J S S JOURNAL OF SEPARATION SCIENCE. Methods Chromatography Electroseparation. Applications Biomedicine Foods Environment

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1 J S S ISSN JSSCCJ 42 (1) (2019) Vol. 42 No. 1 January 2019 D JOURNAL OF SEPARATION SCIENCE 1 19 Methods Chromatography Electroseparation Applications Biomedicine Foods Environment

2 Received: 21 June 2018 Revised: 20 August 2018 Accepted: 20 August 2018 DOI: /jssc REVIEW Recent development trends for chiral stationary phases based on chitosan derivatives, cyclofructan derivatives and chiral porous materials in high performance liquid chromatography Sheng-Ming Xie Li-Ming Yuan Department of Chemistry, Yunnan Normal University, Kunming, P. R. China Correspondence Professor Li-Ming Yuan, Department of Chemistry, Yunnan Normal University, Kunming , P. R. China. The separation of enantiomers by chromatographic methods, such as gas chromatography, high-performance liquid chromatography and capillary electrochromatography, has become an increasingly significant challenge over the past few decades due to the demand of pharmaceutical, agrochemical, and food analysis. Among these chromatographic resolution methods, high-performance liquid chromatography based on chiral stationary phases has become the most popular and effective method used for the analytical and preparative separation of optically active compounds. This review mainly focuses on the recent development trends for novel chiral stationary phases based on chitosan derivatives, cyclofructan derivatives, and chiral porous materials that include metal-organic frameworks and covalent organic frameworks in high-performance liquid chromatography. The enantioseparation performance and chiral recognition mechanisms of these newly developed chiral selectors toward enantiomers are discussed in detail. KEYWORDS chiral recognition, chiral stationary phases, enantioseparation, high-performance liquid chromatography 1 INTRODUCTION Molecular chirality and the stereospecific recognition of optically active molecules are fundamental phenomena in various aspects of chemistry and life science [1,2]. For instance, almost all of the essential components found in the human body, such as nucleic acids, proteins, carbohydrates, enzymes and DNA, are chiral compounds constructed from optically active building blocks. Since the physiological Article Related Abbreviations: ADMPC, amylose tris(3,5-dimethylphenylcarbamate); bdc, 1,4-benzenedicarboxylic acid; bpdc, 4,4 -biphenyldicarboxylate; bpe, 1,2-di(4-pyridyl)ethylene; btc, 1,3,5-benzenetricarboxylic acid; CD, cyclodextrin; CDMPC, cellulose tris(3,5-dimethylphenylcarbamate); CF, cyclofructan; COF, covalent organic framework; CSPs, chiral stationary phases; DMP-CF7, dimethylphenyl-carbamate CF7; IP-CF6, isopropyl carbamate-cf6; MKD, minimum kinetic diameter; RN-CF6, (R)-naphthylethyl carbamate-cf6; TMDPy, 4,4 -trimethylenedipyridine environment within a living organism is chiral, the two enantiomers of an optically active drug usually exhibit different pharmacodynamics and pharmacokinetics. In many optically active drugs, often only one of its enantiomers is responsible for the desired therapeutic effects, while the other enantiomer is less active, inactive or, in sometime cases, produces adverse effects. Therefore, it is now common to perform biological and toxicological tests on all new drug entities using both the racemate and their individual enantiomers [3]. Based on these facts, the drug administration authorities in many countries have announced the required development of optically pure drugs [4]. As the demand for optically pure compounds increases, a large number of important research efforts have been devoted to developing highly efficient, economical and convenient chiral separation methods [5]. Various chromatographic techniques, such as TLC, GC, HPLC, SFC, CE and CEC, are still the most convenient and cost-effective approaches for the separation of enantiomers WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim J Sep Sci 2019;42:6 20.

3 XIE AND YUAN 7 [6]. Among these, HPLC based on chiral stationary phases (CSPs) has become the most widely and effective chromatographic method used for the separation of optically active compounds, especially on a preparative scale. So far, more than 90% of the optically active compounds reported to date can be resolved successfully using this method [7 9]. Over the past three decades, various classical chiral selectors have been used to prepare HPLC CSPs, which can be classified into polysaccharides, Pirkle-type, cyclodextrin, synthetic polymers, macrocyclic glycopeptide antibiotics, proteins, crown ethers, and ligand exchange-based CSPs [1,3, 4,7 15]. Nowadays, there are more than 100 commercially available CSPs used for the separation of different kinds of optically active compounds. However, these commercially available CSPs have some disadvantages, which include: (1) high cost; (2) limited resolution and scope; and (3) difficulties selecting a specific chiral column for the efficient resolution of a specific enantiomer [16]. Hence, the development of novel HPLC CSPs with high efficiency, excellent resolution and low cost is still a research hotspot and continues to attract the attention of many researchers. There are many excellent reviews that have been published on classical CSPs and commercially available CSPs used for the HPLC separation of optically active compounds [1,4,7 10]. This review will mainly focus on the recent development trends of CSPs based on chitosan derivatives, cyclofructan derivatives, and chiral porous materials for HPLC enantioseparation over the last couple of years. 2 CHITOSAN DERIVATIVES AS CHIRAL STATIONARY PHASES FOR HPLC Among the above-mentioned HPLC CSPs, polysaccharide derivatives CSPs, and particularly those based on cellulose and amylose derivatives, such as their phenylcarbamates derivatives, are mostly widely used because these CSPs exhibited high recognition ability towards more than 90% of the racemic compounds reported to date, and cellulose and amylose are the most abundant naturally optically active macromolecules [4,7]. During the development of HPLC CSPs, some other polysaccharide derivatives, including chitin, chitosan, xylan, galactosamin, dextran, curdlan and inulin, have been also explored as CSPs for HPLC enantioseparation. For instance, a series of phenylcarbamate derivatives of xylan have been prepared and used as HPLC CSPs [17]. Some of them (e.g., xylan 2,3- bis(3,5-dimethylphenylcarbamate)) possess a higher chiral recognition ability than the well-known cellulose tris(3,5- dimethylphenylcarbamate) for some racemates. The results showed that the resolution ability of the xylan derivatives significantly depends on the nature, position, and number of substituents on the phenyl moiety. In addition, another important polysaccharide, chitin, is also an abundant chiral natural material obtained from the shells of crustaceans, mollusks, and insects. Chitin consists of N-acetyl-D-glucosamine units connected via a β-(1 4) glycoside bond, and chitosan is derived by the deacetylation of chitin. The chitin and chitosan possess the same primary structure as cellulose except for the groups at the 2-position of the glucose unit, in which there are acetamido and amino groups present in chitin and chitosan, respectively, whereas cellulose has a hydroxyl group. For this reason, a large number of novel derivatives different from cellulose derivatives can be prepared from chitin and chitosan. Therefore, it will be very promising to develop novel HPLC CSPs using chitin and chitosan derivatives as chiral selectors. Early in 1984, the Okamoto group discussed the potential application of chitin and chitosan derivatives as new chiral recognition materials in HPLC [18]. However, over the past decade, no relevant research studies have been reported, which is probably due to the difficulties in derivatizing chitin due to its low solubility in solvents. Until 1996, Cass and coworkers reported the synthesis of chitin bis(arylcarbamate) derivatives and their chiral recognition ability as HPLC CSPs [19]. Subsequently, Franco and co-workers prepared a CSP based on the 3,5-dimethylphenylcarbamate derivative of chitosan bonded on silica gel. Its resolution ability as a CSP for HPLC was evaluated and compared with the 3,5-dimethylphenylcarbamates derivatives of amylose and cellulose [20]. However, the HPLC CSPs comprised of these chitin and chitosan derivatives exhibit very poor chiral recognition ability due to the low modification efficiency of the hydroxy groups in chitin and chitosan according to the experimental data. Subsequently, Okamoto and co-workers prepared many arylcarbamates of chitin and phenylcarbamate-ureas of chitosan using different reaction conditions, such as the use of DMA/LiCl as the solvent [21 25]. When compared with the cellulose and amylose derivatives, some of them (e.g., 3,5-dimethyl-, 3,5-dichloro- of chitin and the completely deacetylated chitosan, etc.) exhibit higher resolving abilities for some optically active compounds. The above-mentioned works have been summarized in detail by Okamoto [4,7]. Over the last few years, the Bai group have also prepared a series of novel chitin and chitosan derivatives by modifying different functional groups at the 2-, 3-, and 6-positions and used the resulting materials as chiral selectors in HPLC enantioseparation. On the basis of the previously reported studies, the type, number and position of the substituents on the phenyl groups of the carbamoylated chitin derivatives significantly affect their enantioseparation capability [21,22]. Bai and co-workers have continued to develop some new CSPs using chitin derivatives bearing halogen groups, including chitin bis(4-trifluoromethoxyphenylcarbamate), chitin bis(3-chloro-4-methylphenylcarbamate) and chitin bis(4-chloro-3-trifluoromethylphenylcarbamate) [26]. These

4 8 XIE AND YUAN CSPs exhibit good enantioseparation capabilities towards tadalafil and its intermediate with two chiral centers. In particular, all the stereoisomers of tadalafil and its intermediate could be baseline separated on the chitin bis(4-trifluoromethoxyphenylcarbamate) CSP (Figure 1). Chitin can be converted to chitosan with an amino group at the 2-position of the glucosamine ring via a deacetylation reaction. Obviously, the amino group of chitosan can be selectively modified due to the reactivity difference observed between the amino and hydroxyl groups in chitosan. Thus, various novel chiral selectors can be prepared by introducing different substituents at 2-position of chitosan. The Okamoto group reported that some chitosan derivatives with different substituents at the 2-, 3-, and 6-position of the glucose unit may be used as CSPs for HPLC enantioseparation [23,24]. More systematic studies including the influence of substituents and the tolerance to various organic solvents on the enantioseparation of many novel CSPs based on chitosan derivatives using highly deacetylated chitosan as a starting material have been reported by the Bai group. For example, a series of chitosan alkyl amide and alkyl urea derivatives were prepared upon introducing an alkyl group, such as cyclobutyl [27], isobutyryl [28 30], n-octyl [31] and n-pentyl [32], at the 2-position of chitosan, and the chiral recognition ability and the solvent tolerance of these chitosan derivatives bearing the same phenylcarbamate groups at the FIGURE 1 The chromatograms of tadalafil (A) and its intermediate (B) separated using a chitin bis(4-trifluoromethoxyphenylcarbamate) CSP. Eluent: (A) n-hexane/ethanol (80:20, v/v) and (B) n-hexane/ethanol (90:10, v/v). Reproduced with permission from [26] 3- and 6-position investigated (Figure 2). It was observed that the chiral recognition performance of these coated-type CSPs was significantly influenced by the nature (e.g., electronwithdrawing and electron-donating properties), position, and number of substituents on the phenyl moieties of the chitosan derivatives and the eluent's composition. The results of the comparison experiments showed that the chitosan derivatives with an electron-withdrawing and/or electron-donating substituent at the 3- and/or 4-position on the phenyl moieties provided a more favorable enantioseparation ability for the tested racemates. Some chitosan derivative CSPs, such as chitosan 3,6-bis(3-chloro-4-methylphenylcarbamate)- (Ncyclobutylformamide) [27], chitosan 3,6-bis(3-methylphenylcarbamate)- (isobutyrylamide) [28], chitosan 3,6-bis(3,4-dichlorophenylcarbamate) (isobutyrylamide) [29], chitosan 3,6-bis(4-methylphenylcarbamate)-2-(isobutylurea) [30], chitosan 3,6-bis(3-chloro-4-methylphenylcarbamate)-2-(isobutylurea) [30], chitosan 3,6-bis(3,4-dichlorophenylcarbamate)- (n-octyl urea) [31] and chitosan 3,6-bis(4-methylphenylcarbamate)-(n-pentylamide) [32], exhibit comparable or even superior enantioseparation capability for many of the tested racemic compounds in comparison with the well-known derivatives cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) and amylose tris(3,5-dimethylphenylcarbamate) (ADMPC). Furthermore, a series of experimental results indicated that though there is an alkyl amide and alkyl urea rather than phenyl urea at the 2-position of the abovementioned chitosan derivatives, these newly developed chitosan derivatives CSPs still exhibit excellent chiral resolution abilities, indicating that the aromatic ring at the 2-position of the chitosan derivatives may not be necessary for chiral recognition. Besides, most of these chitosan derivatives CSPs can work in a wide range of mobile phases, such as chloroform, ethyl acetate and tetrahydrofuran, which cannot be used with those traditional coated-type CSPs based on the cellulose and amylose derivatives (e.g., CDMPC and ADMPC) due to their dissolution or high degree of swelling in these eluents. Therefore, the CSPs of these newly developed chitosan derivatives and the CDMPC and ADMPC CSPs are complementary for the enantiomeric separation of some optically active compounds by HPLC. To investigate the influence of the functional groups at the 2-position of the chitosan derivatives on their enantioseparation properties, the Bai group prepared a variety of chitosan derivative-based chiral selectors with identical substituents at the 3- and 6-positions of the phenylcarbamates used (e.g., 3-methylphenylcarbamate, 4-methylphenylcarbamate or 3,5-dimethylphenylcarbamate), whereas the amino group at the 2-position was modified using different substituents, such as amido or urea groups [33 36] (Figure 3). For example, they synthesized a series of chitosan derivatives by introducing alkoxyformamides at the 2-position of chitosan, including chitosan bis(4-methylphenylcarbamate)-

5 XIE AND YUAN 9 FIGURE 2 A schematic diagram of the chitosan derivatives obtained upon introducing an alkyl group at the 2-position of chitosan and different substituents at the 3- and 6-positions of chitosan, respectively FIGURE 3 A schematic diagram of the chitosan derivatives obtained upon introducing a phenyl group at the 3- and 6-positions of chitosan, and different substituents at the 2-position of chitosan, respectively (n-pentoxyformamide) and chitosan bis(4-methylphenylcarbamate)-(benzoxyformamide), chitosan bis(4-methylphenylcarbamate)-(ethoxyformamide) and chitosan bis(4-methylphenylcarbamate)-(isopropoxyformamide) [33]. The chiral recognition ability of these alkoxyformamylated chitosan derivatives as HPLC chiral selectors was compared, indicating that the enantioselectivity and resolution ability of these materials obviously differed upon varying the substituent at the 2-position of the glucosamine skeleton. Among these chiral selectors, chitosan bis(4-methylphenylcarbamate)-(ethoxyformamide) and chitosan bis(4- methylphenylcarbamate)-(isopropoxyformamide) showed improved enantioseparation capabilities than that of the other two chitosan derivatives. According to their structural features, it seems that the alkoxyformamylated chitosan derivatives containing smaller groups (ethyl and isopropyl groups) at the 2-position of the glucose skeleton show enhanced enantioseparation. However, an interesting experimental result was found upon comparing the enantioseparation capability of another series chitosan derivatives, such as chitosan bis(3,5-dimethylphenylcarbamate)-(alkyl urea)s [34] and chitosan bis(3,5-dimethylphenylcarbamate)- (N-cycloalkylformamide)s [35]. Among the chitosan bis(3,5-dimethylphenylcarbamate)-(alkyl urea) CSPs, two CSPs containing n-butyl and n-dodecyl groups exhibited a slightly lower enantioseparation capability than the CSP containing an n-octyl group. A similar experimental phenomenon was observed with the CSPs of chitosan bis(3,5- dimethylphenylcarbamate)-(n-cycloalkylformamide)s used for the separation of racemates. It was found that chitosan bis(3,5-dimethylphenylcarbamate)-(n-cyclopentylformamide) provided the best chiral recognition ability towards the racemates studied. Thus, with an increase in the ring size (three-, four-, five- and six-membered ring) at the 2-position, the chiral recognition abilities of the four CSPs seemingly showed a trend that initially increased and then decreased. It can be seen that the recognition process of the racemates on these chitosan derivatives-based CSPs is unusually complex. Therefore, it is very difficult to evidently explain the chiral recognition mechanism of these materials. To date, the impact of the molecular weight of cellulose/ amylose on the enantioseparation performance of the corresponding CSPs have been reported in only few studies [37 39]. Bai et al. have also reported some studies on the

6 10 XIE AND YUAN relationship between the molecular weight of the chitosan derivatives and their performance, including the chiral recognition ability and mobile phase tolerance of the CSPs [30,32,36,40 42]. Most of these studies indicated that chitosan derivatives with a lower molecular weight showed better enantioseparation performance, but a higher molecular weight was more preferable to tolerate the organic solvents used. According to the results, the degree of polymerization in chitosan of different molecular weight may affect the suprastructure and polarity of their derivatives, further leading to a great impact on the resolution ability and enantioselectivity of the resulting CSPs in HPLC. In order to balance the enantioseparation capability and mobile phase tolerance of chitosan derivative based CSPs, it is vitally important to adjust the molecular weight of chitosan. In their previous studies, the Bai group reported many biselector CSPs obtained from different chiral materials such as cellulose and amylase [43 46]. Some of these biselector CSPs provide an enhanced resolution ability than the individual CSPs to some extent. As known to all, cellulose and amylose derivatives (CDMPC and ADMPC in particular) used as HPLC CSPs exhibit excellent chiral recognition abilities for a very wide range of optically active compounds. However, the coated-type CSPs containing cellulose and amylose derivatives can dissolve or swell in some organic solvents, such as tetrahydrofuran, chloroform and ethyl acetate. Therefore, this shortcoming restricts their use in many mobile phases. To overcome this shortcoming, the preparation of novel biselector CSPs combining the excellent enantioseparation capability of cellulose and amylose derivatives and the high solvent tolerance of chitin derivatives has been reported [47,48]. The biselector CSPs are prepared by blending different ratios of cellulose and amylose derivatives (e.g., CDMPC and ADMPC) with chitin derivatives, such as chitin bis(3,5-dimethylphenylcarbamate) and chitin bis(3-chloro-4- methylphenylcarbamate). The experimental results show that these coating type biselector CSPs can be used in a wider range of mobile phases and enhance the chiral recognition ability when compared to the corresponding single selector CSPs. Based on the above studies, the results revealed that most of the chitosan derivatives CSPs possess satisfactory chiral recognition and resolving abilities for the racemates studied. Moreover, when compared to the coated-type CSPs derived from cellulose/amylose derivatives, these chiral selectors also have good tolerance against organic solvents, thus widening their practical applications in HPLC enantioseparation. It follows that chitosan derivatives have great potential as commercially available CSPs in practical applications. Further research should be focused on their application towards separating more optically active molecules, in particular, chiral drugs. 3 CYCLOFRUCTANS DERIVATIVES AS CHIRAL STATIONARY PHASES FOR HPLC Polysaccharide-based CSPs exhibit excellent chiral recognition ability and have been widely used for HPLC enantioseparations. Oligosaccharides are a class of saccharide polymers containing a small number (typically three to ten) of monosaccharides linked via a glucoside bond. Various oligosaccharides and their derivatives with abundant chiral recognition sites, such as the linear oligomers of cellulose (cellooligosaccharide) and amylose (maltooligosaccharide), and the cyclic oligomers, such as cyclodextrins (CDs), have been successfully used as CSPs in HPLC, especially for CDs derivatives [4]. Cyclofructans (CFs) are a small group of cyclic oligosaccharides comprised of six or more (usually seven or eight) β-(2 1)-linked D-fructofuranose units, named as CF6, CF7, and CF8, respectively [5]. Each fructofuranose unit contains four stereogenic centers and three hydroxyl groups (3-, 4-, or 6-hydroxyl groups). Although the CFs belong to macrocyclic oligosaccharides exactly like CDs, CFs are quite different in both their structure and behavior. Therefore, it is promising to develop a new type of CSP using CFs as the starting materials. In 2009, Armstrong et al. first prepared a series of CF6- based CSPs bonded to aminopropyl- or epoxy-functionalized silica gel and performed the HPLC enantioseparation of optically active compounds [49]. The cyclofructan CSPs based on aliphatic- and aromatic-functionalized CF6 possess unique and quite different chiral resolving abilities from that of the native CF6, which has rather limited chiral recognition capabilities. When CF6 was lightly derivatized with aliphatic functionalities such as methyl and isopropyl groups, the CF6 derivatives exhibit an extraordinary capability to separate all the chiral primary amines studied in both organic solvents and supercritical CO 2. In contrast, the highly aromaticfunctionalized CF6 CSPs derivatized with larger functional groups (e.g., R-1-(1-naphthyl)ethyl and 3,5-dimethylphenyl, etc.) lose most of their enantioselective capabilities toward chiral primary amines, but they can resolve a broad variety of other racemic compounds, including acids, secondary amines, tertiary amines, alcohols, and others. The CF6-based chiral selectors containing different functional groups on the fructofuranose units exhibit significantly different enantiomeric selectivities and resolving abilities, indicating that the nature and size of the substituents have a significant influence on the chiral recognition abilities of the resulting cyclofructan derivatives. In addition, the CF6-based CSPs possess potential applications for the preparative separation of enantiomers due to their high stability and high loading capability. A total of 4200 μg of N-(3,5-dinitrobenzoyl)- phenylglycine was baseline separated on a (R)-naphthylethyl carbamate-cf6 (RN-CF6) column, as shown in Figure 4.

7 XIE AND YUAN 11 FIGURE 4 The loading test performed on the RN-CF6 column. The analyte was N-(3,5-dinitrobenzoyl)-DL-phenylglycine. The mobile phase was 85ACN/15MEOH/0.3AA/0.2TEA. The injection volumes are 5 (top) and 100 μl (bottom). UV detection: 350 nm. ACN, acetonitrile; MeOH, methanol; AA, acetic acid; TEA, triethylamine. Reproduced with permission from [49] Some other novel chlorinated aromatic derivatives and cationic/basic derivatives of CF6 have also been successively developed as HPLC CSPs [50,51]. Subsequently, Armstrong and co-workers continued to study the cyclofructan containing seven units (CF7) as HPLC chiral selectors. They prepared a series of aromaticfunctionalized CF7 CSPs containing different aromatic substituents, including dimethylphenyl, methylphenyl, naphthylethyl, dichlorophenyl, and chlorophenyl [52]. A large number of racemic compounds, such as chiral acids, amines, metal complexes and neutral compounds, were selected as analytes and separated on the derivatized-cf7 CSPs. It was found that the dimethylphenyl-carbamate CF7 (DMP-CF7) provided the best enantioseparation performance towards the racemic compounds studied in normal phase mode. Among the newly developed cyclofructan-based CSPs, isopropyl carbamate-cf6 (IP-CF6), RN-CF6 and DMP- CF7 exhibited outstanding enantioselectivity and resolving ability towards various racemic compounds. RN-CF6 and DMP-CF7 CSPs show a broad enantiomeric selectivity towards a plethora of chiral analytes [49,52 55], whereas IP-CF6 CSP was particularly well suited for the separation of racemic primary amines [49,56,57]. The three derivatized cyclofructan-based columns have been commercialized, namely, LARIHC CF6-P, LARIHC CF6-RN and LARIHC CF7-DMP. Figure 5 depicts the structures of CF6-P, CF6-RN and CF7-DMP. Afterwards, the same group also carried out numerous comparison studies on the chiral recognition ability of cyclofructan-based CSPs (IP-CF6, RN-CF6 and DMP-CF7, as well as cyclodextrin-based CSPs) for the separation of a wide range of racemic compounds, including some unique and novel compounds such as tetrahydrobenzimidazoles, biaryl atropisomers, ruthenium(ii) polypyridyl complexes, 2-naphthol atropisomers, α-aryl ketones, and spirobrassinin [53,58 68]. The research results showed that most of the chiral analytes studied were well separated on these FIGURE 5 The structures of the cyclofructan-based CSPs used for IP-CF6, RN-CF6 and DMP-CF7 chiral selectors, which have good complementary enantioseparation for some analytes. Furthermore, the dominant interactions participating in the retention mechanism and separation process on IP-CF6, RN-CF6 and DMP-CF7 CSPs were studied using the linear free-energy relationship model [69]. To investigate the influence of the support on the enantioseparation performance of derivatized cyclofructan, a comparison of the HPLC separation of the enantiomers with the CSPs prepared by chemically bonding IP-CF6 on fully and superficially porous particles (FPP and SPP, respectively) was carried out by the Armstrong group [70]. This study indicated that the novel SPP based CSPs prepared from superficially porous silica maintains their enantioselectivity while enhancing the efficiency and decreasing the separations time. The SPP based CSPs greatly improved the resolution performance toward chiral analytes when compared to their FPP analogs. These systematic and detailed research studies provide good guidance for the separation of different types of racemic compounds via the selection of suitable derivatized cyclofructan based HPLC CSPs. 4 CHIRAL POROUS MATERIALS AS CHIRAL STATIONARY PHASES FOR HPLC Over the last few decades, porous solid materials such as metal-organic frameworks, covalent organic frameworks (COFs), porous organic frameworks, and porous organic cages have undergone explosive growth, and have attracted a great deal of attention from chemists, physicists, and materials scientists [71]. Among them, homochiral porous crystal materials have very promising applications in the fields of adsorption, enantioselective separation, asymmetric catalysis, and chiral sensors [5,71 86].

8 12 XIE AND YUAN Many efficient chiral materials (e.g., polysaccharides, chitosan, cyclofructan, cyclodextrin, macrocyclic antibiotics, proteins, crown ethers, etc.) with multiple interaction sites have been used as CSPs in HPLC [1,4,7 16]. The use of polysaccharide-based CSPs continues to dominate the majority of all HPLC separations using CSPs [8]. In recent years, many researchers have made efforts towards developing various novel types of HPLC CSPs using chiral porous materials as chiral selectors. Homochiral MOFs, as an important member of the MOFs family, can be generated from chiral ligands or achiral ligands under spontaneous resolution without any chiral sources. Originally, homochiral MOFs were mainly used for the enantioselective adsorption separation of optically active molecules. Inspired by the studies on enantioselective adsorption, homochiral MOFs have recently attracted an increasing amount of attention as new CSPs for LC. In 2007, Nuzhdin et al. reported the first application of an LC column for the separation of various racemic mixtures over an enantiopure porous MOF, [Zn 2 (1,4-bdc)(L-lac)(dmf)] DMF, used as the stationary phase [87]. A 33 cm long chiral column was prepared by loading a glass tube (8 mm inner diameter) with a suspension of [Zn 2 (bdc)(l-lac)(dmf)] DMF in a 10% solution of DMF in dichloromethane. A series of racemic compounds (alkyl aryl sulfoxides) were directly loaded onto the top of the column, respectively and the complete baseline separation of sulfoxide (PhSOMe) was achieved. This work was the first documented use of an MOF as a CSP for the liquid chromatographic separation of chiral sulfoxides. Furthermore, Tanaka and co-workers reported that a series of chiral sulfoxides could also be successfully separated on a HPLC column using a (R)-CuMOF-1-silica composite as the CSP [88]. (R)-CuMOF-1 was synthesized from a mixture of (R)-2,2 -dihydroxy-1,1 -binaphthalene-6,6 -dicarboxylic acid and Cu(NO 3 ) 2 in DMF. Sixteen chiral sulfoxides were separated on the packed column using hexane/etoh (50/50) and hexane/i-proh (90/10) as the mobile phase. The results revealed that the separation factors (α)ofthepara-substituted phenyl methyl sulfoxides were larger than their orthoand meta-substituted counterparts, which was attributed to steric effects in many cases, and the introduction of both electron-donating ( CH 3 ) and electron-withdrawing ( Cl and NO 2 ) substituents in the aromatic ring of the sulfoxides did not reduce the enantioselectivity. The same elution orders were observed for all the sulfoxides studied, in which the S enantiomers were eluted before the R enantiomers because of the stronger retention of the R enantiomer in the cavity of (R)-CuMOF-1. Further research on the enantioseparation performance of the (R)-CuMOF-1-silica composite and its analogs ((R)- ZnMOF-1 and (R)-CuMOF-2) as CSPs for various kinds of racemic compounds, including sulfoxides, sec-alcohols, β-lactams, benzoins, flavanones and epoxides, was reported by the same research group [89]. On the basis of the previous studies, (R)-ZnMOF-1 and (R)-CuMOF-2 were prepared from a different metal ion (Zn 2+ ) and chiral ligand ((R)-2,2 -dimethoxy-1,1 -binaphthalene-6,6 -dicarboxylic acid), respectively. The chiral recognition abilities of the three homochiral MOF-silica composites were compared. The experimental results showed that the enantioseparation ability of the (R)-CuMOF-1 column was higher than the (R)-ZnMOF-1 column towards all the racemic compounds studied except for a few analytes, whereas the (R)-CuMOF-2 column exhibited very poor enantioselectivity and resolving ability. This may be due to the different channel sizes in the materials (approximately 6.2 Å and 4.4 Å for(r)-cumof-1 and (R)-CuMOF-2, respectively) and the lack of hydrogen bonding interactions between guest molecules and (R)- CuMOF-2, which has no hydroxyl group. In the HPLC enantioseparation of the test solutes, the composition of the mobile phase also played a vital role in the retention and resolution. Afterwards, they reported other two novel pillared homochiral MOF-silica composites with excellent selectivity for the HPLC enantioseparation of chiral sulfoxides, sec-alcohols and flavanones [90]. Padmanaban et al. [91] developed a chiral column employing a chiral-modified UMCM-1 MOF (Bn-ChirUMCM-1) as the stationary phase for HPLC enantioseparation. 1- Phenylethanol was separated on the MOF column, but the 1-phenylethylamine could not be separated, which may be due to the very strong interactions observed between 1-phenylethylamine and the MOF. Recently, Stoddart et al. reported a green and renewable homochiral porous framework material (γ-cd MOF) constructed from γ-cyclodextrin (γ-cd) and alkali metal salts [92]. The γ-cd MOF was utilized as a HPLC CSP to resolve the enantiomers of several chiral analytes, including limonene and 1-phenylethanol. The separation of the 1-phenylethanol enantiomers using CD-MOF was superior to that above-mentioned using Bn-ChirUMCM-1 as a CSP [91]. Subsequently, the Yan group carried out further research and exploration on the practical application of CD MOFs as CSPs for the HPLC separation of enantiomers [93]. A series of chiral aromatic alcohols as analytes were separated on the γ-cd MOF packed column. Twelve chiral aromatic alcohols were well separated on this packed column with good precision and enantioselectivity. The chiral recognition mechanism on the γ-cd MOF was discussed. The γ-cd molecule has a hydrophilic rim and a hydrophobic cavity with a pore size of 1.4 nm, which is bigger than the molecular dimensions of these tested chiral aromatic alcohols. Thus, the chiral aromatic alcohol molecules can enter the pores of the γ-cd MOF and the hydrophobicity of the γ-cd cavity may produce a hydrophobic interaction with the phenyl groups on the chiral aromatic alcohols. During the recognition process, the importance of the hydrogen-bonding interactions

9 XIE AND YUAN 13 between the chiral aromatic alcohols and γ-cd was studied using a comparison experiment. The γ-cd MOF column gave no resolution of (R,S)-1-phenyl-1-butane which has no hydroxyl group, indicating the existence of a hydrogenbonding interaction in the separation process. Therefore, the good chiral recognition capability of γ-cd MOF for chiral aromatic alcohols mainly depends on the micro-environment of the γ-cd MOF and the hydrophobic and hydrogenbonding interactions between aromatic alcohols and γ-cd MOF. Cui and co-workers synthesized two robust homochiral 1,1 -biphenol-based MOFs containing one-dimensional nano-sized channels decorated with chiral dihydroxyl (MOF 1a) or dimethoxy (MOF 1b) groups [94]. A homochiral MOF packed column was prepared using MOF 1a as the CSP for the HPLC separation of racemic amines. Unfortunately, the MOF 1a-packed column failed to separate racemic amines such as 1-phenylethylamine bearing free amine groups, which is similar to that on the chiral-modified UMCM-1 MOF column. However, the free amines after benzoylation can be successfully resolved on the MOF 1a-packed column under the optimized chromatographic conditions. MOF 1b containing methyl-protected biphenol groups was also utilized as a CSP for the separation of these racemic amines as a comparison. These racemic amines could not be resolved on the MOF 1b-packed column, revealing that the hydroxyl groups of the biphenol units in MOF 1a contribute to the chiral separation process. Tang et al. [95] also reported a 3D rigid and stable homochiral MOF {[ZnLBr] H 2 O} n (L = N-(4- pyridylmethyl)-l-leucine) containing a chiral helical channel (aperture size: 9.8 Å) to be used as a CSP in HPLC to separate various racemates using hexane/isopropanol as the mobile phase. The separations of racemates containing different functional groups and with different molecular size (the minimum kinetic diameter, MKD) on the homochiral MOF column were performed, including ibuprofen (7.4 Å), 1-phenyl-1-propanol (7.4 Å), 1-phenylethylamine (7.4 Å), benzoin (9.2 Å), ketoprofen (9.4 Å), and naproxen (9.7 Å). All racemates with smaller MKD than the chiral channels of {[ZnLBr] H 2 O} n (9.8 Å) achieved baseline separation on the MOF column with a short retention time (Figure 6), except for ketoprofen and naproxen. The reason for this may be that the ketoprofen and naproxen molecules find it difficult to enter the chiral helical channels of the crystal due to their MKDs being close to the chiral channels of {[ZnLBr] H 2 O} n. It is worth noting that the homochiral MOF {[ZnLBr] H 2 O} n column can show excellent selectivity for 1-phenylethylamine, which cannot be separated on the above-mentioned homochiral MOF columns. Evidently, the chiral helical channels of homochiral MOF with molecular sieve-like performance and independence of the functional groups play a significant role for resolution of racemates. FIGURE 6 HPLC enantioseparation of racemates on a homochiral MOF-packed column (10 cm long 4.6 mm i.d.): (A) Ibuprofen, (B) 1-phenyl-1-propanol, (C) 1-phenylethylamine and (D) benzoin at 25 C. The inserted molecular models with the lowest energy and the sizes correspond to the MKDs considering the van der Waals radii obtained using Gaussian 03 software. Reproduced with permission from [95] In recent years, our group has made many efforts to develop novel homochiral MOFs stationary phase for HPLC. A variety of homochiral MOFs were chosen as research objects mainly based on the following aspects: The building blocks, the structure, the pore/channel size and solvent stability of the homochiral MOFs. Consequently, an unusual 3D chiral nanoporous MOF [(CH 3 ) 2 NH 2 ][Cd(bpdc) 1.5 ] 2DMA (bpdc = 4,4 -biphenyldicarboxylate) was utilized as a CSP [96]. The homochiral MOF possesses a large pore with a 19.4 by 22.4 Å opening of the hexagonal nanotubes, a righthanded helix, and excellent chemical and solvent stability. A wide range of racemates (e.g., alcohols, ketones, phenols, bases, and amides) were successfully separated on the MOF packed column with high resolution and excellent enantioselectivity employing non-polar or low polar solvents (hexane/dichloromethane or hexane/isopropanol) as the mobile phase. As an inexpensive, commercially available multi-dentate ligand, camphoric acid (H 2 cam) is a very good choice to explore chiral coordination chemistry [73]. Since chiral MOFs constructed from D-(+)-camphoric acid exhibit excellent recognition ability and enantioselectivity as a stationary phase in GC, our group and the Liu group have taken several homochiral MOFs including [Cu 2 (D-Cam) 2 (4,4 -bipyridine)] n,[zn 2 (D-Cam) 2 (4,4 -bipyridine)] n,[co 2 (D-cam) 2 (4,4 - trimethylenedipyridine)], [Cd 2 (D-Cam) 3 ] 2HDMA 4DMA and (Me 2 NH 2 ) 2 [Mn 4 O(D-cam) 4 ] (H 2 O) 5 constructed from D-(+)-camphoric acid and different metal ions as the stationary phases for HPLC separation [97 101]. These

10 14 XIE AND YUAN homochiral MOFs packed columns gave good resolution for the separation of a diverse range of racemates. Natural amino acids, which are inexpensive, nontoxic and readily available, may be ideal enantiopure linkers for the formation of homochiral MOFs [102]. Subsequently, our group synthesized a series of homochiral MOFs built from metal ions (Zn 2+ and Co 2+ ions) and various enantiopure amino acids (L-tyrosine (L-tyr), L-histidine, L-tryptophan (L-trp) and L-glutamic acid), namely [Zn(L-tyr)] n (LtyrZn), [Zn 4 (btc) 2 (Hbtc)(L-His) 2 (H 2 O) 4 ] 1.5H 2 O, {[Zn 2 (Ltrp) 2 (bpe) 2 (H 2 O) 2 ] 2H 2 O 2NO 3 } n, [Co 2 (L-Trp)(INT) 2 (H 2 - O) 2 (ClO 4 )], {[Co(L-trp)(bpe)(H 2 O)] H 2 O NO 3 } n, [Co 2 (4,4 -sulfonyldibenzoate)((l-trp) 2 ] and [Co(L-Glu)(H 2 O) - H 2 O] [102,103]. The fabricated MOFs columns show good chiral recognition abilities towards many optically active compounds, such as alcohols, amines, ketones, ethers, and organic acids. It was observed that these columns exhibit different enantioseparation performance towards the optically active compounds studied, though these homochiral MOFs possess the same chiral ligands (camphoric acid or amino acids), clearly indicating that the framework structure has a significant impact on the enantioselectivity. Some other homochiral MOFs, including [Cu(Smal)(bpy)] n and [Cu(S-mal)(bpe)] n built from S-malic acid, and [In 3 O(obb) 3 (HCO 2 )(H 2 O)] with a helical structure constructed from an achiral building block (4,4 - oxybisbenzoic acid), also exhibit good resolving abilities toward many racemates [ ]. Surprisingly, the MOF, [In 3 O(obb) 3 (HCO 2 )(H 2 O)], without a chiral ligand can separate racemates, showing its helical structure plays a significant role in the chiral recognition process, which was demonstrated in our previous work [107]. The characteristic data and enantioseparation results obtained for the above-mentioned homochiral MOFs in HPLC are summarized in Table 1. As can be seen from Table 1, a large number of homochiral MOFs with different structural features can be used as HPLC CSPs for the separation of various types of racemates, such as sulfoxides, alcohols, ketones, phenols, organic acids, bases, amines, and amides. Some homochiral MOFs (e.g., [Zn 2 (1,4-benzenedicarboxylic acid)(l-lac)(dmf)] DMF, Bn-ChirUMCM-1, γ-cd MOF, MOF 1a and (Me 2 NH 2 ) 2 [Mn 4 O(D-cam) 4 ] (H 2 O) 5 ) can only separate a few optically active compounds, while others exhibit good enantioseparation performance towards a wide range of racemates. The chiral recognition ability of homochiral MOFs mostly depends on the homochiral micro-environment of the MOF crystals. Firstly, the pore/channel size of homochiral MOFs significantly affects the enantioselectivity of optically active molecules. For instance, the homochiral MOF {[ZnLBr] H 2 O} n (aperture size: 9.8 Å) can afford outstanding separation of some enantiomers, which are able to readily enter the chiral channels. However, there is no separation observed for some larger molecules (DL-ketoprofen and (±)- naproxen) because they cannot access the chiral channels of the MOF [95]. Moreover, [(CH 3 ) 2 NH 2 ][Cd(bpdc) 1.5 ] 2DMA with a larger pore size ( Å) than {[ZnLBr] H 2 O} n also exhibits excellent selectivity for a wide range of racemates [96]. Thus, a close match of the dimensions of the chiral channels and size of the optically active guests is required for the recognition process between homochiral MOFs and enantiomers. The homochiral microenvironment of the MOF intraframeworks is generated from the various chiral ligands and metal ions. Most homochiral MOFs assembled from chiral ligands (e.g., camphor acid and amino acids, etc.) and some metal ions (e.g., Zn 2+ and Cu 2+, etc.) show good resolving abilities for various racemates. The functional groups and structures of the analytes also influence their separation performance on MOF CSPs. On the basis of the reported results, we can preliminary speculate that these homochiral MOFs are suitable for separation of some optically active compounds including alcohols, ketones and amides because these optically active molecules can easily interact with the host frameworks via hydrogen bonding, π-π stacking, van der Waals forces or diplole-diplole interactions. Based on the comparison studies report, the design and synthesis of homochiral MOFs can be considered from many viewpoints (chiral ligands, pore/channel size, metal ions and stability) to more efficiently utilize them for the separation of optically active compounds in HPLC. Covalent organic frameworks (COFs) are a new class of crystalline porous materials with highly ordered structures solely constructed from multidentate organic building blocks through strong covalent bonds. Numerous COFs have been reported [108] and some of them have been explored for diverse applications including gas storage, catalysis, sensing, drug delivery, and separation. In particular, COFs materials have great potential as novel chromatographic medias because of their unique properties, such as low density, large specific surface area, permanent porosity, and good stability [109]. However, it is still challenging to design and synthesize homochiral COFs. To date, only several homochiral COFs have been prepared via direct or postsynthetic strategies [ ]. The application of homochiral COFs in chiral separation is still in its infancy and there is only one research paper exploring the homochiral COFs, CTpPa-1, CTpPa-2, and CTpBD for high resolution GC separation of enantiomers including 1-phenylethanol, 1-phenyl-1- propanol, limonene, and methyl lactate [109]. Recently, the Cui group firstly reported the use of 3D homochiral COFs (CCOFs 5 and 6) as CSPs for the HPLC enantioseparation of racemic compounds [118]. The 3D homochiral COFs packed columns offered good enantioselectivity and resolution performance toward racemic alcohols (Figure 7).

11 XIE AND YUAN 15 TABLE 1 List of chiral MOFs used as CSP in HPLC Formula of chiral MOFs [Zn 2 (1,4-benzenedicarboxylic acid)(l-lac)(dmf)] DMF Characteristic data of chiral MOF CSPs Pore/channel diameter (Å) Particle size (μm) (R)-CuMOF-1-silica 6.2 silica gel (particle size, 7 μm) Columns [long i.d. (cm mm)] Particle size (μm) Racemates References 5 < (glass tube) Sulfoxides [87] Sulfoxides, sec-alcohols, β-lactams, benzoins, flavanones, epoxides [88,89] (R)-CuMOF-2-silica Sulfoxides, sec-alcohols, β-lactams, benzoins, flavanones, epoxides [89] (R)-ZnMOF-1-silica Sulfoxides, sec-alcohols, β-lactams, benzoins, flavanones, epoxides [89] (R)-MOF-4-silica Sulfoxides, sec-alcohols, flavanones [90] (R)-MOF-5-silica Sulfoxides, sec-alcohols, flavanones [90] Bn-ChirUMCM-1 < phenylethanol [91] γ-cd MOF Aromatic alcohols [92,93] MOF 1a Amines [94] {[ZnLBr] H 2 O} n Ibuprofen, benzoin, 1-phenyl-1-propanol, 1-phenylethylamine [95] [(CH 3 ) 2 NH 2 ][Cd (bpdc) 1.5 ] 2DMA Alcohols, ketones, phenols, bases, amides [96] [Cu 2 (D-Cam) 2 (4, 4 -bipy)] n Alcohols, naphthols, ketones [97] [Zn 2 (D-Cam) 2 (4, 4 -bpy)] n Alcohols, phenols, aldehydes, ketones, bases, amides [98] [Co 2 (D-cam) 2 (4,4 -trimethylenedipyridine)] Alcohols, amides, ketones, epoxides, bases [99] [Cd 2 (D-Cam) 3 ] 2Hdma 4dma Alcohols, naphthols, ketones, bases [100] (Me 2 NH 2 ) 2 [Mn 4 O(D-cam) 4 ] (H 2 O) Ibuprofen, 1-phenyl-1,2-ethanediol [101] Seven homochiral MOFs based on enantiopure amino acid ligands Alcohols, amines, ketones, ethers, organic acids [102,103] [In 3 O(obb) 3 (HCO 2 )(H 2 O)] ,5-dinitro-N-(1-phenylethyl)benzamide, omeprazole, ibuprofen [104] [Cu(S-mal)(bpy)] n Alcohols, ketones, flavonoids, phenols, amines [105] L-lac, L-lactic acid; L, N-(4-pyridylmethyl)-L-leucine; 4,4 -bipy, 4,4 -bipyridine; H 2 obb, 4,4 -oxybis(benzoic acid); S-mal, S-malic acid.

12 16 XIE AND YUAN FIGURE 7 (A-D) HPLC separation of racemic 1-phenyl-2-propanol, 1-phenyl-1-pentanol, 1-phenyl-1-propanol and 1-(4-bromophenyl)ethanol on the CCOF 5 (blue line) and 6 (red line) packed columns, respectively using hexane/isopropyl alcohol (90:1, v/v) as the mobile phase at a flow rate of 0.2 ml/min. Reproduced with permission from [118] Over the past decade, some homochiral porous materials including MOFs and COFs as chromatographic medias have been successfully applied towards the separation of racemates in HPLC. However, the current research shows that these homochiral porous materials are unable to become commercially available CSPs because of the following factors: (1) the chromatographic performance of the reported homochiral porous material CSPs is not satisfactory and only a very limited number of optically active compounds can be successfully resolved on them; (2) the prepared homochiral porous material CSPs afford poor reproducibility because of the broad particle size distribution and irregular shapes of the homochiral porous materials formed after the manual pestle crushing process; and (3) excellent HPLC CSPs should have high stability in the majority of mobile phases, such as normal phase, reversed phase and polar organic phase under high pressure, which restricts the practical use of homochiral porous materials due to their poor stability in some solvents. Although the development of homochiral porous material CSPs for HPLC enantioseparation is still in its infancy, we believe that homochiral porous materials will gain practical application for chromatographic separations in the near future. 5 CONCLUDING REMARKS HPLC still is the most popular and highly applicable method for both the analytical and preparative resolution of enantiomers. In this review, we have summarized the recent achievements in the novel chiral selectors, including chitosan derivatives, cyclofructan derivatives, and chiral porous materials for HPLC enantioseparation over the past decade. Many CSPs exhibit high enantioselectivity and excellent resolution ability toward a wide range of optically active compounds, and have good perspective in practical application. Some of them have been commercialized, especially for chiral HPLC columns based on the cyclofructan derivatives CSPs. Additionally, there is still great room for the further exploration of homochiral porous materials used as CSPs for HPLC enantioseparation. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos , and ) and the Applied Basic Research Foundation of Yunnan Province (No. 2017FB013). CONFLICT OF INTEREST The authors have declared no conflict of interest. ORCID Li-Ming Yuan REFERENCES 1. Tang, M. L., Zhang, J., Zhuang, S. L., Liu, W. P., Development of chiral stationary phases for high-performance liquid chromatographic separation. Trac Trends Anal. Chem. 2012, 39, Scriba, G. K. E., Chiral recognition in separation science-an update. J. Chromatogr. A 2016, 1467, Lammerhofer, M., Chiral recognition by enantioselective liquid chromatography: mechanisms and modern chiral stationary phases. J. Chromatogr. A 2010, 1217, Shen, J., Okamoto, Y., Efficient separation of enantiomers using stereoregular chiral polymers. Chem. Rev. 2016, 116, Xie, S. M., Yuan, L. M., Recent progress of chiral stationary phases for separation of enantiomers in gas chromatography. J. Sep. Sci. 2017, 40, Francotte, E. R., Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers. J. Chromatogr. A. 2001, 906, Okamoto, Y., Ikai, T., Chiral HPLC for efficient resolution of enantiomers. Chem. Soc. Rev. 2008, 37,