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ISSN 1615-9306 · JSSCCJ 38 (18) 3119–330{ (2015) · Vol. 38 · No. 18 · September 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
18 15
CE-MS interlaboratory study
Methods Chromatography · Electroseparation
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Applications Biomedicine · Foods · Environment
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Jian Tang Limin Pang Jie Zhou Weihua Tang College of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, China Received June 14, 2015 Revised June 19, 2015 Accepted June 21, 2015
Research Article
Enantioseparation tuned by solvent polarity on a -cyclodextrin clicked chiral stationary phase The efficient enantioseparation of 26 racemates has been achieved with the perphenylcarbamoylated cyclodextrin clicked chiral stationary phase by screening the optimum composition of mobile phase in high-performance liquid chromatography. The chromatographic results indicate that both the retention and chiral resolution of racemates are closely related to the polarity of the mobile phases and the structures of analytes. The addition of alcohols can significantly tune the enantioseparation in normal-phase high-performance liquid chromatography. The addition of methanol and the ratio of ethanol/methanol or isopropanol/methanol played a key role on the resolution of flavonoids in ternary eluent systems. The chiral separation of flavonoids with pure organic solvent as mobile phase indicates the preferential order for chiral resolution is methanol>ethanol>isopropanol> n-propanol>acetonitrile. Keywords: Chiral separation / Chiral stationary phases / Cyclodextrins / Highperformance liquid chromatography DOI 10.1002/jssc.201500630
1 Introduction Chiral purity plays a significant role in the interest of pharmaceuticals, agrochemical, and foods, since the pair of enantiomers may exhibit different properties [1,2]. The enantioseparation on chiral stationary phases (CSPs) in HPLC represents one of the most facile and direct methods for chiral analysis. To date, cyclodextrin (CDs, e.g., ␣-, -, or ␥-CD) based chemically bonded CSPs have received prominent attention due to their utility and multimodal adaptability to wide range of chromatographic conditions. The conventional chemical immobilization approaches are less effective due to their poor selectivity of reacting functional groups with pretreated silica, resulting in low enantioselectivities for the resultant CSPs [3]. Cu-catalyzed “click chemistry”, however, shows high selectivity and good tolerance in a large variety of solvent systems [4]. Moreover, 100% conversion can be achieved for the reaction between acetylene and azide groups on surfaces of powdered silica [5]. Click chemistry was successfully introduced into the preparation of CSPs for chromatographic enantioseparations [6–8]. Ng’s group reported the “click” preparation Correspondence: Dr. Weihua Tang, College of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China E-mail: [email protected]
Abbreviations: ACN, acetonitrile; CSPs, chiral stationary phases; EtOH, ethanol; Hex, n-hexane; IPA, isopropanol; MeOH, methanol; NP, normal phase; NPA, n-propanol; i-Oct, isooctane C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of -CD CSPs for enantioseparation of over 40 pairs of enantiomers [9] and a library of neutral and basic drugs in HPLC [10]. The perphenylcarbamoylated -CD clicked CSP with high column efficiency was further developed for the chiral separation of aryl alcohols, flavonoids, adrenergic drugs and other pharmaceuticals in RP-HPLC [11] by taking advantage of potential interactions between them (e.g. hydrogen bonding, steric repulsion, – complexation, dipole–dipole staking, etc.) [12]. In normal-phase (NP) HPLC, the inclusion complexation is absent. This addition of organic modifiers (e.g. alcohols and bases) into the mobile phases, however, can effectively modulate the polarity of CSPs and thus the interactions between CSPs and analytes [11–13]. The enantioselectivities of perphenylcarbamoylated -CD clicked CSP in RP-HPLC have been systemically evaluated by our group. And excellent chiral resolutions were achieved for various flavonoids and aryl alcohols [11]. The chloromethylphenylcarbamated -CD clicked CSPs were further developed to explore their functionalities tuned enantioselectivities [14]. Aiming to explore the versatility and full potential of these perphenylcarbamoylated -CD clicked CSPs for enantioseparation in all range of HPLC modes, we herein explore the enantioselectivity of perphenylcarbamoylated -CD clicked CSP towards 26 model racemates in polar solvents modified NP and polar-organic HPLC. The chiral recognition mechanism with the -CD clicked CSP at different elution modes was explained by correlating the enantioseparation results of racemates with the composition of mobile phases. The solvent polarity of the mobile phase was found to significantly tune the enantioselectivity of CSP. www.jss-journal.com
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Figure 1. Racemates and CD clicked CSP used in this study.
2 Materials and methods 2.1 Materials HPLC-grade solvents including acetonitrile (ACN), methanol (MeOH), ethanol (EtOH), isopropanol (IPA), n-propanol (NPA), n-hexane (Hex) and isooctane (i-Oct) were purchased from Tedia (Phoenix, USA). HPLC-grade TFA and triethylamine (TEA) were obtained from J&K (Shanghai, China). The structures of racemates are depicted in Fig. 1. ArylOH 6, Aryl-OH 10, atenolol, propranolol, isoprenaline and coumachlor were procured from Sigma–Aldrich (St. Louis, MO, USA). Aryl-OH-1, Aryl-OH-2, Aryl-OH-4, Aryl-OH-5, Aryl-OH-7, Aryl-OH-8, Aryl-OH-9, Aryl-OH-11, Aryl-OH-12, 7-methoxyflavanone, naringenin, hesperdin, benzoin and
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4-chlorobenzoin were purchased from J&K (Shanghai, China). The other racemates were obtained from Meryer (Shanghai, China). Deionized water for the experiments was purified by using a Milli-Q system (Millipore, Bedford, MA, USA). 2.2 Preparation of CSP The mono-6A -azido-mono-deoxy-phenylcarbamoylated -CD derivative was prepared according to ref. [9]. Subsequently, the -CD derivative was immobilized onto alkynylfunctionalized silica by “click” chemistry. The clicked CSP was packed into stainless-steel columns (4.6 × 250 mm) with constant pressure (9000 psi) using a packing system (Lab Alliance Scientific)[11]. The column efficiency was determined
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to be 12 000 by using toluene as marker sample and MeOH as mobile phase. The minimum plate height was determined as 20.9 m, corresponding to a theoretical plate number of 48 000 plates/m. 2.3 HPLC procedures Enantioseparations were performed on an Agilent 1260 system, which consisted a G1315D diode array detection (DAD) system, a G1329B quaternary pump, a G1331C automatic injector, a G1316A temperature controller and Agilent Chem Station data manager software (Agilent Technologies, Palo Alto, CA, USA). All separations were performed at 25⬚C unless otherwise specified. The mobile phases for NP mode HPLC were Hex or i-Oct mixed with different alcohols. All solvents and mobile phases were filtered through 0.22 m filter membrane and degassed under ultrasonication before use. The detection wavelength range was 200–300 nm. The sample solutions in IPA were prepared in a concentration of 1–5 mg/mL and stored at 4⬚C. Sample solutions may be further diluted with IPA to 100–500 g/mL before use.
3 Results and discussion 3.1 Enantioseparation in binary eluent systems NP-HPLC In NP-HPLC, the non-polar solvent used in mobile phase can greatly influence the enantioseparations [13, 15]. Two commonly used solvents including Hex and i-Oct were compared (Table 1). For all aryl alcohols, the selectivity and resolution of analytes obtained with Hex/IPA mobile phase were higher than those using i-Oct/IPA. Considered both enantioselectivity and retention time, Hex seemed to be more versatile in tuning the enantioselectivities of the -CD clicked CSP in NP-HPLC. The effect of flow rate of mobile phase on the enantioseparation was demonstrated with flavanone. As shown in Fig. 2, the chiral resolution (Rs ) of flavanone generally increased when the flow rate of mobile phase decreased from 1.4 to 0.2 mL/min, meanwhile longer retention times were observed. The highest Rs of 5.7 was obtained at the flow rate of 0.4 mL/min, while the maximum enantioselectivity (␣) was achieved at 0.8 mL/min. Considering both analysis time and enantioselectivity, flow rate of 1.0 mL/min was chosen for the following study. The effect of column temperature was also examined by taking flavanone and 7-methoxyflavanone as model analytes. As shown in Fig. 3, both racemates exhibited shorter retention with column temperature increased from 10 to 50⬚C. Both of them achieved higher enantioselectivities at lower column temperature (ca. 10⬚C). However, due to the strong peak tailing occurring at low temperature, the maximum Rs of 4.86 and 6.8 was obtained for flavanone at 20⬚C and 7methoxyflavanone at 30⬚C.
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The type of organic modifier is one of the key factors affecting the chiral separation in HPLC [16–18]. It increases the polarity and the density of the mobile phase. As a consequence of its adsorption, the incorporation of alcohol modifiers of different steric size/shape into the chiral cavities of -CD CSPs may result in different chiral discrimination environments, leading to the change of chiral recognition mechanism [19]. Three alcohols including IPA, EtOH and NPA were selected to modulate the polarity of mobile phase. As summarized in Table 1, for the enantioseparation of aryl-alcohols using 20% organic modifiers added to Hex mobile phases, the preferential order for selectivity and resolution was IPA>EtOH>NPA. The retention and resolutions of most racemates were higher with EtOH than those with NPA, probably ascribed to long straight-chain alcohols easily formed cyclic tetramer by OH– OH aggregation [20, 21]. The larger NPA molecules are easier to occupy -CD cavity, resulting in enhanced ability to replace enantiomers. The retention factors of enantiomers thus decreased with NPA in comparison to EtOH. In addition, the longer retention times of enantiomers with IPA in the eluent were probably due to its lower polarity than NPA and EtOH. 3.2 Enantioseparation with methanol as additive for ternary solvent systems in NP-HPLC Methanol is not often used in mobile phase for NP-HPLC due to its high polarity and slight solubility in Hex. Since methanol is miscible with both Hex/EtOH and Hex/IPA, the addition of proper amount of methanol can modulate the polarity of mobile phase as well as the enantioseparations. As summarized in Table 2, for aryl alcohols, their chiral resolutions decreased with the addition of MeOH in comparison to the excellent resolution in binary system (Table 1). However, the ternary eluent system of Hex/IPA/MeOH was favorable for the enantioseparation of flavonoids and coumachlor. They obtained higher Rs values with the addition of methanol in comparison to those obtained in corresponding binary systems (see Fig. 4). Since methanol is a strong polarity regulator, the addition of methanol may enhance the formation of inclusion complexation of enantiomers with -CD cavity, leading to improved resolutions of flavonoids and coumachlor. The Rs of 7-methoxyflavanone was achieved as high as 8.1 with Hex/EtOH/MeOH and 7.6 with Hex/IPA/MeOH. Moreover, benzoin and 4-chlorobenzoin that were not resolvable in either Hex/IPA or Hex/EtOH were partially enantioseparated in Hex/IPA/MeOH. The addition of a proper amount of methanol in mobile phase would enhance or even enable the chiral separation. The composition of Hex/IPA/MeOH and Hex/EtOH/MeOH (Hex content fixed as 70% by volume) was further adjusted for the optimization of enantioseparation of flavonoids (Table 2). The retention times of enantiomers were found to decrease dramatically with methanol content increasing from 5 to 25%. The increased eluting ability of mobile phase
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Table 1. Enantioseparation of racemates in different binary eluent HPLC
Compounds
t1
t2
␣
Rs
Mobile phase
Aryl-OH-1
4.51 4.26 4.67 4.62 4.32 4.36 4.72 4.86 4.35 4.42 4.95 5.19 4.47 4.56 5.23 4.53 4.07 4.06 4.68 3.93 4.09 4.52 4.07 4.01 4.68 5.32 5.54 4.86 5.39 4.85 4.72 5.41 5.34 4.87 5.64 5.85 4.75 4.65 5.00 12.35 6.99 6.58 7.41 8.40 5.67 5.66 6.01 11.99 6.35 6.57 9.69 7.23 7.63 7.502 4.89 4.09 3.98 4.52
4.77 4.41 4.92 5.35 4.72 4.58 5.32 6.11 5.11 4.89 5.78 7.02 5.34 5.34 6.22 6.06 4.89 4.60 5.77 4.01 4.19 5.97 4.87 4.47 5.75 5.47 5.59 5.17 9.62 6.26 6.28 10.24 5.52 4.96 5.83 9.89 6.13 5.74 6.52 27.84 12.72 11.86 13.83 16.66 8.31 7.94 8.92 12.63 6.74 7.04 14.16 8.97 9.79 9.236 5.81 4.56 4.25 5.16
1.71 1.56 1.17 2.20 1.85 1.67 1.39 2.68 2.27 1.94 1.47 3.13 2.37 2.26 1.48 3.03 2.42 2.05 1.73 1.39 1.11 2.96 2.38 1.94 1.72 1.81 1.02 1.83 4.94 2.82 2.99 3.17 1.85 1.66 1.08 2.51 1.96 1.73 1.68 2.69 2.55 2.54 2.38 2.58 2.11 1.91 1.90 1.07 1.13 1.12 1.69 1.44 1.48 1.37 1.54 1.48 1.45 1.50
1.5 1.1 1.3 4.1 2.7 1.2 3.0 5.9 5.2 2.4 4.3 6.4 5.3 2.9 5.5 6.9 5.4 3.0 5.4 0.6 0.5 5.8 4.4 3.7 4.2 0.6 0.3 0.2 6.9 5.9 4.1 5.5 0.7 0.5 0.6 4.2 4.9 3.8 4.3 4.2 6.8 5.6 6.3 4.0 5.6 4.3 5.2 0.4 0.8 0.9 1.9 2.5 2.1 2.2 2.1 1.7 1.6 1.6
Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (50:50, v/v) Hex:EtOH (50:50, v/v) Hex:NPA (50:50, v/v) i-Oct:IPA (50:50, v/v)
Aryl-OH-2
Aryl-OH-3
Aryl-OH-4
Aryl-OH-5
Aryl-OH-6 Aryl-OH-7
Aryl-OH-8 Aryl-OH-9 Aryl-OH-11
Aryl-OH-12
Flavanone
7-Methoxyflavanone
6-Methoxyflavanone
7-Hydroxyflavanone
4-Hydroxyflavanone
Coumachlor
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Figure 2. Effect of flow rate on enantioseparation of flavanone; temperature: 25⬚C; mobile phase: Hex/EtOH = 70:30 v/v.
Figure 3. Temperature-dependent enantioseparation of flavanone and 7-methoxyflavanone with 1 mL/min Hex/EtOH (70:30, v/v).
was attributed to the increased solvent polarity with the addition of methanol. The optimum enantioseparation of 7-methoxyflavanone (Rs = 8.0) and 6-methoxyflavanone (Rs = 6.6) was obtained with Hex/IPA/methanol (70:5:25, v/v/v). 7-Hydroxyflavanone and 4-hydroxyflavanone, however, achieved their best resolution with Hex/IPA/MeOH (70:10:20, v/v/v). All flavanoids exhibited their best resolution with Hex/EtOH/MeOH (70:15:15, v/v/v) when EtOH was used in ternary eluent systems. The representative enantioseparation chromatograms of selected racemates in different eluent systems are summarized in Fig. 5. The enantioseparation of isoprenaline, propranolol, atenolol, and clenbuterol was unsuccessful even with ternary solvent system (Table 1). Partial separation was achieved with the addition of TFA and TEA. The rather low Rs may be attributed to the presence of -chiral center in propranolol and atenolol or the bulky aromatic moieties, resulting in weaker intermolecular interactions. The steric hindrance of bulky aromatic moieties may also account for the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
low enantioseparation of naringenin and hesperetin in NPHPLC. The addition of organic modifiers, therefore, could alter the polarity of mobile phases, resulting in a change of the interactions between CD CSP and enantiomers. The enantioselectivities of CSP were thus tuned by the mobile phases.
3.3 Polar-organic-mode HPLC The capacity of this CSP was finally investigated in polar organic HPLC using protic solvents including MeOH, EtOH, NPA and IPA as well as aprotic solvent ACN as mobile phase [22]. In NP-HPLC, -CD cavity would be occupied by the non-polar solvent and the inclusion complexation thus rarely took place, and the chiral recognition was mainly produced by the interactions of analytes with functional groups at -CD rims. In polar-organic and RP modes, the inclusion complexation exists as an important driving force for enantioseparation. Moreover, the -CD clicked CSP www.jss-journal.com
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Table 2. Methanol tuned enantioseparation of racemates in NPLC
Analytes
t2
␣
Rs
Hex:IPA:MeOH
t2
␣
Rs
Hex:EtOH:MeOH
Aryl-OH-2 Aryl-OH-3 Aryl-OH-4 Aryl-OH-5 Aryl-OH-7 Aryl-OH-11 Flavanone
Hesperetin Naringenin Coumachlor Benzoin 4-Chlorobenzoin Isoprenaline
5.12 4.62 5.14 4.50 4.59 6.50 7.40 6.66 6.22 6.06 5.96 16.28 13.11 11.34 10.64 10.05 10.55 9.01 8.23 7.94 7.74 7.03 6.46 6.44 6.17 6.28 10.98 10.04 9.42 9.14 8.91 27.01 19.44 4.50 6.62 17.61 85.16
2.16 1.69 2.18 1.75 1.80 2.90 2.55 2.39 2.40 2.37 2.77 2.64 2.51 2.45 2.41 2.46 2.43 2.29 2.28 2.26 2.40 1.22 1.19 1.16 1.18 1.19 1.68 1.63 1.58 1.56 1.46 1.07 1.05 1.45 1.05 1.05 1.06
IPA>NPA. It was noteworthy that mobile phase of pure polar organic solvent enabled the chiral separation of flavonoids without the aid of any additive. To get some insight for this phenomenon, aprotic solvent ACN was used as reference. Only partial separation was achieved for all analytes except 7-methoxyflavanone. The
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Figure 4. Enhanced chiral resolutions of flavonoids with MeOH added ternary eluent HPLC.
Figure 5. Representative enantioseparation chromatograms of the studied racemates.
poor resolution may be explained with the aprotic solvent nature of ACN. Since the C–H bonds of ACN have a very high pKa value, it tended to form solvent clusters instead of hydrogen bond network. By comparison, one can reasonably conclude hydrogen bonding formed with protic solvents is beneficial for the chiral resolution of racemates in HPLC. Based on the above discussion, the polarity tuning of mobile phase using polar solvents in polar organic HPLC would not only affect the inclusion complexation but also the interactions between CD CSP and enantiomers. Figure 6. Enantioseparation of flavonoids in polar-organic-mode HPLC.
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4 Concluding remarks
[6] Marechal, A., El-Debs, R., Dugas, V., Demesmay, C. J. Sep. Sci. 2013, 36, 2049–2062.
The enantioselectivity of the perphenylcarbamoylated -CD clicked CSP has been successfully explored for 26 racemates by tuning the polarity of eluent system for both normal-phase and polar-organic HPLC. The enantioseparation properties using this perphenylcarbamoylated -CD clicked CSP were compared and discussed by correlating analyte structures and chromatographic conditions. Satisfactory enantioseparation of most racemates was achieved within 10 min. The addition of methanol can significantly improve the enantioseparation of flavonoids in ternary systems.
[7] Chu, C., Liu, R. Chem. Soc. Rev. 2011, 40, 2177–2188.
We gratefully acknowledged the financial support from the National Natural Science Foundation of China (grant no. 21305066), Program for New Century Excellent Talents in University (NCET-12-0633), Doctoral Fund of Ministry of Education of China (no. 20103219120008), the Jiangsu Province Natural Science Fund for Distinguished Young Scholars (BK20130032), and the Fundamental Research Funds for the Central Universities (30920130111006). The authors have declared no conflict of interests.
[8] Guo, Z., Lei, A., Liang, X., Xu, Q. Chem. Commun. 2006, 43, 4512–4514. [9] Wang, Y., Young, D. J., Tan, T. T., Ng, S. C. J. Chromatogr. A 2010, 1217, 7878–83. [10] Zhang, S., Wang, H., Tang, J., Wang, W., Tang, W. Anal. Methods 2014, 6, 2034–2037. [11] Pang, L., Zhou, J., Tang, J., Ng, S.-C., Tang, W. J. Chromatogr. A 2014, 1363, 119–127. [12] X. H. Lai, W.H. Tang, S.-C. Ng, J. Chromatogr. A 2011, 1218, 5597–5601. ¨ [13] Lammerhofer, M. J. Chromatogr. A 2010, 1217, 814–856. [14] Tang, J., Zhang, S., Lin, Y., Jie Zhou, Pang, L., Nie, X., Zhou, B., Tang, W. Sci. Rep. 2015, 5, 11523. doi:10.1038/srep11523. [15] Lai, X. H., Tang, W., Ng, S.-C., J. Chromatogr. A 2011, 1218, 3496–3501. [16] Perrin, C., Vu, V.A., Matthijs, N., Maftouh, M., Massart, D.L., Vander Heyden, Y., J. Chromatogr. A 2002, 947, 69– 83. [17] Ahmed, K. S., Tazerouti, F., Badjah-Hadj-Ahmed, A., Meklati, B., Chromatographia 2005, 62, 571–579. [18] Aboul-Enein, H. Y. J. Sep. Sci. 2003, 26, 521–524.
5 References
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[22] Ong, T.T., Tang, W., Wayan, I. W., Ng, S.-C.; Chan, H. S. O., Electrophoresis 2005, 26, 3839–3848. [23] Armstrong, D. W., Stalcup, A. M., Hilton, M. L., Duncan, J. D., Faulkner Jr, J. R., Chang, S. C., Anal. Chem. 1990, 62, 1610–1615.
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ISSN 1615-9306 · JSSCCJ 38 (18) 3119–330{ (2015) · Vol. 38 · No. 18 · September 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
18 15
CE-MS interlaboratory study
Methods Chromatography · Electroseparation
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Applications Biomedicine · Foods · Environment
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Jian Tang Limin Pang Jie Zhou Weihua Tang College of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, China Received June 14, 2015 Revised June 19, 2015 Accepted June 21, 2015
Research Article
Enantioseparation tuned by solvent polarity on a -cyclodextrin clicked chiral stationary phase The efficient enantioseparation of 26 racemates has been achieved with the perphenylcarbamoylated cyclodextrin clicked chiral stationary phase by screening the optimum composition of mobile phase in high-performance liquid chromatography. The chromatographic results indicate that both the retention and chiral resolution of racemates are closely related to the polarity of the mobile phases and the structures of analytes. The addition of alcohols can significantly tune the enantioseparation in normal-phase high-performance liquid chromatography. The addition of methanol and the ratio of ethanol/methanol or isopropanol/methanol played a key role on the resolution of flavonoids in ternary eluent systems. The chiral separation of flavonoids with pure organic solvent as mobile phase indicates the preferential order for chiral resolution is methanol>ethanol>isopropanol> n-propanol>acetonitrile. Keywords: Chiral separation / Chiral stationary phases / Cyclodextrins / Highperformance liquid chromatography DOI 10.1002/jssc.201500630
1 Introduction Chiral purity plays a significant role in the interest of pharmaceuticals, agrochemical, and foods, since the pair of enantiomers may exhibit different properties [1,2]. The enantioseparation on chiral stationary phases (CSPs) in HPLC represents one of the most facile and direct methods for chiral analysis. To date, cyclodextrin (CDs, e.g., ␣-, -, or ␥-CD) based chemically bonded CSPs have received prominent attention due to their utility and multimodal adaptability to wide range of chromatographic conditions. The conventional chemical immobilization approaches are less effective due to their poor selectivity of reacting functional groups with pretreated silica, resulting in low enantioselectivities for the resultant CSPs [3]. Cu-catalyzed “click chemistry”, however, shows high selectivity and good tolerance in a large variety of solvent systems [4]. Moreover, 100% conversion can be achieved for the reaction between acetylene and azide groups on surfaces of powdered silica [5]. Click chemistry was successfully introduced into the preparation of CSPs for chromatographic enantioseparations [6–8]. Ng’s group reported the “click” preparation Correspondence: Dr. Weihua Tang, College of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China E-mail: [email protected]
Abbreviations: ACN, acetonitrile; CSPs, chiral stationary phases; EtOH, ethanol; Hex, n-hexane; IPA, isopropanol; MeOH, methanol; NP, normal phase; NPA, n-propanol; i-Oct, isooctane C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of -CD CSPs for enantioseparation of over 40 pairs of enantiomers [9] and a library of neutral and basic drugs in HPLC [10]. The perphenylcarbamoylated -CD clicked CSP with high column efficiency was further developed for the chiral separation of aryl alcohols, flavonoids, adrenergic drugs and other pharmaceuticals in RP-HPLC [11] by taking advantage of potential interactions between them (e.g. hydrogen bonding, steric repulsion, – complexation, dipole–dipole staking, etc.) [12]. In normal-phase (NP) HPLC, the inclusion complexation is absent. This addition of organic modifiers (e.g. alcohols and bases) into the mobile phases, however, can effectively modulate the polarity of CSPs and thus the interactions between CSPs and analytes [11–13]. The enantioselectivities of perphenylcarbamoylated -CD clicked CSP in RP-HPLC have been systemically evaluated by our group. And excellent chiral resolutions were achieved for various flavonoids and aryl alcohols [11]. The chloromethylphenylcarbamated -CD clicked CSPs were further developed to explore their functionalities tuned enantioselectivities [14]. Aiming to explore the versatility and full potential of these perphenylcarbamoylated -CD clicked CSPs for enantioseparation in all range of HPLC modes, we herein explore the enantioselectivity of perphenylcarbamoylated -CD clicked CSP towards 26 model racemates in polar solvents modified NP and polar-organic HPLC. The chiral recognition mechanism with the -CD clicked CSP at different elution modes was explained by correlating the enantioseparation results of racemates with the composition of mobile phases. The solvent polarity of the mobile phase was found to significantly tune the enantioselectivity of CSP. www.jss-journal.com
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Figure 1. Racemates and CD clicked CSP used in this study.
2 Materials and methods 2.1 Materials HPLC-grade solvents including acetonitrile (ACN), methanol (MeOH), ethanol (EtOH), isopropanol (IPA), n-propanol (NPA), n-hexane (Hex) and isooctane (i-Oct) were purchased from Tedia (Phoenix, USA). HPLC-grade TFA and triethylamine (TEA) were obtained from J&K (Shanghai, China). The structures of racemates are depicted in Fig. 1. ArylOH 6, Aryl-OH 10, atenolol, propranolol, isoprenaline and coumachlor were procured from Sigma–Aldrich (St. Louis, MO, USA). Aryl-OH-1, Aryl-OH-2, Aryl-OH-4, Aryl-OH-5, Aryl-OH-7, Aryl-OH-8, Aryl-OH-9, Aryl-OH-11, Aryl-OH-12, 7-methoxyflavanone, naringenin, hesperdin, benzoin and
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4-chlorobenzoin were purchased from J&K (Shanghai, China). The other racemates were obtained from Meryer (Shanghai, China). Deionized water for the experiments was purified by using a Milli-Q system (Millipore, Bedford, MA, USA). 2.2 Preparation of CSP The mono-6A -azido-mono-deoxy-phenylcarbamoylated -CD derivative was prepared according to ref. [9]. Subsequently, the -CD derivative was immobilized onto alkynylfunctionalized silica by “click” chemistry. The clicked CSP was packed into stainless-steel columns (4.6 × 250 mm) with constant pressure (9000 psi) using a packing system (Lab Alliance Scientific)[11]. The column efficiency was determined
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to be 12 000 by using toluene as marker sample and MeOH as mobile phase. The minimum plate height was determined as 20.9 m, corresponding to a theoretical plate number of 48 000 plates/m. 2.3 HPLC procedures Enantioseparations were performed on an Agilent 1260 system, which consisted a G1315D diode array detection (DAD) system, a G1329B quaternary pump, a G1331C automatic injector, a G1316A temperature controller and Agilent Chem Station data manager software (Agilent Technologies, Palo Alto, CA, USA). All separations were performed at 25⬚C unless otherwise specified. The mobile phases for NP mode HPLC were Hex or i-Oct mixed with different alcohols. All solvents and mobile phases were filtered through 0.22 m filter membrane and degassed under ultrasonication before use. The detection wavelength range was 200–300 nm. The sample solutions in IPA were prepared in a concentration of 1–5 mg/mL and stored at 4⬚C. Sample solutions may be further diluted with IPA to 100–500 g/mL before use.
3 Results and discussion 3.1 Enantioseparation in binary eluent systems NP-HPLC In NP-HPLC, the non-polar solvent used in mobile phase can greatly influence the enantioseparations [13, 15]. Two commonly used solvents including Hex and i-Oct were compared (Table 1). For all aryl alcohols, the selectivity and resolution of analytes obtained with Hex/IPA mobile phase were higher than those using i-Oct/IPA. Considered both enantioselectivity and retention time, Hex seemed to be more versatile in tuning the enantioselectivities of the -CD clicked CSP in NP-HPLC. The effect of flow rate of mobile phase on the enantioseparation was demonstrated with flavanone. As shown in Fig. 2, the chiral resolution (Rs ) of flavanone generally increased when the flow rate of mobile phase decreased from 1.4 to 0.2 mL/min, meanwhile longer retention times were observed. The highest Rs of 5.7 was obtained at the flow rate of 0.4 mL/min, while the maximum enantioselectivity (␣) was achieved at 0.8 mL/min. Considering both analysis time and enantioselectivity, flow rate of 1.0 mL/min was chosen for the following study. The effect of column temperature was also examined by taking flavanone and 7-methoxyflavanone as model analytes. As shown in Fig. 3, both racemates exhibited shorter retention with column temperature increased from 10 to 50⬚C. Both of them achieved higher enantioselectivities at lower column temperature (ca. 10⬚C). However, due to the strong peak tailing occurring at low temperature, the maximum Rs of 4.86 and 6.8 was obtained for flavanone at 20⬚C and 7methoxyflavanone at 30⬚C.
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The type of organic modifier is one of the key factors affecting the chiral separation in HPLC [16–18]. It increases the polarity and the density of the mobile phase. As a consequence of its adsorption, the incorporation of alcohol modifiers of different steric size/shape into the chiral cavities of -CD CSPs may result in different chiral discrimination environments, leading to the change of chiral recognition mechanism [19]. Three alcohols including IPA, EtOH and NPA were selected to modulate the polarity of mobile phase. As summarized in Table 1, for the enantioseparation of aryl-alcohols using 20% organic modifiers added to Hex mobile phases, the preferential order for selectivity and resolution was IPA>EtOH>NPA. The retention and resolutions of most racemates were higher with EtOH than those with NPA, probably ascribed to long straight-chain alcohols easily formed cyclic tetramer by OH– OH aggregation [20, 21]. The larger NPA molecules are easier to occupy -CD cavity, resulting in enhanced ability to replace enantiomers. The retention factors of enantiomers thus decreased with NPA in comparison to EtOH. In addition, the longer retention times of enantiomers with IPA in the eluent were probably due to its lower polarity than NPA and EtOH. 3.2 Enantioseparation with methanol as additive for ternary solvent systems in NP-HPLC Methanol is not often used in mobile phase for NP-HPLC due to its high polarity and slight solubility in Hex. Since methanol is miscible with both Hex/EtOH and Hex/IPA, the addition of proper amount of methanol can modulate the polarity of mobile phase as well as the enantioseparations. As summarized in Table 2, for aryl alcohols, their chiral resolutions decreased with the addition of MeOH in comparison to the excellent resolution in binary system (Table 1). However, the ternary eluent system of Hex/IPA/MeOH was favorable for the enantioseparation of flavonoids and coumachlor. They obtained higher Rs values with the addition of methanol in comparison to those obtained in corresponding binary systems (see Fig. 4). Since methanol is a strong polarity regulator, the addition of methanol may enhance the formation of inclusion complexation of enantiomers with -CD cavity, leading to improved resolutions of flavonoids and coumachlor. The Rs of 7-methoxyflavanone was achieved as high as 8.1 with Hex/EtOH/MeOH and 7.6 with Hex/IPA/MeOH. Moreover, benzoin and 4-chlorobenzoin that were not resolvable in either Hex/IPA or Hex/EtOH were partially enantioseparated in Hex/IPA/MeOH. The addition of a proper amount of methanol in mobile phase would enhance or even enable the chiral separation. The composition of Hex/IPA/MeOH and Hex/EtOH/MeOH (Hex content fixed as 70% by volume) was further adjusted for the optimization of enantioseparation of flavonoids (Table 2). The retention times of enantiomers were found to decrease dramatically with methanol content increasing from 5 to 25%. The increased eluting ability of mobile phase
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Table 1. Enantioseparation of racemates in different binary eluent HPLC
Compounds
t1
t2
␣
Rs
Mobile phase
Aryl-OH-1
4.51 4.26 4.67 4.62 4.32 4.36 4.72 4.86 4.35 4.42 4.95 5.19 4.47 4.56 5.23 4.53 4.07 4.06 4.68 3.93 4.09 4.52 4.07 4.01 4.68 5.32 5.54 4.86 5.39 4.85 4.72 5.41 5.34 4.87 5.64 5.85 4.75 4.65 5.00 12.35 6.99 6.58 7.41 8.40 5.67 5.66 6.01 11.99 6.35 6.57 9.69 7.23 7.63 7.502 4.89 4.09 3.98 4.52
4.77 4.41 4.92 5.35 4.72 4.58 5.32 6.11 5.11 4.89 5.78 7.02 5.34 5.34 6.22 6.06 4.89 4.60 5.77 4.01 4.19 5.97 4.87 4.47 5.75 5.47 5.59 5.17 9.62 6.26 6.28 10.24 5.52 4.96 5.83 9.89 6.13 5.74 6.52 27.84 12.72 11.86 13.83 16.66 8.31 7.94 8.92 12.63 6.74 7.04 14.16 8.97 9.79 9.236 5.81 4.56 4.25 5.16
1.71 1.56 1.17 2.20 1.85 1.67 1.39 2.68 2.27 1.94 1.47 3.13 2.37 2.26 1.48 3.03 2.42 2.05 1.73 1.39 1.11 2.96 2.38 1.94 1.72 1.81 1.02 1.83 4.94 2.82 2.99 3.17 1.85 1.66 1.08 2.51 1.96 1.73 1.68 2.69 2.55 2.54 2.38 2.58 2.11 1.91 1.90 1.07 1.13 1.12 1.69 1.44 1.48 1.37 1.54 1.48 1.45 1.50
1.5 1.1 1.3 4.1 2.7 1.2 3.0 5.9 5.2 2.4 4.3 6.4 5.3 2.9 5.5 6.9 5.4 3.0 5.4 0.6 0.5 5.8 4.4 3.7 4.2 0.6 0.3 0.2 6.9 5.9 4.1 5.5 0.7 0.5 0.6 4.2 4.9 3.8 4.3 4.2 6.8 5.6 6.3 4.0 5.6 4.3 5.2 0.4 0.8 0.9 1.9 2.5 2.1 2.2 2.1 1.7 1.6 1.6
Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) Hex:NPA (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (80:20, v/v) Hex:EtOH (80:20, v/v) i-Oct:IPA (80:20, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (70:30, v/v) Hex:EtOH (70:30, v/v) Hex:NPA (70:30, v/v) i-Oct:EtOH (70:30, v/v) Hex:IPA (50:50, v/v) Hex:EtOH (50:50, v/v) Hex:NPA (50:50, v/v) i-Oct:IPA (50:50, v/v)
Aryl-OH-2
Aryl-OH-3
Aryl-OH-4
Aryl-OH-5
Aryl-OH-6 Aryl-OH-7
Aryl-OH-8 Aryl-OH-9 Aryl-OH-11
Aryl-OH-12
Flavanone
7-Methoxyflavanone
6-Methoxyflavanone
7-Hydroxyflavanone
4-Hydroxyflavanone
Coumachlor
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Figure 2. Effect of flow rate on enantioseparation of flavanone; temperature: 25⬚C; mobile phase: Hex/EtOH = 70:30 v/v.
Figure 3. Temperature-dependent enantioseparation of flavanone and 7-methoxyflavanone with 1 mL/min Hex/EtOH (70:30, v/v).
was attributed to the increased solvent polarity with the addition of methanol. The optimum enantioseparation of 7-methoxyflavanone (Rs = 8.0) and 6-methoxyflavanone (Rs = 6.6) was obtained with Hex/IPA/methanol (70:5:25, v/v/v). 7-Hydroxyflavanone and 4-hydroxyflavanone, however, achieved their best resolution with Hex/IPA/MeOH (70:10:20, v/v/v). All flavanoids exhibited their best resolution with Hex/EtOH/MeOH (70:15:15, v/v/v) when EtOH was used in ternary eluent systems. The representative enantioseparation chromatograms of selected racemates in different eluent systems are summarized in Fig. 5. The enantioseparation of isoprenaline, propranolol, atenolol, and clenbuterol was unsuccessful even with ternary solvent system (Table 1). Partial separation was achieved with the addition of TFA and TEA. The rather low Rs may be attributed to the presence of -chiral center in propranolol and atenolol or the bulky aromatic moieties, resulting in weaker intermolecular interactions. The steric hindrance of bulky aromatic moieties may also account for the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
low enantioseparation of naringenin and hesperetin in NPHPLC. The addition of organic modifiers, therefore, could alter the polarity of mobile phases, resulting in a change of the interactions between CD CSP and enantiomers. The enantioselectivities of CSP were thus tuned by the mobile phases.
3.3 Polar-organic-mode HPLC The capacity of this CSP was finally investigated in polar organic HPLC using protic solvents including MeOH, EtOH, NPA and IPA as well as aprotic solvent ACN as mobile phase [22]. In NP-HPLC, -CD cavity would be occupied by the non-polar solvent and the inclusion complexation thus rarely took place, and the chiral recognition was mainly produced by the interactions of analytes with functional groups at -CD rims. In polar-organic and RP modes, the inclusion complexation exists as an important driving force for enantioseparation. Moreover, the -CD clicked CSP www.jss-journal.com
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Table 2. Methanol tuned enantioseparation of racemates in NPLC
Analytes
t2
␣
Rs
Hex:IPA:MeOH
t2
␣
Rs
Hex:EtOH:MeOH
Aryl-OH-2 Aryl-OH-3 Aryl-OH-4 Aryl-OH-5 Aryl-OH-7 Aryl-OH-11 Flavanone
Hesperetin Naringenin Coumachlor Benzoin 4-Chlorobenzoin Isoprenaline
5.12 4.62 5.14 4.50 4.59 6.50 7.40 6.66 6.22 6.06 5.96 16.28 13.11 11.34 10.64 10.05 10.55 9.01 8.23 7.94 7.74 7.03 6.46 6.44 6.17 6.28 10.98 10.04 9.42 9.14 8.91 27.01 19.44 4.50 6.62 17.61 85.16
2.16 1.69 2.18 1.75 1.80 2.90 2.55 2.39 2.40 2.37 2.77 2.64 2.51 2.45 2.41 2.46 2.43 2.29 2.28 2.26 2.40 1.22 1.19 1.16 1.18 1.19 1.68 1.63 1.58 1.56 1.46 1.07 1.05 1.45 1.05 1.05 1.06
IPA>NPA. It was noteworthy that mobile phase of pure polar organic solvent enabled the chiral separation of flavonoids without the aid of any additive. To get some insight for this phenomenon, aprotic solvent ACN was used as reference. Only partial separation was achieved for all analytes except 7-methoxyflavanone. The
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Figure 4. Enhanced chiral resolutions of flavonoids with MeOH added ternary eluent HPLC.
Figure 5. Representative enantioseparation chromatograms of the studied racemates.
poor resolution may be explained with the aprotic solvent nature of ACN. Since the C–H bonds of ACN have a very high pKa value, it tended to form solvent clusters instead of hydrogen bond network. By comparison, one can reasonably conclude hydrogen bonding formed with protic solvents is beneficial for the chiral resolution of racemates in HPLC. Based on the above discussion, the polarity tuning of mobile phase using polar solvents in polar organic HPLC would not only affect the inclusion complexation but also the interactions between CD CSP and enantiomers. Figure 6. Enantioseparation of flavonoids in polar-organic-mode HPLC.
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4 Concluding remarks
[6] Marechal, A., El-Debs, R., Dugas, V., Demesmay, C. J. Sep. Sci. 2013, 36, 2049–2062.
The enantioselectivity of the perphenylcarbamoylated -CD clicked CSP has been successfully explored for 26 racemates by tuning the polarity of eluent system for both normal-phase and polar-organic HPLC. The enantioseparation properties using this perphenylcarbamoylated -CD clicked CSP were compared and discussed by correlating analyte structures and chromatographic conditions. Satisfactory enantioseparation of most racemates was achieved within 10 min. The addition of methanol can significantly improve the enantioseparation of flavonoids in ternary systems.
[7] Chu, C., Liu, R. Chem. Soc. Rev. 2011, 40, 2177–2188.
We gratefully acknowledged the financial support from the National Natural Science Foundation of China (grant no. 21305066), Program for New Century Excellent Talents in University (NCET-12-0633), Doctoral Fund of Ministry of Education of China (no. 20103219120008), the Jiangsu Province Natural Science Fund for Distinguished Young Scholars (BK20130032), and the Fundamental Research Funds for the Central Universities (30920130111006). The authors have declared no conflict of interests.
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