Chiral separation by counter-current chromatography

Accepted Manuscript Title: Chiral separation by counter-current chromatography Author: Xin-Yi Huang, Duo-Long Di PII: DOI: Reference:

S0165-9936(15)00040-0 http://dx.doi.org/doi:10.1016/j.trac.2015.01.009 TRAC 14394

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Trends in Analytical Chemistry

Please cite this article as: Xin-Yi Huang, Duo-Long Di, Chiral separation by counter-current chromatography, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi:10.1016/j.trac.2015.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chiral separation by counter-current chromatography Xin-Yi Huang, Duo-Long Di * Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Science, Lanzhou 73000，China

HIGHLIGHTS  Chiral separation by counter-current chromatography  Benefits and limitations of chiral separation by counter-current chromatography  Novel methods for chiral separation by counter-current chromatography  Challenges of applications of chiral separation by counter-current chromatography ABSTRACT Counter-current chromatography (CCC) is a new, efficient, productive chromatographic technique based on continuous liquid-liquid partition. Recently, it attracted more attention in chiral separation due to its high load capacity, cheap liquid stationary phase and low solvent consumption, when compared to traditional chiral separation techniques. This review presents advances and applications of chiral separation using high-speed CCC in recent years. We summarize the major benefits and the limitations of chiral separation by CCC. We introduce in detail some novel methods, which can improve the resolution of enantiomers and are easily achieved on classical CCC apparatus. We also outline challenges and future perspectives in developing chiral separation by CCC. Keywords:

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Biphasic chiral recognition Chiral chromatography Chiral selector Chiral separation Counter-current chromatography Liquid-liquid partition Multiple dual-mode elution Recycling elution mode Separation efficiency Solvent system

Abbreviations: 2-PPA, 2-phenylpropionic acid; BCR, Biphasic chiral recognition; BRCE, Biphasic recognition chiral extraction; CCC, Counter-current chromatography; CE, Capillary electrophoresis; ChMWat; Chloroform/methanol/water; EbuWat, Ethyl acetate/butanol/water; GC, Gas chromatography; HEMWat, Hexane/ethyl acetate/methanol/water; HPCCC, High-performance counter-current chromatography; HPLC, High-performance liquid chromatography; HSCCC, High-speed counter-current chromatography; MDM, Multiple dual-mode elution; MTBE, Methyl t-butyl ether; NAP, 2-(6-methoxy-2-naphthyl)propionic acid; REM, Recycling elution mode * Corresponding author. Tel.: +86 931 4968248; Fax: +86 931 8277088. E-mail address: [email protected] (D.-L. Di).

1. Introduction Chiral separation is the main method to obtain individual enantiomers. Counter-current chromatography (CCC) processes are powerful preparative techniques due to their high capacity and low cost of solvent. They can achieve chiral separation through establishing a chiral

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environment by adding a chiral selector. According to the literature, the first attempt at chiral separation with CCC was by Prelog’s group [1]. They selected (R,R)-di-5-nonyltartrate as a chiral selector and employed a rotation locular CCC machine and a 1,2-dichloroethane-water solvent system to achieve chiral separation of racemic norephedrine. Although this experiment accomplished a partial separation, it still got high-purity enantiomers and proved that the CCC technique can be used in chiral separation. However, because of the low theoretical plates of CCC and the absence of highly selective chiral selectors, chiral CCC is developing slowly and is not as popular as other technologies, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Foucault [2] specifically reviewed the progress of chiral separation by CCC and centrifugal partition chromatography in 2001. Some other reviews [3–5] also mentioned chiral separation by CCC. High-speed CCC (HSCCC) is a kind of CCC based on the hydrodynamic equilibrium system, which is designed using a J-type synchronous planetary motion to improve the separation efficiency. The higher retention of the stationary phase with higher flow rate was achieved by this system, rather than the I-type centrifuge and the locular CCC devices mentioned above, so this CCC system was named HSCCC. Then, a novel J-type CCC device was developed with increased rotational speed and g-force field, which finally led to better stationary-phase retention, increased efficiency and shorter run times than the initial HSCCC, so this was named high-performance CCC (HPCCC). At present, HPCCC is a powerful and widely used instrument because of its higher separation efficiency and shorter separation time than other CCC systems. In recent years, studies on chiral separation by CCC aroused considerable interest among researchers and a number of related articles were published. This review will give a brief summary of recent progress in research on applications of CCC to chiral separation, discuss the advantages and the disadvantages of the chiral separation by CCC, and highlight novel approaches and excellent examples of applications.

2. Application Recently, chiral separation by CCC saw increased activity with an increase in the number of publications. Table 1 is a brief summary of some relevant examples reported in the literature of applications of J-type CCC for chiral separation, which summarize the solvent systems, chiral selectors and racemates in the applications, because the choices of solvent system and chiral selector are

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key factors that affect separation in a chiral separation by CCC. Table 1 classifies and analyzes the published articles and the results were shown in Figs. 1–3. Figs. 1–3 show that the applications of J-type CCC to chiral separation have zoomed in the past five years. Publications in 2010–14 are the 4.67 times as many as in 2005–09. β-cyclodextrin derivatives are the chiral selectors most widely used in CCC because of their high stereoselectivity and chiral recognition ability. Meanwhile, due to a broad range of hydrophobicity, classical hexane/ethyl acetate/methanol/water (HEMWat)-based solvent systems are popular in chiral separation by CCC.

3. Advantages and disadvantages The successful application of CCC technique to chiral separation provides a new methodology for preparing single enantiomers. Compared with conventional chromatography techniques, because its free solid stationary phase and continuous liquid-liquid partition design, CCC has many distinct advantages in chiral separation. First, CCC has high sample loading capacity. Since the solid support phase is superseded by a liquid stationary phase and the target compounds can dissolve adequately in it, the injection volume of CCC in a single run is much larger than conventional chromatography techniques, such as HPLC, CE, and gas chromatography (GC). Also, the loading capacity can be further increased through some special modes, such as pH-zone-refine mode [7]. Second, CCC has a cheaper liquid stationary phase and a low cost per-unit solvent. Tong et al [20] contrasted HSCCC with HPLC in chiral separation of (±)-2-phenylpropionic acid. The results showed that, in separating the same quantity of racemate, although needing more time, solvent and chiral selector, CCC is more economical than HPLC because it does not require, first, an expensive solid chiral stationary phase and, second, as high a grade reagent as HPLC. Third, CCC uses a simple multilayer coil as column, so a suitable chiral selector and two-phase solvent system can achieve separation of a variety of chiral racemates. Fourth, the method is easy to scale up. On the same instrument, it can easily be achieved from the analytical to preparative scale by increasing the amount of chiral selector.

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Fifth, the racemate can be completely recovered, since there is no irreversible adsorption and the column requires no extensive rinsing, so CCC is a versatile, cost-economical alternative method for chiral separation. It has great potential in terms of preparative separation of racemate. Currently, chiral separation has become an important research focus in CCC. Although there are so many advantages, few applications of CCC to chiral separation were reported because of the main disadvantage – the relatively low separation efficiency of CCC. As the maximum number of theoretical plates of CCC can reach only thousands [4], it requires the chiral selector to have high enantioselectivity for the target racemate and a separation factor (α) generally greater than 1.40 [2]. However, in practice, this was rarely achieved because the α values of most chiral reagents are not quite high enough. This low separation efficiency has stunted the development and the applications of chiral separation by CCC.

4. Application of novel methods In recent years, chiral separation by CCC became increasingly attractive due to its merits, especially in preparation. More and more researchers paid attention to this field and some novel methods, such as recycling elution mode (REM), multiple dual-mode elution (MDM) and biphasic chiral recognition (BCR) were used in CCC to overcome its deficiency in chiral separation. Those novel methods made up for the low number of theoretical plates of CCC to some extent, improved the resolution of enantiomers and greatly expanded the scope of CCC in chiral separation. Below, we briefly describe those methods and their specific applications. 4.1. Recycling elution mode (REM) REM is a method previously used in preparative LC [27]. It can improve the separation factor through recycling the elution in the column, just like lengthening the column. This method has been applied in HSCCC for separation of some natural compounds [28–30] and racemates [17,20,26], which have quite low separation factors. In HSCCC, REM can be easily achieved through connecting the outlet of the detector with the inlet of the mobile-phase pump through a tube and a valve. The elution from the column returns to the suction side of the mobile-phase pump; in this way, the solutes can separated in the column again, and the resolution increases after each cycle. Because of the serious broadening of the solute peaks with the increasing number of

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cycles, this method is hardly fit for the simultaneous separation of several compounds [28]. But it is admirably suited for chiral separation because it can improve the separation efficiency and the purity for target enantiomers without requiring any extra consumption of chiral selector and solvent. In literature [17], Tong et al discussed some technical details that require attention for successful application of REM in a real HSCCC chiral separation, such as the volume of pipeline connecting the outlet of the detector with the inlet of the pump, the selection of the time to begin recycling the elution, and the air bubble in the mobile phase. Although REM in CCC indeed shows much advantage in achieving effective chiral separation, it also has some disadvantages that should not be ignored, the chief one being peak extension. Because of the liquid stationary phase of CCC, the peak extension between separation cycles in CCC is more serious than in HPLC, and limits its practical application. Consequently, the racemate should be easily dissolved in the mobile phase and the partition coefficient of enantiomers should be less than 1, so that the target solute can have a narrow peak. More separation cycles could therefore be carried out in recycling chiral separations by CCC [17]. Meanwhile, due to mixing of elution in the pump, some loss of separation efficiency occurs in passing through the pump. REM is an appropriate choice when the improvement in resolution is greater than the loss of resolution due to mixing in the pump per cycle. In practice, it means that the volume of the column must be much larger than the volume of the pump [27]. 4.2. Multiple dual-mode elution (MDM) MDM is an extension of dual-mode elution. Dual-mode CCC makes full use of the CCC characteristics of the both liquid stationary and mobile phases. During the separation process, the stationary phase changes to mobile phase and simultaneously switches the elution direction at a certain time [31]. MDM was developed on the basis of dual-mode elution and involves a series of consecutive dual-mode steps [32]. It can improve the separation factor of target solutes by eluting the solutes back and forth in the column through a series of switches in operation between normal and reversed-phase mode elution. This methodology can therefore be used in chiral separation by CCC [12,22]. Rubio et al. introduced a modified MDM method that can be used in chiral separation by HSCCC [12]. The fundamental difference between conventional MDM and this modified MDM is that, in the modified MDM, the rotation is stopped every time elution goes in the opposite direction to the initial direction. They found that modified MDM can further improve the resolution of the enantiomers compared to conventional MDM if the chiral recognition occurs only in the stationary phase. This phenomenon has also been well documented [22]. Rubio et

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al explained that it is because, in MDM elution, the redistribution of enantiomers counteracted the separation after the phase reversal but the modified MDM could be useful to minimize this unwanted effect. In MDM separation, the relative volume of the stationary and mobile phases changes when the elution mode is switched, so the enantiomers in the column are redistributed accordingly. Moreover, the ratio of chiral selector to racemate also changes due to the fresh chiral selector in the new mobile phase, and the amount of chiral selector in the new mobile phase allows the two enantiomers to react with the chiral selector. All these circumstances tend to reduce the separation efficiency of the two enantiomers, so the resolution decreases during the changed elution mode and it counteracts the separation achieved during classical elution mode [12]. Modified MDM can reduce this disadvantageous effect on separation. During the phase-inversion elution, the new mobile phase merely pushes the previous stationary phase back, so, when the second phase reversal is performed and rotation restarted, the column is filled with the initial phase ratio and the enantiomers keep on separating from the point where it was in the last cycle. This procedure simulates a physical extension of the length of the CCC column, so that the resolution can be improved accordingly [12]. 4.3. Biphasic chiral recognition (BCR) BCR is a chiral separation technique that can remarkably improve the separation factor of enantiomers by adding lipophilic and hydrophilic chiral selectors with oppositely preferential recognition direction in the organic phase and aqueous phase, respectively. It presents much stronger chiral separation ability than monophasic recognition chiral extraction and greatly improves enantioselectivity for enantiomers, since both lipophilic and hydrophilic chiral selectors simultaneously play a role in the separation. This technique has been widely used in chiral extraction and has been called biphasic recognition chiral extraction (BRCE) [33]. Normally, tartaric-acid and cyclodextrin derivatives are used as lipophilic and hydrophilic chiral selectors in the BRCE [34]. This technique was later introduced into chiral separation by HSCCC [13,24]. In contrast with other chiral CCC methods, the biphasic recognition CCC improves the separation factor of enantiomers without increasing consumption of solvent and time. It can raise the loading quantity of racemate and improve the purity of the single enantiomers obtained. However, BCR can also bring some defects. First, it is much more difficult to find two different chiral selectors with oppositely preferential recognition ability and respectively soluble in organic and aqueous phase for a given racemate than that of typical chiral CCC separation. Second, the consumption of chiral selector increases due to its presence in the mobile phase, and a further purification step is required to remove the

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chiral selector and obtain the pure enantiomers in isolated form. This may lead to more consumption of chiral selector and an extra step in the purification process [13]. Although BCR has its shortcomings, it is still an effective alternative technique for chiral CCC separation. 4.4. Comparison of the three methods The three methods described above were developed by fully utilizing their advantage of intrinsic flexibility, which is given by the liquid nature of both stationary and mobile phases in CCC. By artificially extending the length of the CCC column operationally (REM and MDM) or enhancing the enantioselectivity through cooperation of oppositely chiral recognition ability of the two chiral selectors (BCR), the separation factor of enantiomers can be improved and the deficiency of a low number of theoretical plates of CCC overcome to some extent. The developer of CCC applications can select a suitable method to meet different experimental requirements and to cope with different practices in real chiral separation. Table 2 briefly summarizes and compares the three methods.

5. Principles Some caution is needed in selecting the conditions to be employed in achieving successful chiral separation by CCC. The following are some simple principles for developing a usable chiral CCC method, as below. First, look for an effective and appropriate chiral selector, which should have a sufficiently high enantioselectivity for the target racemate. Meanwhile, it also requires the chiral selector to have good solubility in one phase of the biphasic solvent system and to be insoluble in the other phase in order to avoid leakage of chiral selector between the two phases, which would lead to a decrease in chiral separation efficiency and even cause failure in chiral separation by CCC. Second, select a suitable solvent system. Besides meeting the ordinary requirements, the solvent system should provide reasonable solubility for both chiral selector and racemate, and provide a distribution ratio and a separation time suitable for racemate. At the same time, it should have no adverse effect on the enantioselectivity of the chiral selector.

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Third, choose an appropriate stationary phase. Generally, the phase in which the chiral selector is dissolved is used as the stationary phase to save consumption of chiral selector. The enantiomers can be collected at the outlet and do not require any further purification.

6. Conclusions and perspectives Chiral separation by CCC is gaining increasing attention from researchers and is becoming more and more active and attractive. It provides a useful alternative technique for routine use in separation and purification of racemate in the laboratory to meet the demands for chiral separation in terms of speed, reproducibility and low costs. Although it has undoubtedly led to much exciting progress in recent years, chiral separation is still a challenge for CCC due to its low-efficiency separation. To promote the development of chiral CCC, research should not only focus on borrowing new ideas and new chiral selectors from other separation techniques, but also make efforts on the following aspects. First, increase separation efficiency. Continual innovation and improvement have proceeded since HSCCC instruments were developed by Ito. Lately, some useful tests were done with newly-designed CCC instruments, such as the non-synchronous preparative CCC device [35], and the assembly spiral disk and its improved design [36]. These novel designs can provide higher retention of the stationary phase and more efficient peak resolution than conventional CCC, and greatly improve the separation speed and efficiency. At the same time, some novel techniques and methods were introduced in chiral separation by CCC to enhance chiral resolution, such as REM [17], MDM [12,22] and BCR [13,24], and these have proved to have good effect, so they deserve more attention and effort in further research. Second, exploration of chiral selector. Only some kinds of chiral reagent can be successfully used in CCC for chiral separation, because they need to meet both requirements simultaneously (i.e., particular solubility in one of the liquid phases and high enantioselectivity for given target enantiomers). Usually, the selection of chiral selector refers to other chromatographic separation techniques and is not well suited to CCC, with the major trends in development of chiral selectors being towards high sensitivity and specificity. It is therefore necessary to develop a novel chiral selector for chiral separation by CCC. Ma et al [37] described some strategies for chiral selector development for HSCCC, including attaching a long hydrophobic chain to the asymmetric carbon of the chiral selector and chemically bonding the chiral selector onto small hydrophobic particles. Meanwhile, the invention of new chiral materials, {e.g., chiral ionic liquid [38], chiral polyoxometalate [39], chiral metal-organic framework [40,41], and chiral nanomaterials [42–44]} also contributed to broadening the choice of chiral selector. Novel techniques and methods of synthesis are increasingly being developed for chiral materials, demonstrating improved selectivity and other features,

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and continually deepening understanding of the chiral recognition mechanism, all of which can rapidly advance choice and create suitable chiral selectors for given enantiomers. Third, the influence of solvent systems, which paly a very important role in chiral CCC because the separation totally depends on the difference in partition coefficient of the enantiomers in two phase solvents. In a separation by CCC, selection of the solvent system is comparable to simultaneously choosing the column and the eluent in HPLC [45]. At present, the solvent system is merely treated as stationary with mobile phases in the column, but it should play a greater role. For example, Ma et al. [37] introduced the idea that the violent molecular movement of the chiral selector dissolved in the liquid stationary phase may reduce chiral selectivity based on steric affinity. They therefore recommended using a viscous stationary phase, such as an aqueous-aqueous polymer phase system to suppress the molecular movement of the chiral selector in the liquid stationary phase. In chiral HSCCC, the chiral selector is dissolved or distributed in one phase of the solvent system and this provides chiral recognition for the enantiomers. Usually, the solvent system is composed of several solvents and the different solvents may have different influences on the separation of enantiomers, such as competition for the binding on the stereo discriminating sites, change of solubility and retention of chiral selector and/or enantiomers, the solvation and complex secondary equilibria of chiral selector and/or enantiomers. The investigation of various solvents for their general behavior in chiral separations is able to induce a considerable increase of separation efficiency and is undoubtedly essential in the development of chiral CCC. Although chiral separation by CCC is still in its infancy, it has immense potential, especially in preparative applications. With improvements in instrument design and efficiency, development of new chiral selectors and knowledge of the solvent system, CCC could become a tool, routinely used in preparative chiral separation in the future. Acknowledgements Financial support of the National Natural Science Foundation of China (NSFC Nos. 20775083 and 21175142) is acknowledged. X.-Y Huang is also grateful for the financial support from CAS for him to undertake an academic visit abroad and he would also like to express his appreciation to the Advanced Bioprocessing Centre at Brunel University, UK, for affording the opportunity for him to study at Brunel. References

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to functional applications, Coordin. Chem. Rev. 257 (2013) 702-717. [40] M. Padmanaban, P. Müller, C. Lieder, K. Gedrich, R. Grünker, V. Bon, I. Senkovska, S. Baumgärtner, S. Opelt, S. Paasch, E. Brunner, F. Glorius, E. Klemm, S. Kaskel, Application of a chiral metal-organic framework in enantioselective separation, Chem. Commun. 47 (2011) 12089-12091. [41] Y. Peng, T. Gong, K. Zhang, X. Lin, Y. Liu, J. Jiang, Y. Cui, Engineering chiral porous metal-organic frameworks for enantioselective adsorption and separation, Nat. Commun. 5 (2014) DOI:10.1038/ncomms5406. [42] N. Shukla, M.A. Bartel, A.J. Gellman, Enantioselective Separation on Chiral Au Nanoparticles, J. Am. Chem. Soc. 132 (2010) 8575-8580. [43] R.J. Moon, A. Martini, J. Nairn, J. Simonsen, J. Youngblood, Cellulose nanomaterials review: structure, properties and nanocomposites, Chem. Soc. Rev. 40 (2011) 3941-3994. [44] Y. Yin, D. Talapin, The chemistry of functional nanomaterials, Chem. Soc. Rev. 42 (2013) 2484-2487. [45] J.B. Friesen, G. F. Pauli, Rational development of solvent system families in counter-current chromatography, J. Chromatogr. A 1151 (2007) 51-59.

Captions Fig. 1. Chart of chiral separation by J-type CCC publications over time. Fig. 2. Pie chart of chiral selectors used in the articles reviewed. Fig. 3. Pie chart of the families of solvent systems used in the articles reviewed.

Table 1. Chiral separations by J-type counter-current chromatography Target racemate

Solvent system

Chiral selector

Ref.

N-(3,5-dinitrobenzoyl)-(±)-phenylglycin

N-hexane/ethyl acetate/methanol/l0 mm hydrochloric acid (8:2:5:5, v/v)

N-dodecanoyl-L-proline-3,5-dimet

[6]

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e,

N-hexane/ethyl acetate/methanol/l0 mm hydrochloric acid (6:4:5:5, v/v)

N-(3,5-dinitrobenzoyl)-(±)-phenylalanin

(for preparative separation of N-(3,5-dinitrobenzoyl)-(±)-leucine) and

e, N-(3,5-dinitrobenzoyl)-(±)-valine and

methyl tert-butyl ether/water (for pH-zone-refining CCC) [7]

hylanilide

[7]

N-(3,5-dinitrobenzoyl)-(±)-leucine 7-des-Methyl-ormeloxifene

Ethyl acetate / methanol/ triethylammonium acetate buffer (10:3:7, v/v)

Β-cyclodextrin

[8]

Gemifloxacin

N-butanol/ethyl-acetate/20 mm

(+)-(18-crown-6)-tetracarboxylic

[9]

bis(2-hydroxyethyl)aminotris(hydroxymethyl) methane acetate buffer

acid

(6:5:10, v/v) Chlorpheniramine

Ethyl acetate/methanol/water (10:1:9, v/v)

Carboxymethyl‐β‐cyclodextrin

[10]

Α‐methylbenzylamine

Chloroform/methanol/water (4:3:1, v/v)

L‐(+)‐tartaric acid

[11]

(±)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydr

*n-hexane/ethyl acetate/methanol/water (9:1:9:1, v/v) and

N,N-diethyl-(S)-naproxenamide

[12]

ophenanthren-4-yl)-3,5-dinitrobenzami

Methyl tert-butyl ether/50 mm phosphate buffer (ph6.0)

(−)-2-ethylhexyl tartrate and

[13]

de and N-(3,5-dinitrobenzoyl)-(±)-leucine Α-cyclohexylmandelic

N-hexane/methyl tert-butyl ether/water (9:1:10, v/v)

hydroxypropyl-β-cyclodextrin were respectively employed as lipophilic and hydrophilic chiral selectors N-(3,5-dinitrobenzoyl)-(±)-leucine and

Ethoxynonafluorobutane/isopropanol/water (25:35:40, v/v)

N-(3,5-dinitrobenzoyl)-tert-butyl-(±)-leu

N-perfluoroundecanoyl-L-proline-

[14]

3,5-dimethylanilide

cinamide Lomefloxacin hydrochloride

Ethyl acetate/methanol/water (10:1:10, v/v)

Sulfated-β-cyclodextrin

[15]

Ofloxacin

Ethyl acetate/methanol/water (10:1:9, v/v)

L-(+)-tartaric acid

[16]

Hydroxypropyl-β-cyclodextrin

[17]

(R, S)-naproxen

−1

N-hexane/ethyl acetate/0.1 mol L

phosphate buffer solution (ph2.67)

(8:2:10, v/v)

Page 15 of 17

Phenylsuccinic acid

−1

N-hexane/methyl tert-butyl ether/0.1 mol l

phosphate buffer solution

Hydroxypropyl-β-cyclodextrin

[18]

Di-n-hexyl l-tartrate

[19]

Hydroxypropyl-β-cyclodextrin

[20]

Di-n-butyl l-tartrate

[21]

Hydroxypropyl-β-cyclodextrin

[22]

[23]

(ph2.51) (0.5:1.5:2, v/v) Propranolol, pindolol and alprenolol

−1

Chloroform/0.05 mol l

−1

acetate buffer containing 0.10 mol l

boric acid

(1:1, v/v), 2-phenylpropionic acid

−1

N-hexane/ethyl acetate/0.1 mol l

phosphate buffer solution ph2.67

(5:5:10 for isocratic elution and 8:2:10 for recycling elution, v/v) Propafenone

Chloroform/0.05 mol l

−1

acetate buffer pH 3.4 (1:1, v/v) −1

2-(6-methoxy-2-naphthyl)propionic

N-hexane/ethyl acetate/0.1 mol L

acid (NAP) and 2-phenylpropionic

for NAP and 7:3:10 for 2-PPA, v/v)

phosphate buffer pH 2.67 (7.5:2.5:10

acid(2-PPA) Trans-δ-viniferin

N-hexane/ethyl acetate/water (5:5:10, v/v)

Hydroxypropyl-β-cyclodextrin

Phenylsuccinic acid

N-hexane/methyl tert-butyl ether/water (0.5:1.5:2, v/v)

D-isobutyl

tartrate

and

[24]

hydroxypropyl-β-cyclodextrin were respectively

employed

as

lipophilic and hydrophilic chiral selectors Aromatic α -hydroxyl acids Oxybutynin

N-butanol/water (1:1, v/v) or hexane/n-butanol/water (0.5:0.5:1, v/v) −1

N-hexane/methyl tert-butyl ether/0.1 mol l

phosphate buffer solution pH

N-n-dodecyl-l-proline

[25]

Hydroxypropyl-β-cyclodextrin

[26]

5.0 (6:4:10, v/v) * In this article, two different solvent systems were used to achieve the chiral separation of the two racemates, respectively: n-hexane/ethyl acetate/methanol/water for ((±)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide and methyl t-butyl ether (MTBE)/phosphate buffer for N-(3,5-dinitrobenzoyl)-(±)-leucine.

Page 16 of 17

Table 2. Brief summary and comparison of the three methods Item REM MDM

BCR

Chiral selector

Mainly distributed in

Mainly distributed in

Two different chiral

distribution

organic or aqueous

organic or aqueous

selectors added in

phase

phase

organic and aqueous phase respectively

The way improve the

The enantiomers

The enantiomers

The enantiomers

separation factor of

eluted with the

eluted back and forth

respectively bind with

enantiomers

recycling mobile

in the column through

preferential

phase through a

the repeated switch

recognition chiral

closed loop

of phase role and

selector in different

Merit

circulation direction

phases

Improve the

Improve the

Improve the

resolution of

resolution of

resolution of

enantiomers; without

enantiomers

enantiomers; without

extra consumption of

extra consumption of

solvent and chiral

solvent and time

selector Defect

Extend the

Extend the

Require two different

separation time with

separation time with

chiral selectors; need

the recycling cycles;

the switched steps;

further purification

serious peak

increase

process

extension

consumption of solvent and chiral selector

The scope of

Not merely in chiral

Not merely in chiral

application

separation

separation

Chiral separation

Page 17 of 17