heptakis (2,3-di- O -acetyl-6- O -sulfo)- b -CD and

the enantiomers of propranolol in nonaqueous electrolytes ( 55 )

( S )-propranolol forms a tighter complex with the CD as the side

chain of the molecule is inserted deeper into the CD cavity

com-pared to ( R )-propranolol Differences in the structures of the

diastereomeric complexes have also been obtained by molecular modeling calculations of the b -CD complexes with the enantiom-ers of naproxen ( 56 )

Fig 3 Structures of the complexes of ( R )-propranolol with ( a ) heptakis (2,3-di-O-acetyl-6-O-sulfo)- b -CD in nonaqueous

background electrolyte and ( b ) heptakis (2,3-di-O-methyl-6-O-sulfo)- b -CD in aqueous background electrolyte as derived

from ROESY NMR experiments The arrows indicate the observed intermolecular NOE upon irradiation of the respective

protons (Adapted with permission by John Wiley & Sons from ref 49 © 2010)

Furthermore, the use of buffer additives may also affect the structure of analyte–CD complexes Thus, it has been concluded from NMR measurements and molecular modeling that the enantiomers of the dipeptide Ala-Tyr in the protonated state are inserted deeper into the cavity of b -CD in the presence of urea compared to the absence of urea ( 57 ) This is also re fl ected in

b -CD-mediated CE enantioseparations where higher peak tion is observed when urea is added to the background electrolyte Another example is the complex between b -CD and naproxen when adding 1,2-dibromoethane ( 56 ) In the presence of 1,2-dibromoethane naproxen enters the CD cavity from the wider secondary side, while inclusion from the narrower primary side of

b -CD occurs in the absence of 1,2-dibromoethane

Macrocyclic glycopeptides are also called macrocyclic antibiotics due to their medical applications They were introduced in separa-tion sciences by Dan Armstrong and coworkers ( 58 ) The most prominent representatives of this group are vancomycin, ristocetin, teicoplanin, and the teicoplanin aglycone, but other compounds have also been evaluated as chiral selectors for enantioseparations ( 59, 60 ) The common structural feature of this class of com-pounds is a heptapeptide as a set of interconnected macrocycles each composed of two aromatic rings and a peptide sequence Vancomycin contains three macrocycles, while teicoplanin and ris-tocetin A are composed of four The macrocycles form a three-dimensional, C-shaped basket-like structure as shown for vancomycin in Fig 4 The carbohydrate moieties are positioned at the surface Ionizable groups such as a carboxylic acid group or amino groups are present Thus, a large number of interactions between analyte molecules and the glycopeptide antibiotics are possible including hydrogen bonds, p – p , dipole–dipole, and ionic interactions depending on the experimental conditions ( 61 ) The detailed recognition mechanism on a molecular basis has not been studied in detail yet Mechanisms deduced from structure–separation studies using various classes of analytes were not always conclusive and even contradictory in some instances ( 60, 61 ) For example, the amino function in the aglycone basket proved to be vital for enantioseparations of amino acids as demonstrated by the addition

of Cu 2+ ions, while it did not signi fi cantly affect the separation of other enantiomers The presence of the carbohydrate moieties had

a detrimental effect in amino acid enantiomer separations when compared to the enantioseparation by the aglycone In contrast, the sugar moieties were required for enantioseparations of other compounds such as b -blockers Detailed NMR studies or X-ray crystallographic studies with respect to chiral separations have not been published to date except for vancomycin and the trip-

eptide ligand N a , N w -diacetyl- L -Lys- D -Ala- D -Ala ( 62, 63 ) (Fig 4b ),

3.3 Macrocyclic

Glycopeptides

vancomycin and small ligands such as D -lactic acid, N -acetyl- D -Ala

or N -acetyl glycine ( 64 ) , as well as balhimycin and D -Ala- D -Ala ( 65 ) Molecular modeling studies have been carried out to ratio-nalize the enantioseparation of nonsteroidal anti-in fl ammatory drugs and N-derivatized amino acids by vancomycin in CE ( 66 )

O

O HO

OHOH

O O

CH3

NH2OH

CH3Cl

Cl

H N O

N H

a

b

H N O

N H O

H2NOC

H N

OH O

NH O COOH

Fig 4 ( a ) Structure of vancomycin and ( b ) X-ray crystal structure of the complex with N a , N w -diacetyl- L -Lys- D -Ala- D -Ala (in orange) The X-ray crystal structure image was generated with Accelrys Discovery Studio Visualizer 2.5 software from the coordinates from the Brookhaven Protein Data Bank ( www.rcbs.org/pdb , fi le 1FVM)

In HPLC macrocyclic glycopeptide selectors form the second most important group of chiral stationary phases after the polysac-charide derivatives Commercial columns are sold by Astec and Supelco under the trade names Chirobiotic V™ (vancomycin), Chirobiotic T™ (teicoplanin), Chirobiotic R™ (ristocetin), and Chirobiotic TAG™ (teicoplanin aglycone); vancomycin and teicopla-nin are also available with new binding chemistry as Chirobiotic V2™ and Chirobiotic T2™ Macrocyclic glycopeptides as chiral selectors in separation sciences have been summarized ( 59, 60, 67, 68 )

The stereoselective interactions of chiral compounds with proteins are a well-known phenomenon in nature Consequently, proteins have been used as chiral selectors in separation sciences as pio-neered by Stig Allenmark, Jun Haginaka, and others ( 69– 71 ) The stereoselective binding of drugs by human serum albumin (HSA) has been investigated The protein has two major binding sites termed site 1 (warfarin–azapropazone site) and site 2 (indole–ben-zodiazepine site) as well as several minor sites binding a variety of drugs and other compounds (Fig 5 ) ( 72, 73 ) Due to the com-plexity of the protein selectors, a number of molecular interactions including hydrogen bonds, p – p , dipole, and ionic interactions con-tribute to the complexation of analytes The crystal structure of HSA co-complexed with myristate and the enantiomers of warfarin revealed that both enantiomers were bound to site 1 in almost identical conformations, making many of the same interactions with the amino acid side chains which accounts for the relative lack

of enantioselectivity for the warfarin enantiomers ( 74 ) The

co-crystallization of ( S )-propranolol and the catalytic domain of

cel-lobiohydrolase revealed the importance of ionic interactions between the protonated amino group of propranolol and two glu-tamic acid residues for the complexation ( 75 ) Exchanging one of the glutamate residues by glutamine led to a loss of enantioselec-tivity in HPLC separations of propranolol indicating that both glu-tamate residues are essential for the chiral recognition of the drug Besides NMR and molecular modeling studies for the elucidation

of the recognition mechanism of turkey ovomucoid ( 76 ) , nant exchange of amino acids or derivatization of amino acids have been used to identify individual amino acids involved in the bind-ing of drugs to human and chicken a 1 -acid glycoprotein ( 77, 78 ) For a comprehensive summary of studies published on the chiral recognition of substances by proteins, see ( 79 )

Commercial chiral columns include Chiral AGP™ ( a 1 -acid coprotein, Chiral Technologies and Regis), Resolvosil BSA™ (bovine serum albumin, Macherey-Nagel), Chiral HSA™ (HSA, Chiral Technologies and Regis), Ultron ES-OVM™ (ovomucoid, Shinwa Chemical), or Chiral CBH™ (cellobiohydrolase I, Chiral Technologies and Regis) Recent reviews on the use of protein chi-ral stationary phases by HPLC can be found in ( 69– 71 )

3.4 Proteins

Pirkle-type selectors are also termed brush-type or donor–acceptor phases They are named after William H Pirkle, one of the pioneers

of their development The stationary phases are based on molecule chiral selectors capable of donor–acceptor interactions including hydrogen-bonding, p – p interactions (face-to-face or face-to-edge), and dipole–dipole stacking Rigid and bulky moieties as steric barriers may further amplify chiral recognition Commercial columns include Whelk-O1™ (Regis), ULMO™ (Regis), DACH-DNB™ (Regis), or Chirex™ (Phenomenex) The Whelk-O1 phase may be the most widely used Pirkle-type chiral stationary phase Its development has been reviewed ( 80 ) The selector combines

p -donor (tetrahydrophenanthrene) and p -acceptor phenyl) as well as hydrogen-bonding sites (amide) and is assumed

(3,5-dinitro-to possess a cleft-like binding site resulting from the perpendicular orientation of the phenyl and tetrahydrophenanthrene moieties The preferentially bound enantiomer interacts via face-to-face p – p interactions with the dinitrophenyl moiety and hydrogen bonds with the amide function Further face-to-edge p – p interactions

3.5 Pirkle-Type Chiral

Selectors

Fig 5 Overview of the binding of ligands by human serum albumin as de fi ned by crystallography showing the subdivision into domains I–III as well as the locations of the drug binding sites 1 and 2 Human serum albumin is shown as ribbon model, while the ligands are represented by space- fi lling models (For the colored version of the fi gure, see the online ver- sion of the reference Reproduced by permission of Elsevier from ref 72 © 2005)

enhance the af fi nity between selector and preferentially bound selectand ( 81 ) The binding mode of the pivaloylamide of ( R )-1- (4-bromophenyl)ethylamine by the ( S , S )- and ( R , R )-enantiomers

of the Whelk-O1 selector according to a X-ray crystallographic study ( 81 ) is shown in Fig 6 Both complexes are formed via a face-to-face p – p interaction between the dinitrobenzyl ring of the selec-tor and the bromophenyl ring of the selectand and a hydrogen bond between the benzamide NH and the carbonyl group of the selector In the case of the more stable homochiral complex, the selectand resides inside the cleft of the selector and exhibits addi-tional stabilization via a face-to-edge p – p interaction between the bromophenyl ring and the tetrahydrophenanthroline moiety of the selector This is not the case for the heterochiral complex due to a spatial offset The complex geometry was con fi rmed by NMR ( 81 ) and molecular modeling studies ( 82 ) Furthermore, the chiral rec-ognition of analytes by the Whelk-O1 selector in the presence of solvent has been analyzed by molecular dynamics simulations ( 83 ) Chiral ion-exchange stationary phases are often considered a sub-group of the brush-type (Pirkle-type) phases They interact with ionizable analytes via ionic interactions, but p – p interactions and hydrogen-bonding contribute to the stabilization of the complex Popular chiral anion-exchange phases for the separation of anionic racemates are based on cinchona alkaloids ( 84, 85 ) They have been commercialized under the trade names Chiralpak QN-AX™ and Chiralpak QD-AX™ (Chiral Technologies) and contain qui-nine and quinidine carbamates, respectively, as chiral selectors Furthermore, chiral cation exchangers for the separation of basic analytes exhibit sulfonic acid or carboxylic acid residues ( 86 ) Recently, zwitterionic ion exchangers combining cinchona alka-loids with sulfonic acid or carboxylic acid functions have been

3.6 Chiral Ion

Exchange

Fig 6 X-ray crystallographic structures of ( R )-1-(4-bromophenyl)ethylamine in ( a ) the homochiral complex with

( R , R )-enantiomer of the Whelk-O1 selector and ( b ) the heterochiral complex with the ( S , S )-enantiomer of the selector

(Reproduced by permission of Elsevier from ref 81 © 2005)

developed, expanding the use of this type of chiral selector to acidic, basic, as well as zwitterionic analytes ( 87, 88 ) The applica-tions of chiral ion-exchange phases have been summarized ( 89 ) The chiral recognition mechanism of cinchona alkaloid-based anion-exchange selectors has been studied extensively by various techniques including chromatography ( 85, 90 ) , NMR spectros-copy ( 90– 94 ) , X-ray crystallography ( 90, 91, 93– 95 ) , and molecu-lar dynamics simulations ( 91 ) NMR investigations of the quinine selector revealed that after protonation of the quinuclidine nitro-gen and when forming a complex with an acidic analyte, the con-formation of the selector transforms preferentially into the

“anti-open” conformation with the quinuclidine ring pointing away from the quinoline ring This results in a cleft allowing a negatively charged analyte to freely access the protonated quinucli-dine N for the primary ionic interaction p – p interactions between aromatic moieties of the solutes and the quinoline ring of the selec-tor as well as hydrogen bonds with the carbamate group may sta-bilize the complex as illustrated in Fig 7 Quinidine with the 8 R ,9 S

con fi guration and quinine with the 8 S ,9 R con fi guration form

pseudo-enantiomeric complexes with the enantiomers of N

-(3,5-dinitrobenzoyl)-leucine as shown by X-ray crystallography which explains the reversed enantiomer elution order of analytes observed

in chromatography for the two chiral selectors ( 90 ) Synthetic polymers used as chiral stationary phases are obtained by polymerization of chiral monomers ( 96– 98 ) or triphenylmethacry-late ( 99 ) The latter results in a helically chiral polymer Hydrogen bonding and p – p interactions along with steric factors contribute

to the chiral discrimination of analytes Although these chiral tors are not often used, a number of commercial products are avail-able including Chiralpak OT(+)™ (Daicel), ChiraSpher™ (E Merck), P-CAP™ and P-CAP-DP™ (Astec), as well as Kromasil CHI-DMB™ and Kromasil CHI-TBB™ (Eka)

Molecularly imprinted polymers (MIPs) are synthetic polymers produced by polymerization of functional monomers and cross-linkers in the presence of a non-covalently bound template Thus, such chiral selectors possess a predetermined selectivity for a given analyte or a group of structurally related compounds ( 100, 101 ) Chiral recognition is determined by the steric arrangement of the interacting groups Despite the fact that very high enantioselectivi-ties have been achieved using MIPs as stationary phases in HPLC and CEC, the selectors cannot compete with non-target-speci fi c chiral selectors at present MIPs typically suffer from poor chro-matographic ef fi ciency, peak tailing, and poor loading capacity

3.7 Synthetic

Polymers

3.8 Molecularly

Imprinted Polymers

Chiral crown ethers used in separation sciences as selectors rate chiral moieties such as binaphthyl or tartaric acid units in a poly-ether macrocycle They form complexes with protonated primary amines so that their use is essentially limited to this group of analytes although some exceptions have been reported The discrimination

incorpo-of amino acid enantiomers by boxylic acid ((+)-18C6H4, Fig 8a ) has been studied by NMR ( 102,

103 ) and X-ray crystallography ( 104– 106 ) Analyte complexation is due to the formation of hydrogen bonds between the protonated amine with oxygen atoms of the macrocycle For chiral recognition, (+)-18C6H4 has to adopt an asymmetric C1-type conformation exhibiting a bowl-like shape with the NH and C a H protons of the amino acid interacting with the oxygen atoms of the ring system as

NO2

NO2H-bond

ion-pairing

π - π interaction H-bond

steric interaction/

hydrophobic interaction

Fig 7 ( a ) Possible interactions between quinine-based chiral selectors and N -(3,5-dinitrobenzoyl)amino acids and X-ray

crystallographic structures of the complexes of ( b ) b -chloro- tert -butylcarbamoylquinine with N ( S )-leucine and ( c ) b -chloro- tert -butylcarbamoylquinidine with N -(3,5-dinitrobenzoyl)-( R )-leucine (For the colored version

-(3,5-dinitrobenzoyl)-of ( b ) and ( c ), see the online version -(3,5-dinitrobenzoyl)-of the reference Reproduced by permission -(3,5-dinitrobenzoyl)-of The American Chemical Society from

ref 90 © 2002)

well as the carboxylate groups The asymmetric C1-type shape is assumed to originate from a conformational sequence of successive rotations in the macrocycle ( 105 ) In complexes with D - and L -amino acids, speci fi c differences have been observed For example, in a com-bined NMR and molecular modeling study, it was shown that the stronger bound D -phenylglycine exhibits more favorable complex geometry and an additional hydrogen bond (Fig 8b ) compared to the weaker bound L -enantiomer (Fig 8c ) ( 102 ) Comparable results have been obtained from X-ray crystallographic studies showing dis-tinct differences in the complexes with D - and L -amino acids ( 104 ) Commercial HPLC columns based on (3,3 ¢ -diphenyl-1,1 ¢ -binaphthyl)-20-crown-6 (Crownpak CR(+)™ and Crownpak CR(−)™ from Daicel) and (18-crown-6)-2,3,11,12-tetracarboxylic acid (ChiroSil RCA(+)™ and ChiroSil RCA(−)™ from Regis) are available in both enantiomeric forms of the selector as indicated by the plus or minus sign Enantioseparations by HPLC ( 107, 108 ) and by CE ( 109, 110 ) have been summarized

The principle of chiral ligand-exchange was introduced by Danakov ( 111 ) It is based on the reversible chelate coordination of a chiral analyte into the sphere of a metal ion which is immobilized by complexation with a chelating selector resulting in a selectand–metal ion–selector complex The resulting diastereomeric chelates possess different thermodynamic stabilities or formation rates Amino acid derivatives are typically employed as chelating agents

3.10 Ligand-Exchange

Fig 8 Structure of ( a ) (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and complexes with ( b ) D -phenylglycine and ( c )

L -phenylglycine The complexes of the structures were generated from NOE data and molecular dynamics calculations

hydrogen bonds are displayed by dotted lines (For the color version of the fi gure, the reader is referred to the online version

of ref 102 ( b ) and ( c ) are reproduced by permission of The Royal Society of Chemistry from ref 102 © 2001)

along with divalent metal ions such as Cu 2+ , Zn 2+ , or Ni 2+ although

D -gluconic acid, D -saccharic acid, or L -threonic acid have also been used as complexation agents ( 112 ) The method is restricted to analytes bearing two or three electron-donating groups such as amino acids, hydroxy acids, or amino alcohols

In HPLC chiral ligand-exchange is performed either by tion of the metal ion and the ligand into the mobile phase or using

addicommercial columns based on immobilized ligands such as N , N

-dioctyl- L -alanine (Chiralpak MA(+)™ by Chiral Technologies) or

L -hydroxyproline (Nucleosil Chiral-1™ by Macherey-Nagel or Chiralpak WH™ by Chiral Technologies) The Cu(II)-complex of 4-hydroxyproline with amino acid enantiomers has been studied

by molecular modeling ( 113 ) Enantioseparations by HPLC, CE, and CEC have been summarized ( 112, 114, 115 )

Calixarenes are basket-shaped synthetic molecules composed of phenol units linked by methylene groups Chirality is introduced

by modi fi cations of the parent (achiral) calixarene by chiral cules such as amino acids, ephedrine, quinine alkaloids, or cyclo-dextrins Inclusion into the calixarene cavity as well as p – p or ionic interactions with the chiral side chains may be assumed to contrib-ute to the chiral recognition of analytes by the selectors Only few studies applied calixarenes for enantioseparations in GC ( 116 ) , HPLC ( 117– 120 ) , and CE ( 121, 122 )

Aptamers are single-stranded RNA or DNA oligonucleotides obtained in vitro by the iterative process of systematic evolution of ligands by exponential enrichment (SELEX) ( 123, 124 ) They possess a complex three-dimensional shape containing structural motifs such as stems, loops, bulges, hairpins, triplexes, or quadru-plexes and can bind a large variety of target compounds with an

af fi nity, speci fi city, and selectivity comparable to antibodies

Af fi nities for small molecules are characterized by nanomolar to micromolar dissociation constants although constants in the pico-molar range have also been reported Enantioselectivities higher than 10,000 have been reported ( 125 ) Typically, enantiospeci fi c aptamers are raised against single enantiomers, but it has also been shown that it is possible to obtain enantioselective aptamers using

a racemate as target ( 126 ) Binding of the target molecules occurs via an adaptive confor-mational change of the aptamer in a so-called induced- fi t process, resulting in a tight aptamer–target complex ( 127 ) The aptamer folds from a relatively disordered structure into a de fi ned binding pocket encapsulating the target molecule as illustrated in Fig 9 for the binding of L -argininamide by a DNA aptamer ( 128 ) Hydrogen- bonding, electrostatic interactions, stacking interactions, or hydro-phobic interactions contribute depending on the structure of the target From a kinetic standpoint, the binding mechanism has been

3.11 Calixarenes

3.12 Aptamers

conformational change of the aptamer from the inactive form to the active species The second step refers to the rate-limiting slower process of the binding between the partners including structural changes mediated by the induced- fi t process

Aptamers have been used as chiral selectors in recent years for enantioseparations by HPLC ( 130– 132 ) , CE ( 133, 134 ) , MEKC ( 135 ) , and CEC ( 136 ) Reviews can be found in ( 137, 138 ) Chiral micelles are formed from chiral surfactants above the critical micelle concentration in aqueous media or by so-called polymeric chiral micelles ( 139 ) They act as pseudostationary phases in elec-trokinetic chromatography A large number of chiral surfactants are available including bile acid derivatives or surfactants derived from amino acids or carbohydrates Polymeric micelles or molecu-lar micelles are obtained by suitably functionalized monomeric sur-factants derived from amino acids or dipeptides

The interaction between molecular micelles containing chiral dipeptide headgroups such as poly(sodium N -undecanoyl- L,L -

leucylleucinate) or poly(sodium N -undecanoyl- L,L -leucylvalinate) and the enantiomers of 1,1 ¢ -binaphthyl-2,2 ¢ -diyl hydrogen phos-phate (BNDHP) has been studied by nuclear Overhauser enhance-ment spectroscopy (NOESY) The chiral selector adopts a folded conformation creating a chiral pocket into which the BNDHP is inserted The N-terminal amino acid of the dipeptide headgroup was the primary site of chiral recognition ( 140, 141 ) The H3/H3 ¢ and H4/H4 ¢ protons of BNDHP interacted strongly with the amino acid moiety, while the H6/H6 ¢ and H7/H7 ¢ protons

primarily interacted with the hydrocarbon chain In contrast, dansyl amino acids appeared to interact primarily with the

C-terminal amino acid of poly( N -undecanoyl- L -leucine- L -valine) ( 142 ) Tröger’s base and propranolol seemed to bind to the chiral pocket of the molecular micelle as well ( 143 )

In 31 P-NMR experiments of the BNDHP enantiomers in the presence of sodium cholate above the critical micelle concentra-

tion, a larger up fi eld shift was observed for the S -enantiomer which correlates with the larger residence times of the S -enantiomer in

the micelles as determined by MEKC ( 144 ) Moreover, 1 H NMR indicated that the naphthyl moieties appear to be “inserted” into the hydrophobic micelle This also led to chiral discrimination as indicated by the relatively large differences of the chemical shifts of

the corresponding signals of the S - and R -enantiomers of BNDHP

Recent reviews on the application of chiral micelles in rations can be found in ( 139, 145– 147 )

Another class of recently developed chiral selectors are chiral ionic liquids These are ionic compounds that are liquid at or close to room temperature Either the cation or the anion or both may be chiral ( 148 ) Ionic and ion-pair interactions between analyte and selector predominate Besides applications in spectroscopic tech-niques, chiral ionic liquids have been used in separation sciences as stationary phases in GC ( 149, 150 ) and in HPLC as stationary phase ( 151 ) or as mobile phase additives in HPLC ( 152 ) Due to their high aqueous solubility, chiral ionic liquids have been used as chiral background electrolyte or chiral selector in CE ( 153– 155 ) including MEKC ( 156 ) and ligand-exchange ( 152 )

References

3.14 Chiral Ionic

Liquids

1 Chankvetadze B (1997) Separation

selectiv-ity in chiral capillary electrophoresis with

charged selectors J Chromatogr A

792:269–295

2 Lämmerhofer M (2010) Chiral recognition

by enantioselective liquid chromatography:

mechanisms and modern chiral stationary

phases J Chromatogr A 1217:814–856

3 Ali KK, Aboul-Enein HY (2006) Mechanistic

principles in chiral separations using liquid

chromatography and capillary

electrophore-sis Chromatographia 63:295–307

4 Berthod A (2006) Chiral recognition

mecha-nisms Anal Chem 78:2093–2099

5 Berthod A (2010) Chiral recognition in

sepa-ration methods Springer, Heidelberg

6 Easson LH, Stedman E (1933) Studies on the

relationship between chemical constitution

and physiological action V Molecular

dis-symmetry and physiological activity Biochem

7 Danakov VA (1997) The nature of chiral recognition: is it a three-point interaction? Chirality 9:99–102

8 Topiol S (1989) A general criterion for ular recognition: implications for chiral inter- actions Chirality 1:69–79

9 Dodziuk H, Kozinski W, Ejchart A (2004) NMR studies of chiral recognition by cyclo- dextrins Chirality 16:90–105

10 Uccello-Barretta G, Vanni L, Balzano F (2010) Nuclear magnetic resonance approaches for the rationalization of chro- matographic enantiorecognition processes

J Chromatogr A 1217:928–940

11 Del Rio A (2009) Exploring enantioselective molecular recognition mechanisms with chemoinformatic techniques J Sep Sci 32:1566–1584

12 Lipkowitz KB (2001) Atomistic modeling

of enantioselection in chromatography

13 Hesse G, Hagel R (1973) Eine vollständige

Racemattrennung durch

Elutions-graphie an Cellulose-tri-acetat

Chromato-graphia 6:277–280

14 Okamoto Y, Kawashima M, Hatada K (1986)

Chromatographic resolution XI Controlled

chiral recognition of cellulose

triphenylcar-bamate derivatives supported on silica gel

J Chromatogr 363:173–186

15 Okamoto Y, Aburatani R, Fukumoto T,

Hatada K (1987) Useful chiral stationary

phases for HPLC Amylose

tris(3,5-dimeth-ylphenylcarbamate) and

tris(3,5-dichlorophe-nylcarbamate) supported on silica gel Chem

Lett 16:1857–1860

16 Chen X, Yamamoto C, Okamoto Y (2007)

Polysaccharide derivatives as useful chiral

sta-tionary phases in high-performance liquid

chro-matography Pure Appl Chem 79:1561–1573

17 Okamoto Y, Ikai T (2008) Chiral HPLC for

ef fi cient resolution of enantiomers Chem Soc

Rev 37:2593–2608

18 Ikai T, Yamamoto C, Kamigaito M, Okamoto

Y (2008) Immobilized-type chiral packing

materials for HPLC-based on polysaccharide

derivatives J Chromatogr B 875:2–11

19 Ika T, Okamoto Y (2009) Structure control

of polysaccharide derivatives for ef fi cient

sepa-ration of enantiomers by chromatography

Chem Rev 109:6077–6101

20 Yamamoto C, Yashima E, Okamoto Y (2002)

Structural analysis of amylose

tris(3,5-dime-thylphenylcarbamate) by NMR relevant to its

chiral recognition mechanism in HPLC J Am

Chem Soc 124:12583–12589

21 Ma S, Shen S, Lee H, Yee N, Senanayake C,

Na fi e LA, Grinberg N (2008) Vibrational

cir-cular dichroism of amylose carbamate: structure

and solvent-induced conformational changes

Tetrahedron Asymmetry 19:2111–2114

22 Ma S, Shen S, Lee H, Eriksson M, Zeng X,

Xu J, Fandrick K, Yee N, Senanayake C,

Grinberg N (2009) Mechanistic studies on

the chiral recognition of polysaccharide-based

chiral stationary phases using liquid

chroma-tography and vibrational circular dichroism

Reversal of elution order of N-substituted

alpha-methyl phenylalanine esters

J Chromatogr A 1216:3784–3793

23 Yamamoto C, Yashima E, Okamoto Y (1999)

Computational studies on chiral

discrimina-tion mechanism of phenylcarbamate

deriva-tives of cellulose Bull Chem Soc Jpn

72:1815–1825

24 Kasat RB, Wang NHL, Franses EI (2007)

Effects of backbone and side chain on the

molecular environments of chiral cavities in

polysaccharide-based biopolymers cromolecules 8:1676–1685

25 Wenslow RM, Wang T (2001) Solid-state NMR characterization of amylose tris(3,5- dimethylphenylcarbamate) chiral stationary- phase structure as a function of mobile-phase composition Anal Chem 73:4190–4195

26 Kasat RB, Zvinevich Y, Hillhouse HW, Thomson KT, Wang N-HL, Franses EI (2006) Direct probing of sorbent-solute interactions for amylose tris(3,5-dimethylphenylcarbam- ate) using infrared spectroscopy, x-ray diffrac- tion, solid-state NMR, and DFT modeling

J Phys Chem B 110:14114–14122

27 Wang T, Wenslow RM (2003) Effects of hol mobile-phase modi fi ers on the structure and chiral selectivity of amylose tris(3,5-dim- ethylphenylcarbamate) chiral stationary phase

alco-J Chromatogr A 1015:99–110

28 Tamura K, Sam NSM, Ikai T, Okamoto Y, Yashima E (2011) Synthesis and chiral recog- nition ability of poly(phenylenevinylene)- encapsulated amylose derivative Bull Chem Soc Jpn 84:741–747

29 Kasat RB, Wee SY, Loh JX, Wang N-HL, Franses EI (2008) Effect of the solute molec- ular structure on its enantioresolution on cellulose tris(3,5-dimethylphenylcarbamate)

J Chromatogr B 875:81–92

30 Kasat RB, Wang N-HL, Franses EI (2008) Experimental probing and modeling of key sorbent-solute interactions of norephedrine enantiomers with polysaccharide-based chi- ral stationary phases J Chromatogr A 1190: 110–119

31 Kasat RB, Fransies EI, Wang N-HL (2010) Experimental and computational studies of enantioseparation of structurally similar com- pounds on amylose tris(3,5-dimethylphenyl- carbamate) Chirality 22:565–579

32 Ye YK, Bai S, Vyas S, Wirth MJ (2007) NMR and computational studies of chiral discrimi- nation by amylose tris(3,5-dimethylphenyl- carbamate) J Phys Chem B 111:1189–1198

33 West C, Guenegou G, Zhang Y, Morin-Allory

L (2011) Insights into chiral recognition mechanisms in supercritical fl uid chromatog- raphy II Factors contributing to enantiomer separation on tris-(3,4-dimethylphenylcar- bamate) of amylose and cellulose stationary phases J Chromatogr A 1218:2033–2057

34 Biwer A, Antranikian G, Heinzle E (2002) Enzymatic production of cyclodextrins Appl Microbiol Biotechnol 59:609–617

35 Rekharsky MV, Inoue Y (1998) Complexation thermodynamics of cyclodextrins Chem Rev 98:1875–1917

36 Schurig V (2002) Chiral separations using gas

chromatography Trends Analyt Chem 21:

647–661

37 Schurig V (2010) Use of derivatized

cyclo-dextrins as chiral selectors for the separation

of enantiomers by gas chromatography Ann

Pharm Fr 68:82–98

38 Han SM (1997) Direct enantiomeric

separa-tions by high performance liquid

chromatog-raphy using cyclodextrins Biomed

Chromatogr 11:259–271

39 Mitchell CR, Armstrong DW (2004)

Cyclodextrin-based chiral stationary phases

for liquid chromatography: a twenty-year

overview In: Gübitz G, Schmid MG (eds)

Chiral separations methods and protocols, vol

243, Methods in molecular biology Springer,

New York, pp 61–112

40 Muderawan IW, Ong TT, Ng SC (2006) Urea

bonded cyclodextrin derivatives onto silica for

chiral HPLC J Sep Sci 29:1849–1871

41 Fanali S (2000) Enantioselective

determina-tion by capillary electrophoresis with

cyclo-dextrins as chiral selectors J Chromatogr A

875:89–122

42 Schmitt U, Branch SK, Holzgrabe U (2002)

Chiral separations by cyclodextrin-modi fi ed

capillary electrophoresis—determination of the

enantiomeric excess J Sep Sci 25:959–974

43 Scriba GKE (2008) Cyclodextrins in capillary

electrophoresis enantioseparations—recent

developments and applications J Sep Sci

31:1991–2011

44 Del Valle EMM (2004) Cyclodextrins and

their uses: a review Process Biochem 39:

1033–1046

45 Buschmann H-J, Knittel D, Schollmeyer E

(2001) New textile applications of

cyclodex-trins J Incl Phenom Macrocycl Chem

40:169–172

46 Dodziuk H (ed) (2006) Cyclodextrins and

their complexes Wiley-VCH, Weinheim

47 Hazai E, Hazai I, Demko L, Kovacs S, Malik

D, Akli P, Hari P, Szeman J, Fenyvesi E, Benes

E, Szente L, Bikadi Z (2010) Cyclodextrin

knowledgebase a web-based service managing

CD-ligand complexation data J Comput

Aided Mol Des 24:713–717

48 Chankvetadze B, Burjanadze N, Maynard

DM, Bergander K, Bergenthal D, Blaschke G

(2002) Comparative enantioseparations with

native b -cyclodextrin and

heptakis-(2-O-methyl-3,6-di-O-sulfo)- b -cyclodextrin in

cap-illary electrophoresis Electrophoresis

23:3027–3034

49 Servais A-C, Rousseau A, Fillet M, Lomsadze

K, Salgado A, Crommen J, Chankvetadze B

(2010) Separation of propranolol enantiomers

by CE using sulfated b -CD derivatives in aqueous and non-aqueous electrolytes: com- parative CE and NMR study Electrophoresis 31:1467–1474

50 Schneider H-J, Hacket F, Rüdiger V, Ikeda H (1998) NMR studies of cyclodextrins and cyclodextrin complexes Chem Rev 98: 1755–1785

51 Chankvetadze B (2004) Combined approach using capillary electrophoresis and NMR spectroscopy for an understanding of enanti- oselective recognition mechanisms by cyclo- dextrins Chem Soc Rev 33:337–347

52 Chankvetadze B, Fillet M, Burjanadze N, Bergenthal D, Bergander K, Luftmann H, Crommen J, Blaschke G (2000) Enantioseparation of aminoglutethimide with cyclodextrins in capillary electrophoresis and studies of selector-selectand interactions using NMR spectroscopy and electrospray ionization mass spectrometry Enantiomer 5:313–322

53 Servais A-C, Rousseau A, Fillet M, Lomsadze

K, Salgado A, Crommen J, Chankvetadze B (2010) Capillary electrophoretic and nuclear magnetic resonance studies on the opposite

af fi nity pattern of propranolol enantiomers towards various cyclodextrins J Sep Sci 33: 1617–1624

54 Chankvetadze B, Lomsadze K, Burjanadze N, Breitkreutz J, Pintore G, Chessa M, Bergander

K, Blaschke G (2003) Comparative separations with native b -cyclodextrin, ran- domly acetylated b -cyclodextrin and heptakis-(2,3-di-O-acetyl)- b -cyclodextrin in capillary electrophoresis Electrophoresis 24:1083–1091

55 Lomsadze K, Salgado A, Calvo E, Lopez JA, Chankvetadze B (2011) Comparative NMR and MS studies on the mechanism of enanti- oseparation of propranolol with heptakis(2,3- diacetyl-6-sulfo)- b -cyclodextrin in capillary electrophoresis with aqueous and non-aqueous electrolytes Electrophoresis 32:1156–1163

56 Wei Y, Wang S, Chao J, Wang S, Cong C, Shuang S, Paau MC, Choi MMF (2011) An evidence for the chiral discrimination of naproxen enantiomers: a combined experi- mental and theoretical study J Phys Chem C 115:4033–4040

57 Waibel B, Schreiber J, Meier C, Hammitzsch

M, Baumann K, Scriba GKE, Holzgrabe U (2007) Comparison of cyclodextrin-dipeptide inclusion complexes in the absence and pres- ence of urea by means of capillary electropho- resis, nuclear magnetic resonance and molecular modeling European J Org Chem 2007: 2921–2930