Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition Printed Edition of the Special Issue Published in Molecules www.mdpi.com/journal/molecules Maria Elizabeth Tiritan, Madalena Pinto and Carla Sofia Garcia Fernandes Edited by Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition Special Issue Editors Maria Elizabeth Tiritan Madalena Pinto Carla Sofia Garcia Fernandes MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Special Issue Editors Maria Elizabeth Tiritan Cooperativa de Ensino Superior Polit ́ ecnico e Universit ́ ario (CESPU) Universidade do Porto Portugal Madalena Pinto Universidade do Porto Portugal Carla Sofia Garcia Fernandes Universidade do Porto Portugal Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Molecules (ISSN 1420-3049) (available at: https://www.mdpi.com/journal/molecules/special issues/Chiral separation recognition). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03936-238-7 ( H bk) ISBN 978-3-03936-239-4 (PDF) Cover image courtesy of Maria Elizabeth Tiritan. c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Preface to ”Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition” . . ix Maria Elizabeth Tiritan, Madalena Pinto and Carla Fernandes Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition Reprinted from: Molecules 2020 , 25 , 1713, doi:10.3390/molecules25071713 . . . . . . . . . . . . . . 1 Diana Ibrahim and Ashraf Ghanem On the Enantioselective HPLC Separation Ability of Sub-2 μ m Columns: Chiralpak R © IG-U and ID-U Reprinted from: Molecules 2019 , 24 , 1287, doi:10.3390/molecules24071287 . . . . . . . . . . . . . . 7 Ali Fouad, Montaser Sh. A. Shaykoon, Samy M. Ibrahim, Sobhy M. El-Adl and Ashraf Ghanem Colistin Sulfate Chiral Stationary Phase for the Enantioselective Separation of Pharmaceuticals Using Organic Polymer Monolithic Capillary Chromatography Reprinted from: Molecules 2019 , 24 , 833, doi:10.3390/molecules24050833 . . . . . . . . . . . . . . 19 Anamarija Kneˇ zevi ́ c, Jurica Novak and Vladimir Vinkovi ́ c New Brush-Type Chiral Stationary Phases for Enantioseparation of Pharmaceutical Drugs Reprinted from: Molecules 2019 , 24 , 823, doi:10.3390/molecules24040823 . . . . . . . . . . . . . . 35 Lie-Ding Shiau Chiral Separation of the Phenylglycinol Enantiomers by Stripping Crystallization Reprinted from: Molecules 2018 , 23 , 2901, doi:10.3390/molecules23112901 . . . . . . . . . . . . . . 51 Andreea Elena Bodoki, Bogdan-Cezar Iacob, Laura Elena Gliga, Simona Luminita Oprean, David A. Spivak, Nicholas A. Gariano and Ede Bodoki Improved Enantioselectivity for Atenolol Employing Pivot Based Molecular Imprinting Reprinted from: Molecules 2018 , 23 , 1875, doi:10.3390/molecules23081875 . . . . . . . . . . . . . . 63 Joana Teixeira, Maria Elizabeth Tiritan, Madalena M. M. Pinto and Carla Fernandes Chiral Stationary Phases for Liquid Chromatography: Recent Developments Reprinted from: Molecules 2019 , 24 , 865, doi:10.3390/molecules24050865 . . . . . . . . . . . . . . 83 Yangfeng Peng, Cai Feng, Sohrab Rohani and Quan He Improved Resolution of 4-Chloromandelic Acid and the Effect of Chlorine Interactions Using ( R )-(+)-Benzyl-1-Phenylethylamine as a Resolving Agent Reprinted from: Molecules 2018 , 23 , 3354, doi:10.3390/molecules23123354 . . . . . . . . . . . . . . 121 Fei Xiong, Bei-Bei Yang, Jie Zhang and Li Li Enantioseparation, Stereochemical Assignment and Chiral Recognition Mechanism of Sulfoxide-Containing Drugs Reprinted from: Molecules 2018 , 23 , 2680, doi:10.3390/molecules23102680 . . . . . . . . . . . . . . 133 Yuichi Uwai Enantioselective Drug Recognition by Drug Transporters Reprinted from: Molecules 2018 , 23 , 3062, doi:10.3390/molecules23123062 . . . . . . . . . . . . . . 145 v Ankur Gogoi, Nirmal Mazumder, Surajit Konwer, Harsh Ranawat, Nai-Tzu Chen and Guan-Yu Zhuo Enantiomeric Recognition and Separation by Chiral Nanoparticles Reprinted from: Molecules 2019 , 24 , 1007, doi:10.3390/molecules24061007 . . . . . . . . . . . . . . 159 Giovanna Brancatelli, Enrico Dalcanale, Roberta Pinalli and Silvano Geremia Probing the Structural Determinants of Amino Acid Recognition: X-Ray Studies of Crystalline Ditopic Host-Guest Complexes of the Positively Charged Amino Acids, Arg, Lys, and His with a Cavitand Molecule Reprinted from: Molecules 2018 , 23 , 3368, doi:10.3390/molecules23123368 . . . . . . . . . . . . . . 191 Allan Ribeiro da Silva, Deborah Araujo dos Santos, Marcio Weber Paix ̃ ao and Arlene Gon ̧ calves Corrˆ ea Stereoselective Multicomponent Reactions in the Synthesis or Transformations of Epoxides and Aziridines Reprinted from: Molecules 2019 , 24 , 630, doi:10.3390/molecules24030630 . . . . . . . . . . . . . . 203 Farid Chebrouk, Khodir Madani, Brahim Cherfaoui, Leila Boukenna, M ́ onica V ́ alega, Ricardo F. Mendes, Filipe A. A. Paz, Khaldoun Bachari, Oualid Talhi and Artur M. S. Silva Hemi-Synthesis of Chiral Imine, Benzimidazole and Benzodiazepines from Essential Oil of Ammodaucus leucotrichus subsp. leucotrichus Reprinted from: Molecules 2019 , 24 , 975, doi:10.3390/molecules24050975 . . . . . . . . . . . . . . 227 Solida Long, Diana I. S. P. Resende, Anake Kijjoa, Artur M. S. Silva, Ricardo Fernandes, Cristina P. R. Xavier, M. Helena Vasconcelos, Em ́ ılia Sousa and Madalena M. M. Pinto Synthesis of New Proteomimetic Quinazolinone Alkaloids and Evaluation of Their Neuroprotective and Antitumor Effects Reprinted from: Molecules 2019 , 24 , 534, doi:10.3390/molecules24030534 . . . . . . . . . . . . . . 237 Jinsong Xuan and Yingang Feng Enantiomeric Tartaric Acid Production Using cis -Epoxysuccinate Hydrolase: History and Perspectives Reprinted from: Molecules 2019 , 24 , 903, doi:10.3390/molecules24050903 . . . . . . . . . . . . . . 255 Carla Fernandes, Maria Let ́ ıcia Carraro, Jo ̃ ao Ribeiro, Joana Ara ́ ujo, Maria Elizabeth Tiritan and Madalena M. M. Pinto Synthetic Chiral Derivatives of Xanthones: Biological Activities and Enantioselectivity Studies Reprinted from: Molecules 2019 , 24 , 791, doi:10.3390/molecules24040791 . . . . . . . . . . . . . . 267 Anna Poryvai, Terezia Vojtylov ́ a-Jurkoviˇ cov ́ a, Michal ˇ Smahel, Natalie Kolderov ́ a, Petra Tom ́ aˇ skov ́ a, David S ́ ykora and Michal Kohout Determination of Optical Purity of Lactic Acid-Based Chiral Liquid Crystals and Corresponding Building Blocks by Chiral High-Performance Liquid Chromatography and Supercritical Fluid Chromatography Reprinted from: Molecules 2019 , 24 , 1099, doi:10.3390/molecules24061099 . . . . . . . . . . . . . . 303 Emilija Petronijevic and Concita Sibilia Enhanced Near-Field Chirality in Periodic Arrays of Si Nanowires for Chiral Sensing Reprinted from: Molecules 2019 , 24 , 853, doi:10.3390/molecules24050853 . . . . . . . . . . . . . . 315 Bang-Jin Wang, Ai-Hong Duan, Jun-Hui Zhang, Sheng-Ming Xie, Qiu-E Cao and Li-Ming Yuan An Enantioselective Potentiometric Sensor for 2-Amino-1-Butanol Based on Chiral Porous Organic Cage CC3-R Reprinted from: Molecules 2019 , 24 , 420, doi:10.3390/molecules24030420 . . . . . . . . . . . . . . 325 vi About the Special Issue Editors Maria Elizabeth Tiritan has a degree in chemistry and PhD in organic chemistry. Currently, she is Assistant Professor at the Faculty of Pharmacy of University of Porto and Team Leader of the Drug Research line at IINFACTS. Her current interests in research include exploiting new enantioselective analytical methods to quantify and identify metabolites in the biodegradation of chiral drugs in environmental matrices; synthesis of new chiral compounds for diverse biological activities, including potential antitumor and antimicrobial agents; as well as structure–activity–properties relationships. She is also involved in the design of new chiral selectors for enantiomeric separation by liquid chromatography and membranes. https://orcid.org/0000-0003-3320-730X; https://sigarra.up.pt/ffup/pt/func geral.formview?p codigo=481977; https://iinfacts.cespu.pt/. Madalena Pinto is a retired Full Professor from the Faculty of Pharmacy (FFUP) and Researcher and Team Leader of the Group of Natural Products and Medicinal Chemistry at the Interdisciplinary Center for Marine and Environmental Research (CIIMAR) University of Porto, Portugal. Her research areas focus on medicinal chemistry, involving the synthesis and molecular modification of pharmacologically active compounds based on natural models of plant and marine sources, as well as the development of substances with antifouling activity. She has a special interest in chirality, namely in synthesis and bioenantioselectivity studies of chiral bioactive compounds, as well as studies of structure–activity–properties relationships; molecular recognition of artificial receptors as new chiral stationary phases for liquid chromatography is another area of interest. ORCID: http://orcid.org/0000-0002-4676-1409; CIIMAR: https://www2.ciimar.up.pt/team.php?id=226; https://www2.ciimar.up.pt/research.php?team=27; Linkedin: linkedin.com/in/madalena-pinto-14334836; Webpage: http://madalenapinto.com/. Carla Sofia Garcia Fernandes is a researcher at the Research Center CIIMAR and Assistant Professor of Organic and Medicinal Chemistry in the Faculty of Pharmacy of the University of Porto, Portugal. She obtained her B.S. in pharmaceutical sciences at the University of Porto in 2002; M.S. in quality control - scientific area in drug substances and medicinal plants, from the same university in 2006; and her PhD in pharmaceutical and medicinal chemistry from the same university in 2012. Current research interests include design of chiral compounds with potential biological activity, development of synthetic methodologies for obtaining bioactive chiral compounds, studies of enantioselectivity and structure–activity relationships, synthesis of chiral stationary phases, analytical and preparative enantiomeric separation of diverse racemic mixtures by liquid chromatography and membranes, and chiral recognition mechanisms. ORCID: 0000-0003-0940-9163 URL https://sigarra.up.pt/ffup/pt/func geral.formview?p codigo=424646. vii Preface to ”Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition” The importance of producing chiral compounds in enantiomerically pure form is well recognized by academics and industries. Currently, the demand for efficient methodologies to produce chiral compounds with a high degree of enantiomeric purity requires continuous advances in enantioselective synthesis, chiral analyses, preparative enantioseparation, as well as chiral recognition studies. This book includes both fundamental studies and applications in a multidisciplinary research field that considered commercial chiral compounds with industrial applications, bioactive compounds and pharmaceuticals, and new compounds with promising biological activities. Nineteen papers published in the Special Issue entitled ”Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition” in Molecules are gathered in this edition. The recent developments and innovative approaches in enantiomeric separation, both on the analytical and preparative scale, and in enantioselective synthesis are presented. Many different aspects of chiral recognition, including chiral sensors, recognition in biological systems, and in analytical methods are described.. The editors acknowledge all authors that contributed the papers included in this book. Ai-Hong Duan; Ali Fouad; Allan Ribeiro da Silva; Anake Kijjoa; Anamarija Kneˇ zevi ́ c; Andreea Elena Bodoki; Ankur Gogoi; Anna Poryvai; Arlene Gon ̧ calves Corrˆ ea; Artur M. S. Silva; Artur M. S. Silva; Ashraf Ghanem; Ashraf Ghanem; Bang-Jin Wang; Bei-Bei Yang; Bogdan-Cezar Iacob; Brahim Cherfaoui; Cai Feng; Carla Fernandes; Concita Sibilia; Cristina P. R. Xavier; David A. Spivak; David S ́ ykora; Deborah Araujo dos Santos; Diana I. S. P. Resende; Diana Ibrahim; Ede Bodoki; Em ́ ılia Sousa; Emilija Petronijevic; Enrico Dalcanale; Farid Chebrouk; Fei Xiong; Filipe A. A. Paz; Giovanna Brancatelli; Guan-Yu Zhuo; Harsh Ranawat; Jie Zhang; Jinsong Xuan; Joana Ara ́ ujo; Joana Teixeira; Jo ̃ ao Ribeiro; Jun-Hui Zhang; Jurica Novak; Khaldoun Bachari; Khodir Madani; Laura Elena Gliga; Leila Boukenna; Li Li; Lie-Ding Shiau; Li-Ming Yuan M. Helena Vasconcelos; Madalena M. M. Pinto; Marcio Weber Paix ̃ ao; Maria Elizabeth Tiritan; Maria Let ́ ıcia Carraro; Michal Kohout; Michal ˇ Smahel; M ́ onica V ́ alega; Montaser Sh. A. Shaykoon; Nai-Tzu Chen; Natalie Kolderov; Nicholas A. Gariano; Nirmal Mazumder; Oualid Talhi; Petra Tom ́ aˇ skov; Qiu-E Cao; Quan He; Ricardo F. Mendes; Ricardo Fernandes; Roberta Pinalli; Samy M. Ibrahim; Sheng-Ming Xie; Silvano Geremia; Simona Luminita Oprean; Sobhy M. El-Adl; Sohrab Rohani; Solida Long; Surajit Konwer; Terezia Vojtylov ́ a-Jurkov ̆ ıcov; Vladimir Vinkovi ́ c; Yangfeng Peng; Yingang Feng; Yuichi Uwai. Maria Elizabeth Tiritan, Madalena Pinto, Carla Sofia Garcia Fernandes Special Issue Editors ix molecules Editorial Enantioselective Synthesis, Enantiomeric Separations and Chiral Recognition Maria Elizabeth Tiritan 1,2,3, * , Madalena Pinto 2,3 and Carla Fernandes 2,3 1 CESPU, Instituto de Investigaç ã o e Formaç ã o Avançada em Ci ê ncias e Tecnologias da Sa ú de (IINFACTS), Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal 2 Laborat ó rio de Qu í mica Org â nica e Farmac ê utica, Departamento de Ci ê ncias Qu í micas, Faculdade de Farm á cia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; madalena@ ff .up.pt (M.P.); cfernandes@ ff .up.pt (C.F.) 3 Centro Interdisciplinar de Investigaç ã o Marinha e Ambiental (CIIMAR), Edif í cio do Terminal de Cruzeiros do Porto de Leix õ es, Av. General Norton de Matos s / n, 4050-208 Matosinhos, Portugal * Correspondence: elizabeth.tiritan@iucs.cespu.pt or beth@ ff .up.pt; Tel.: + 351-913499293 Received: 1 April 2020; Accepted: 2 April 2020; Published: 8 April 2020 Chirality is a geometric property associated with the asymmetry of tridimensional features that accompanies our daily life at macroscopic as well as microscopic molecular levels. Chirality is a hallmark of many natural small molecules, and it is intrinsically associated with chiral building blocks as D-sugars and L-amino acids, intervening in chemical procedures of living cells, for example, as enzymes and receptors constituent proteins. Interestingly, free D-amino acids, which are naturally occurring, are important biomarkers with diagnostic value that demonstrate the importance of chiral analyses [ 1 ]. Nevertheless, the importance of chirality is recognized across many related areas as witnessed in wide-ranging fields such as chemistry, physics, biochemistry, material science, pharmacology, and many others (Figure 1). Figure 1. Results analysis for Scopus query “chiral” in titles, keywords, or the abstract section of articles between 2009 and 2019. Though chirality has a major position in chemistry, compared with other fields, due to the importance of chiral compounds in their pure enantiomeric form, there is a need for the development of analytic methods capable of controlling the enantiomeric ratio, and to understand the behavior of chiral compounds in biological systems and in other matrices in which chirality is also present. Currently, Molecules 2020 , 25 , 1713; doi:10.3390 / molecules25071713 www.mdpi.com / journal / molecules 1 Molecules 2020 , 25 , 1713 there is a very high demand for e ffi cient methodologies to obtain chiral bioactive compounds with a high degree of enantiomeric purity, which boosts the continuous advances in enantioselective synthesis, chiral analyses, preparative enantioseparation, as well as in chiral recognition studies. The number of publications with chirality as a subject has increased in the last decade and disclosed considerable growth in the last year, demonstrating the importance of the research in this field (Figure 2). Figure 2. Results analysis for Scopus query “chiral” in titles, keywords, or abstract sections of articles between 2009 and 2019 (Limited to Chemistry). These demands are related to drug discovery and development, safety in medication, food and environmental quality, materials for fine chemical industry such as chiral building blocks, among others. To meet these needs, it is essential that the international scientific community must work intensively to ensure e ff ective production and quality of analyses of chiral compounds for a diversity of applications. For this reason, Molecules recognized the need to propose the Special Issue “Enantioselective Synthesis, Enantiomeric Separations, and Chiral Recognition”. This Special Issue is aimed at o ff ering an opportunity to all the contributors to make their results and techniques more visible, and to present the most recent findings. This Special Issue has received remarkably positive feedback, with many contributions submitted by numerous geographically diverse scientists, resulting in a collection of 19 publications, including six exhaustive review articles [ 2 – 7 ], and thirteen original articles [ 8 – 20 ]. Among the contributing authors, we can find countries of origin such as Algeria, Australia, Brazil, Canada, China, Croatia, Czech Republic, Egypt, India, Italy, Japan, Portugal, Romania, Russia, and Taiwan. The published articles include findings related to the analytical chiral stationary phases (CSPs) for liquid chromatography (LC), currently the better choice for chiral quality control and determination of enantiomeric ratios. Faster, more e ffi cient, and sensitive methods are urgently needed for chiral analysis, and can be achieved within small particle sizes (sub-2 μ m) of the chromatographic support. The ability of the recently commercialized sub-2 μ m CSP with di ff erent substituents for the fast enantioseparation of a set of drugs was demonstrated in an original article [ 8 ]. New selectors for CSPs are always required to show the response in the continuous progress of chiral analyses, and there is a need for better and low cost CSPs. In this context a new brush-Type CSP for LC was reported for enantioseparation of several drugs including nonsteroidal anti-inflammatory drugs and 3-hydroxybenzodiazepine [ 10 ]; and a new colistin sulfate CSP for nano-LC reported enantioseparation for α - and β -blockers, anti-inflammatory, antifungal, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative-hypnotic, antihistaminic, anticancer, and antiarrhythmic drugs [ 9 ]. Additionally, an exhaustive review concerning recent 2 Molecules 2020 , 25 , 1713 developments in CSPs for LC includes many di ff erent types of selectors, showing that it continues to be a field of research with great importance [2]. Methodologies regarding innovation in the preparative scale were also comprised in this Special Issue. For example, one article presents the purification of R -phenylglycinol from the phenylglycinol enantiomers by stripping crystallization, a new separation technology, which combines melt crystallization and vaporization to produce a crystalline product due to the three-phase transformation [ 11 ]. The classical preparative scale approach through diastereomeric salts formation, widely used in the pharmaceutical industry, is also presented with the resolution of 4-chloromandelic acid using the ( R )-( + )-benzyl-1-phenylethylamine; with diastereomeric salts exhibiting significant di ff erences in solubility and in thermodynamic properties. These di ff erences originate from the distinct supramolecular interactions in the crystal lattice of the pair of diastereomeric salts. In addition to well-recognized hydrogen-bonding, CH / π interactions and aromatic group packing, halogen involved interactions, such as Cl . . . Cl and Cl / π were observed as significant contributions to the chiral discrimination [13]. The approach to achieve bioactive enantiomers by enantioselective synthesis is reported in two original publications and two reviews. One article reports the syntheses of a small library of proteomimetic quinazolinone-derived compounds and investigates their action on neurodegenerative disorders as well as the search of their potential as tumor cell growth inhibitors, giving evidence for the influence of stereochemistry on the bioactivity of diverse derivatives. Here, the enantiomeric ratio was determined by a chiral LC [ 17 ]. In another original article, the hemi-synthesis of chiral imine, benzimidazole, and benzodiazepine structures is reported by the condensation of ( S )-( − )-perillaldehyde, the major phytochemical of the Ammodaucus leucotrichus subsp. leucotrichus essential oil, with di ff erent amine derivatives of 2,3-diaminomaleonitrile, o -phenylenediamine, and 3-[(2-aminoaryl)amino]dimedone. The chiral analyses confirm the formation of unique enantiomers and diastereomeric mixtures [ 16 ]. Small ring heterocycles, such as epoxides and aziridines, present in several natural products, are frequently involved as highly versatile building blocks in the synthesis of numerous bioactive products and pharmaceuticals. Multicomponent reactions (MCRs) have been explored in the synthesis and ring opening of these heterocyclic units. An exhaustive review about the recent advances in MCRs discuss the synthesis and applications of epoxides and aziridines to prepare other heterocycles, emphasizing the stereoselectivity of the reactions [ 7 ]. Synthesis of chiral derivatives of xanthones, an important class of bioactive compounds, as well the enantioselectivity in their biological activities, was also exhaustively revised [3]. Industrial production by biocatalyse using the cis -epoxysuccinic acid hydrolases (CESHs) was summarized, as well the perspective on the future research and applications of CESH in enantiomeric tartaric acid production [6]. Additional work concerning chiral recognition are also included in this Special Issue, such as stereochemistry assignment and chiral recognition mechanisms of sulfoxide-containing drugs [ 14 ], the structural determination of the crystal structures of three complexes between the Tiiii cavitand as host and positively charged amino acids (Arg, Lys, and His) as guests [ 15 ]; a revision concerning enantioselective drug recognition by transporters [ 4 ], and another article about enantiomeric recognition and separation by chiral nanoparticles [ 5 ]. Molecular imprinting technology is a well-established tool for the synthesis of highly selective biomimetic molecular recognition platforms. One article reports the improvement in chiral selectivity of the important β -blocker atenolol by the addition of a metal pivot versus the traditional molecular imprinted polymer formulation [12]. Finally, original works related to special materials as chiral liquid crystals and components for chiral sensing are presented. For the proper function of liquid crystals-based devices, not only chemical but also optical purity of materials is strongly desirable, since any impurity could be detrimental to the self-assembly of the molecules. One article demonstrated that LC with UV detection and supercritical fluid chromatography with UV and mass spectrometry detection enables full control over the chemical and enantioselectivity of the synthesis of a novel type of lactic acid-based chiral liquid crystals and 3 Molecules 2020 , 25 , 1713 the corresponding chiral building blocks [ 18 ]. Regarding chiral sensing, one article reports a path to enhanced near-field optical chirality, by means of symmetric Si nanowires arrays, which support leaky waveguide modes that enhance the near-field optical chirality of circularly polarized excitation in the shorter wavelength part of the visible spectrum, which is of interest for many chiral molecules [ 19 ]. Another article reports an enantioselective potentiometric sensor composed of a polyvinyl chloride membrane electrode modified with CC3-R porous organic cages material, used for the recognition of enantiomers of 2-amino-1-butanol [20]. This Special Issue is accessible thought the following link: https: // www.mdpi.com / journal / molecules / special_issues / Chiral_separation_recognition. As Guest Editors for this Special Issue, we would like to thank all the authors and co-authors for their contributions and all the reviewers for their e ff ort in the careful and rapid evaluation of the manuscripts. Last but not least, we would like to appreciate the hard work done by the editorial o ffi ce of the Molecules journal, as well as their kind assistance in preparing this Special Issue. Funding: This work was supported by the Strategic Funding UID / Multi / 04423 / 2019 through national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF), through the COMPETE—Programa Operacional Factores de Competitividade (POFC) program in the framework of the program PT2020; Project No. POCI-01-0145-FEDER-028736, co-financed by COMPETE 2020, under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and the project CHIRALBIOACTIVE-PI-3RL-IINFACTS-2019. Conflicts of Interest: The authors declare no conflict of interest. References 1. Kimura, R.; Tsujimura, H.; Tsuchiya, M.; Soga, S.; Ota, N.; Tanaka, A.; Kim, H. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 5 molecules Article On the Enantioselective HPLC Separation Ability of Sub-2 μ m Columns: Chiralpak ® IG-U and ID-U Diana Ibrahim and Ashraf Ghanem * ,† Chirality Program, Faculty of Science and Technology, University of Canberra, ACT 2601, Australia; diana.ibrahim@canberra.edu.au * Correspondence: ashraf.ghanem@canberra.edu.au; Tel.: +61-(02)-6201-2089 or 0429319993; Fax: +61-(02)-6201-2328 † Dedicated to Prof. Frantisek Svec on the occasion of his 75th birthday. Received: 4 February 2019; Accepted: 29 March 2019; Published: 2 April 2019 Abstract: Silica with a particle size of 3–5 μ m has been widely used as selector backbone material in 10–25 cm HPLC chiral columns. Yet, with the availability of 1.6 μ m particles, shorter, high-efficiency columns practical for minute chiral separations are possible to fabricate. Herein, we investigate the use of two recently commercialized sub-2 μ m columns with different substituents. Thus, Chiralpak ® IG-U and ID-U were used in HPLC for the fast enantioseparation of a set of drugs. Chiralpak ® IG-U [amylose tris (3-chloro-5-methylphenylcarbamate)] has two substituents on the phenyl ring, namely, a withdrawing chlorine group in the third position and a donating group in the fifth position. Chiralpak ® ID-U [amylose tris (3-chlorophenylcarbamate)] has only one substituent on the phenyl ring, namely a withdrawing chlorine group. Their applications in three liquid chromatography modes, namely, normal phase, polar organic mode, and reversed phase, were demonstrated. Both columns have similar column parameters (50 mm length, 3 mm internal diameter, and 1.6 μ m particle size) with the chiral stationary phase as the only variable. Improved chromatographic enantioresolution was obtained with Chiralpak ® ID-U. Amino acids partially separated were reported for the first time under an amylose-based sub-2-micron column. Keywords: Chiralpak ® ID-U; Chiralpak ® IG-U; mobile phase modifiers; polar organic and reversed phase modes; sub-2 μ m particles 1. Introduction In nature and chemical systems, enantiomeric distinction and chiral recognition are fundamental occurrences [ 1 ]. This phenomenon has had a profound impact on a plethora of scientific fields, though the pharmaceutical industry significantly drives developments in chirotechnologies to cater to the demands of drug discovery [ 2 , 3 ]. There is no option when it comes to chiral considerations; all enantiomers must be tested in isolation of each other before being introduced to the market [ 3 ]. As a result, high performance liquid chromatography (HPLC) has emerged as the workhorse for racemate resolution [ 4 ]. HPLC enantiomer separation using chiral stationary phases (CSPs) is known to be one of the most convenient and versatile methods for the separation of chiral drugs [4]. In the last few decades, numerous CSPs have been developed and become commercially available [ 5 , 6 ]. CSPs filled in conventional columns of 4.0–4.6 mm internal diameter (i.d.) are the most widely used for analytical scale enantioseparation for industrial applications [ 5 , 6 ]. Nonetheless, conventional chiral columns are expensive; they consume large volumes of hazardous solvents and have long analysis times, and due to the dimensions of these large columns they are of limited throughput [ 6 ]. One of the possible solutions to enhance the speed of the analysis is to use columns filled with a CSP of smaller particles (sub-2 μ m) and hence a smaller theoretical plates height [7]. Molecules 2019 , 24 , 1287; doi:10.3390/molecules24071287 www.mdpi.com/journal/molecules 7 Molecules 2019 , 24 , 1287 Sub-2 μ m totally porous particles can be used to speed up analysis without loss in efficiency, as the optimal flow rate is inversely proportional to particle diameter [ 8 ]. The main limitation of using totally porous particles is the induction of high back pressure across the column induced by the friction of the mobile phase percolating through the particles generating heat, which hinders their usage within conventional HPLC systems [ 9 ]. Studies suggest that small i.d. columns can be used to minimize the frictional heating effect since heat dissipation is faster within such a narrow-bore column compared to conventional 4.6 mm i.d. columns [ 10 ]. Narrow-bore columns have a lower internal volume (2.1 mm i.d.) than the standard HPLC columns and thus achieve fast analysis [ 10 , 11 ]. They operate at lower flow rates (0.1–0.5 mL/min) with much reduced peak volumes, resulting in reduced mobile phase consumption and increased sensitivity [11,12]. Mobile phases can be modified to achieve higher enantioselective separation of racemates via improvement of complementary interactions between functional groups on the chiral selector and the analyte structure [ 13 ]. Pirkle and Welch have studied modifier effects on chiral selectivity and found that the influence of the mobile phase modifier was dependent upon the analyte structure [ 13 – 15 ]. Tambute and co-workers have also examined the use of modifiers and concluded that selectivity in their system depends on the steric hindrance of the alcohol modifier [ 14 – 16 ]. Researchers believe that the mobile-phase modifiers not only compete for chiral bonding sites with chiral solutes but can also alter the steric environment of the chiral grooves on the CSP by binding to the achiral sites at or close to the groove [ 13 , 17 ]. Enantioselective resolution is mainly due to the overall combination of all types of bonding [ 18 ]. Thus, not only the steric but also the substitutes of a certain chiral compound and the CSP should be taken into consideration to elucidate chiral recognition mechanisms [19]. Here we evaluate and compare the enantiorecognition abilities of two amylose-based sub-2 μ m CSPs towards 28 compounds, as they differ in the substituents on the phenyl ring. Recently commercialized Chiralpak ® IG-U [amylose tris (3-chloro-5-methylphenylcarbamate)] possesses an extra donating methyl group in the fifth position compared to the prototype Chiralpak ® ID-U [amylose tris (3-chlorophenylcarbamate)]. This investigation was performed using an operational instrument at an HPLC system pressure of 500 bar at which frictional heating is not very significant. Hence, thermal gradients inside the column were not expected to affect the efficiency. 2. Experimental 2.1. Instrumentation The mobile phase for the HPLC was filtered through a Millipore membrane filter (0.2 μ m) and degassed before use. The HPLC system consisted of a Waters binary pump, Model 1525, (Milford, MA, USA), equipped with a dual wavelength absorbance detector, Model 2487, an autosampler, Model 717 plus, and an optical rotation detector (JM Science Inc., Grand Island, NY, USA) operating at room temperature. The UV-detector was set at 254 nm. Chiralpak ® IG-U and ID-U (50 mm column length, 3.0 mm i.d, and 1.6 μ m silica gel) were supplied by Daicel (Tokyo, Japan). 2.2. Chemicals and Reagents All compounds and solvents (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The choice of compounds was arbitrary and guided by preliminary investigations. The compounds were, namely: beta-blockers (propranolol and atenolol), alpha-blockers (naftopidil), anti-inflammatory compounds (carprofen, naproxen, flurbiprofen, ketoprofen, and indoprofen), anticancers (ifosfamide), sedative hypnotics (aminoglutethimide), antiarrhythmic drugs (tocainide), norepinephrine-dopamine reuptake inhibitors (nomifensine), catecholamines (normetanephrine and epinephrine), antihistamines (chlorpheniramine), flavonoids (flavanone and 6-hydroxyflavanone), miscellaneous (1-acenaphthenol, 1-indanol, 4-hydroxy-3-methoxymandelic acid, propafenone HCL, cizolirtine, and 1-phenyl-2,2,2-trifluoroethanol), amino acids (glutamic acid, tyrosine, and phenylalanine) and antifungals (miconazole and sulconazole). 8 Molecules 2019 , 24 , 1287 2.3. Procedures Mobile phases were filtered through a membrane Sartorius Minisart RC 15 0.2 μ m pore size filter (Goettingen, Germany), further used for analysis without dilution, and degassed before use. The chromatographic measurements were performed at a flow rate of 0.5 mL/min at a temperature of 25 ◦ C. All measurements were performed in triplicate with an injection volume of 1 μ L. Stock solutions of samples were prepared at a concentration of 1 mg/mL using HPLC-grade 2-propanol as a solvent. 3. Results and Discussion The potential of the sub-2 μ m CSPs to separate the racemic compounds listed above under normal-phase, reversed-phase, and polar organic solvents have been investigated. The influence of the mobile phase composition on the separation ( α ), resolution (Rs), and retention time (RT) of enantiomers has been examined using (1) non-polar solvents (n-alkanes) containing a polar alcohol modifier, namely, ethanol (EtOH), 2-propanol (2-PrOH), and n -butanol ( n -BuOH), and (2) polar solvents, namely, methyl tert-butyl ether (MtBE), acetonitrile (ACN)