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Cyclodextrin clicked chiral stationary phases with functionalities-tuned enantioseparations in high performance liquid chromatography
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Cyclodextrin clicked chiral stationary phases with functionalities-tuned enantioseparations in high performance liquid chromatography Yuzhou Lin, Jie Zhou, Jian Tang, Weihua Tang ∗ College of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, People’s Republic of China
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Article history: Received 29 April 2015 Received in revised form 13 June 2015 Accepted 17 June 2015 Available online xxx Keywords: Cyclodextrin Chiral stationary phase Click chemistry Enantioselectivity
a b s t r a c t In this work, two cyclodextrin (CD) chiral stationary phases (CSPs) have been developed by clicking per-4-chloro-3-methylphenylcarbamoylated mono-6A -azido--CD (CSP1) and per-5-chloro2-methylphenylcarbamoylated mono-6A -azido--CD (CSP2) onto alkynylated silica support. The enantioslectivies of the as-obtained new CSPs have been evaluated using 29 model racemates including aromatic alcohols, flavonoids, -blocker and FMOC-amino acids in both reversed-phase (RP) and normal-phase (NP) high performance liquid chromatography (HPLC). The CD functionalities tuned enantioselectivities were elucidated in different HPLC elution modes. Higher chiral resolutions were achieved in RP-elution mode with the aid of the inclusion complexation in comparison to NP-elution mode. The - stacking interaction and dipole–dipole interaction provided by phenylcarbamate moieties can also contribute to the enantioseparation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chiral analysis and purification has become a prerequisite process prior to the clinical use of drugs [1]. High-performance liquid chromatography (HPLC) is a versatile approach for both chiral analysis and manufacturing [2–4]. Enantioseparation can be achieved by the utilization of chiral mobile phases (CMPs) [5,6] or chiral stationary phases (CSPs) [7,8]. The CSPs have aroused prominent interests due to their practicability and chemical stability in different modes of HPLC. Chemically bonded cyclodextrin (CD) CSPs are developed by immobilizing CD derivatives onto silica support [9,10]. The ‘click chemistry’ [11] has recently evolved as a facile and efficient synthesis approach in the field of biological science and material science, e.g. the preparation of CSPs [12–15]. The introduction of an electron-donating methyl group or an electron-withdrawing halogen at the 3- and/or 4-position of the phenyl ring on phenylcarbamated polysaccharide (cellulose and amylose) CSPs was found to improve their chiral recognition ability towards many racemates [16]. Now, various carbamates including chlorinated and methylsubsituted phenylcarbamates substituted CDs CSPs has been reported in [17–19], but only own one electrondonating group or one electron-withdrawing group.
∗ Corresponding author. Tel.: +86 25 8431 7311. E-mail address: [email protected] (W. Tang).
As a continuation of our research in developing chemically bonded perphenylcarbamoylated CD CSPs [20,21], we herein report per-4-chloro-3-methylphenylcarbamoylated -CD clicked CSP1 and per-5-chloro-2-methylphenylcarbamoylated -CD clicked CSP2. The enantioselectivities of these CSPs were evaluated with 29 racemates including aryl alcohols, flavanoids, adrenergic drugs and FMOC-amino acids in both normal-phase (NP), and reversedphase (RP) modes. The enantioseparation was tuned by optimizing the composition of mobile phases.
2. Experimental 2.1. Chemicals and materials Reagents including -CD, 4-chloro-3-methylaniline and 5chloro-2-methylphenyl isocyanate were purchased from Energy Chemical (Shanghai, China). Kromasil spherical silica gel (5 m, ˚ was obtained from Eka Chemicals (Bohus, Sweden). HPLC100 A) grade methanol (MeOH), acetonitrile (ACN), n-hexane (HEX), 2-propanol (IPA) and ethanol (EtOH) were purchased from Tedia (USA). HPLC-grade trifluoroacetic acid (TFA) and triethylamine (TEA) were purchased from J&K (Shanghai, China). The structures of racemates and CSPs are depicted in Fig. 1. Among of them, A6 and A9 were purchased from Sigma–Aldrich (St. Louis, MO), the other racemates were procured from J&K (Shanghai, China).
http://dx.doi.org/10.1016/j.chroma.2015.06.051 0021-9673/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Y. Lin, et al., Cyclodextrin clicked chiral stationary phases with functionalities-tuned enantioseparations in high performance liquid chromatography, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.06.051
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Fig. 1. Chemical structure of 29 analytes and CSPs studied.
2.2. Instruments NMR spectra were collected on Bruker AVANCE 500 (500 MHz, Bruker Daltonics, Bremen, Germany). Fourier transform infrared spectra (FTIR) were recorded on Thermo Scientific Nicolet iS-10 FT-IR (Thermo Fisher Scientific, Waltham, MA). Elemental analysis was conducted on Vario EL-III CHONS record (Elementar Analysensysteme GmbH, Frankfurt, Germany). HPLC experiments were performed at Agilent 1260 system (Agilent Technologies, Palo Alto,
CA) equipped with G1315D diode array detection (DAD) system, G1329B quaternary pump, G1331 C automatic injector, G1316A temperature controller and Agilent ChemStation data manager software (Version No.C.01. 04). 2.3. HPLC procedures In HPLC, all analytes were prepared as 200 g/ml in MeOH for RP mode and 200 g/ml in IPA for NP mode. Each sample solution was
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Table 1 Optimized enantioseparations of selected racemates in RP-elution mode. Analytes
A1 A2a A3 A4 A5 A6b A7 A8 A9 A10 A11 B1 B2 B3 B4 B5 B6 B7 B8 B9 C1 C2 C3 C4 D1 D2 D3 D4
CSP1
CSP2
t1
t2
˛
Rs
Condition
– 4.7 6.1 6.8 8.0 7.0 12.9 – 3.0 16.0 16.2 6.5 10.5 9.9 9.7 5.3 6.2 5.9 5.7 9.0 4.2 11.7 7.5 4.7 8.3 31.8 – –
– 5.6 6.6 7.8 9.6 10.4 17.0 – 3.1 28.1 17.3 9.4 18.9 17.0 11.6 6.6 8.4 12.9 6.1 10.7 4.7 12.3 7.7 5.1 9.7 89.6 – –
– 1.55 1.17 1.26 1.31 1.88 1.41
– 2.50 1.50 2.50 3.17 5.16 0.79 – 0.45 5.68 1.01 4.45 4.92 5.85 2.45 3.82 3.68 7.62 0.95 2.01 1.28 0.71 0.56 0.65 2.11 4.83 – –
MeOH/H2 O(20/80;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(10/90;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(70/30;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(90/10;v/v) MeOH/TEAA(70/30;v/v) MeOH/TEAA(60/40;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(70/30;v/v) ACN/TEAA(80/20;v/v) MeOH/TEAA(70/30;v/v) ACN/TEAA(80/20;v/v) ACN/TEAA(80/20;v/v)
1.8 1.93 1.08 1.78 2.10 2.00 1.54 1.49 1.66 3.32 1.18 1.27 1.21 1.07 1.06 1.20 1.25 3.00 – –
␣
Rs
Condition
– 2.9 – – – –
– 3.1 – – – –
– 1.06 – – –
– – – – 11.5 23.6 14.3 – – 9.1 4.7 – – – – – – 4.5
– – – – 13.7 26.8 16.7 – 10.3 5.7 – – – – – – 7.3
– – – – 1.28 1.16 1.21 – – 1.22 1.52 – – – – – 3.00
– 0.79 – – – – – – – –
– –
– –
MeOH/H2 O(20/80;v/v) MeOH MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(10/90;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(70/30;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(90/10;v/v) MeOH/TEAA(70/30;v/v) MeOH/TEAA(60/40;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(90/10;v/v) ACN/TEAA(80/20;v/v) MeOH/TEAA(70/30;v/v) ACN/TEAA(80/20;v/v) ACN/TEAA(80/20;v/v)
t1
t2
– –
2.01 1.42 1.67 – 1.51 2.59 – – – – – 4.38 – –
Conditions: flow rate 1 ml/min, analytes’ concentration 200 g/ml unless specified, injection volume 10 L. a Analyte concentration 400 g/ml. b Analyte concentration 400 g/ml, 15 L injection volume.
injected 10 L otherwise specified. The detection wavelength range was 200–300 nm. All mobile phases were degassed with ultrasonication and all samples were filtered through a 0.22 m membrane before use. The CSPs were prepared and packed into columns in the lab (see Supplementary Information). 3. Results and discussion From elemental analysis, CSP1 exhibited a carbon content of 12.0% and nitrogen content of 0.364% while 12.3 and 0.35% for CSP2, which were greatly improved in comparison to 6.9% (C%) and 0.271% (N%) for alkynyl silica. The surface loading of CSP1 and CSPs was calculated to be 0.18 and 0.21 mol/ m2 according to literature [21]. The column efficiency was 10794 and 10130 plates/m for CSP1 and CSP2, respectively. 3.1. Evaluation of enantioselectivities of clicked CSPs in RP-elution mode In reversed-phase mode, CSP1 could baseline resolve 16 racemates among 28 selected analytes (Rs > 1.5), but CSP2 could only separate five compounds (See Table 1). For analytes A2-A4 containing electron-withdrawing halide groups (i.e.,–F, -Cl, and -Br) in aromatic moieties, CSP1 exhibited an enantioselectivity following the order: A2 > A4 > A3. B2 and B3 achieved better enantioseparation than B4 and B5 by CSPs, indicating the position of substituent on aromatic ring played an important role in enantioseparation. A5 was much better resolved than A3, attributed to the contribution of double bond to conjugation in CSP1. A7 was partially separated by CSP1 while A8 showed no separation since the larger steric hindrance of the ˛-substitute. A direct comparison study on the enantioselectivities of CSP1 with ACN/H2 O or MeOH/H2 O mobile phases was conducted by
using A3, A4, A5 and A10 as model analytes (See Fig. S2). For enantioseparations of analytes with both MeOH/H2 O and ACN/H2 O, all Rs were found to decrease with increased percentage of organic solvents (including both methanol and ACN) in the mobile phases. Faster elution was also observed with increased organic solvents percentage since the increased competition between the organic solvent and analytes in entering CD cavity when forming inclusion complexation. Enantioselectivities of CSP1 with MeOH/H2 O were found to be higher than those with ACN/H2 O, but with longer retention. The alkalescent -blockers (C1–C4) could not be resolved with both CSPs. Even by adding TEAA buffer into mobile phase, we could only obtain partial enantioseparation. However, enhanced enantioseparations were achieved for B8 and B9 on CSP1 with the addition of TEAA (1%) in mobile phase (see Fig. 2). D1 could be separated by both CSP1 and CSP2 at the presence of TEAA (1%, pH 4) in mobile phase. And Rs as high as 4.83 was obtained for D2 on CSP1 with MeOH/TEAA (1%, pH 4) (70/30, v/v). The rest FMOCamino acids could not be resolved with both CSPs under the mobile phases as explored. 3.2. Evaluation of the enantioselectivities of CD clicked CSPs in NP-elution mode In RP-elution mode, the inclusion complexation between CD and the analytes is considered as the main driving force for enantioseparations. But under NP-elution mode, such contribution of inclusion complexation is absent due to the prevailing inclusion of weak polar solvent inside CD’s cavity [22]. The enantioselectivities of our clicked CSPs at NP-elution mode were explored (see Table 2). As shown in Table 2, the retentions of enantiomers were shortened with the increased percentage of polar solvent in both
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Fig. 2. Enhanced resolution of B8 and B9 with CSP1 using 1% TEAA containing mobile phase.
ethanol/HEX and IPA/HEX mobile phases. In most cases, ethanol afforded both higher enantioselectivity and chiral resolution than IPA with the same percentage. In NP-elution mode, CD cavity is occupied by nonpolar HEX, the intermolecular interactions between CD functionalities and enantiomers are the dominant driving forces for enantioseparation. With ethanol used as organic modifier, all selected racemates were well resolved, with their best Rs obtained at 20% ethanol percentage. The highest ␣ values, however, were all obtained at 40% ethanol except A4 at 20% ethanol. Considering all aromatic alcohols could not be resolved by CSP2 and only two of them (A4 and A5) were separated by CSP1 in NPelution mode. In addition, Naringenin and Hesperetin were unable to be separated with IPA as organic modifier on CSP1, but they turned out to be easily resolved when ethanol was applied (see Fig. 3). Interestingly, Coumachlor could not be separated with both IPA and ethanol by CSP1. However, CSP2 exhibited great potential
to resolve it using ethanol as organic modifier. Moreover, the addition of TFA and TEA into the mobile phase could further improve the enantioseparations no matter what type of organic modifier was used. Based on the results discussed as above, our two chloromethylphenylcarbamoylated CD clicked CSPs exhibited quite different enantioselectivities for the model racemates. Similar substituents position-tuned enantioselectivities was earlier observed for chloromethylphenylcarbarmated cellulose CSPs [16], where the meta- and para-disubstituted derivatives were found to show higher chiral recognition than ortho- and metaor para-disubstituted ones. The reason was explained with more acidic N-H protons of meta- and para-disubstituted phenylcarbamate celluloses would interact more strongly with appropriate enantiomers via hydrogen-bonding. This may explain the higher enantioselectivities of CSP1 than CSP2 in our CD cases.
Table 2 Enantioseparations of aromatic alcohols and flavonoids with CSP1 or CSP2 using IPA/HEx or EtOH/HEX as mobile phases. Entry
CSP
Organic modifier/HEX 20/80;v/v
CSP1
A5
CSP1
B1
CSP1
B1
CSP2
B2
CSP1
B3
CSP1
B4
CSP2
B5
CSP2
B6
CSP1
B6
CSP2
B7
CSP1
B7
CSP2
40/60;v/v
50/50;v/v
Condi-tion
␣
Rs
t2
␣
Rs
t2
␣
Rs
␣
Rs
4.72 4.75 4.12 4.21 6.88 7.25
1.15 2.08 1.08 1.29 1.63 2.81
1.09 2.93 0.43 1.50 4.29 6.10
4.46 3.99 3.92 3.63 10.88 6.39
1.35 1.39 1.30 1.17 2.48 2.95
1.90 2.23 1.07 1.19 4.94 5.72
4.09 3.61 3.63 3.35 10.07 5.88
1.48 1.53 1.41 1.38 2.55 3.71
1.66 1.68 0.96 0.79 4.78 5.44
3.98 3.43 3.61 3.22 9.63 5.59
1.57 1.29 1.50 1.14 2.46 2.24
1.76 1.23 1.05 0.47 4.74 5.25
I II I II I II
5.05 4.72 13.56 16.86 9.56 10.61
1.17 2.50 1.83 2.66 1.88 2.84
1.20 1.38 6.23 7.63 4.39 7.42
4.83 4.42 28.04 13.50 19.11 9.16
1.21 1.50 2.58 3.14 2.87 3.60
1.20 1.33 4.55 7.35 5.34 7.18
4.61 4.16 23.82 11.60 17.49 8.30
1.25 1.89 2.64 3.47 2.96 4.78
1.14 1.22 4.48 7.12 5.18 6.84
4.53 4.03 21.7 10.5 16.7 7.84
1.22 1.30 2.67 3.17 3.01 3.34
1.11 1.14 4.36 7.01 5.21 6.83
I II I II I II
6.87 5.95 5.41 5.04 21.72 18.08
1.13 1.21 1.07 1.27 1.34 1.68
1.01 1.03 0.55 0.80 2.61 3.77
6.10 5.14 5.16 4.60 18.83 11.32
1.39 1.16 1.07 1.17 1.82 1.68
0.68 0.89 0.54 0.67 2.23 3.32
5.47 4.66 4.85 4.31 12.91 8.43
1.12 1.18 1.07 1.16 1.81 1.76
0.62 0.74 0.51 0.65 1.96 2.98
5.23 4.43 4.77 4.18 10.5 7.12
1.11 1.15 1.10 1.15 1.81 1.68
0.58 0.64 0.50 0.64 1.87 2.79
I II I II I II
12.02 9.80 28.50 28.95
1.10 1.20 2.46 3.97
0.74 1.68 6.96 7.49
8.09 6.78 45.40 18.09
1.09 1.22 6.16 3.97
0.55 1.41 4.01 6.71
6.25 5.42 31.19 13.23
1.09 1.22 6.17 4.39
0.47 1.18 3.89 6.22
5.52 4.03 25.0 10.9
1.05 1.22 3.75 4.08
0.32 0.98 3.97 5.93
I II I II
13.42 11.07
1.48 1.70
2.02 4.36
9.10 7.77
1.49 1.75
2.03 3.53
7.13 6.16
1.49 1.79
1.74 3.07
6.28 5.43
1.49 1.79
1.59 2.78
I II
t2 A4
30/70;v/v
t2
Note: condition I: IPA/HEX, condition II: EtOH/HEX.
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Fig. 3. Enantioseparations of naringenin, hesperetin and coumachlor with different organic modifiers.
4. Conclusion Two chloromethylphenylcarbamoylated CD clicked CSPs have been successfully developed and evaluated their enantioselectivities using 29 analytes in both RP- and NP-elution mode in HPLC. The 4-chloro-3-methylphenylcarbamoylated CD clicked CSP1 exhibited relatively better enantioseparation ability than 5-chloro-2-methylphenylcarbamoylated CD clicked CSP2 in both HPLC modes. Higher enantioselectivities were achieved in RPelution mode. The composition of mobile phases exerted significant impact on the enantioselectivities of the clicked CD CSPs. And the intermolecular interactions between CD and analytes played an important role for the chiral separation. This comparison study may provide some insight in molecular design of functionalized CD clicked CSPs for high-performance enantioseparation in HPLC. Acknowledgements 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 A Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.06. 051 References [1] M.E. Bosch, A.J.R. Sánchez, F.S. Rojas, C.B. Ojeda, Recent advances in analytical determination of thalidomide and its metabolites, J. Pharm. Biomed. Anal. 46 (2008) 9–17. [2] P. López-Ram-de-Víu, J.A. Gálvez, M.D. Díaz-de-Villegas, High-performance liquid chromatographic enantioseparation of unusual amino acid derivatives with axial chirality on polysaccharide-based chiral stationary phases, J. Chromatogr. A 1390 (2015) 78–85. [3] X. Lai, W. Tang, S.-C. Ng, Novel, -cyclodextrin chiral stationary phases with different length spacers for normal-phase high performance liquid chromatography enantioseparation, J. Chromatogr. A 1218 (2011) 3496–3501. [4] S. Mao, Y. Zhang, S. Rohani, A.K. Ray, Chromatographic resolution and isotherm determination of (R,S)-mandelic acid on Chiralcel-OD column, J. Sep. Sci. 35 (2012) 2273–2281.
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Short communication
Cyclodextrin clicked chiral stationary phases with functionalities-tuned enantioseparations in high performance liquid chromatography Yuzhou Lin, Jie Zhou, Jian Tang, Weihua Tang ∗ College of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 13 June 2015 Accepted 17 June 2015 Available online xxx Keywords: Cyclodextrin Chiral stationary phase Click chemistry Enantioselectivity
a b s t r a c t In this work, two cyclodextrin (CD) chiral stationary phases (CSPs) have been developed by clicking per-4-chloro-3-methylphenylcarbamoylated mono-6A -azido--CD (CSP1) and per-5-chloro2-methylphenylcarbamoylated mono-6A -azido--CD (CSP2) onto alkynylated silica support. The enantioslectivies of the as-obtained new CSPs have been evaluated using 29 model racemates including aromatic alcohols, flavonoids, -blocker and FMOC-amino acids in both reversed-phase (RP) and normal-phase (NP) high performance liquid chromatography (HPLC). The CD functionalities tuned enantioselectivities were elucidated in different HPLC elution modes. Higher chiral resolutions were achieved in RP-elution mode with the aid of the inclusion complexation in comparison to NP-elution mode. The - stacking interaction and dipole–dipole interaction provided by phenylcarbamate moieties can also contribute to the enantioseparation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chiral analysis and purification has become a prerequisite process prior to the clinical use of drugs [1]. High-performance liquid chromatography (HPLC) is a versatile approach for both chiral analysis and manufacturing [2–4]. Enantioseparation can be achieved by the utilization of chiral mobile phases (CMPs) [5,6] or chiral stationary phases (CSPs) [7,8]. The CSPs have aroused prominent interests due to their practicability and chemical stability in different modes of HPLC. Chemically bonded cyclodextrin (CD) CSPs are developed by immobilizing CD derivatives onto silica support [9,10]. The ‘click chemistry’ [11] has recently evolved as a facile and efficient synthesis approach in the field of biological science and material science, e.g. the preparation of CSPs [12–15]. The introduction of an electron-donating methyl group or an electron-withdrawing halogen at the 3- and/or 4-position of the phenyl ring on phenylcarbamated polysaccharide (cellulose and amylose) CSPs was found to improve their chiral recognition ability towards many racemates [16]. Now, various carbamates including chlorinated and methylsubsituted phenylcarbamates substituted CDs CSPs has been reported in [17–19], but only own one electrondonating group or one electron-withdrawing group.
∗ Corresponding author. Tel.: +86 25 8431 7311. E-mail address: [email protected] (W. Tang).
As a continuation of our research in developing chemically bonded perphenylcarbamoylated CD CSPs [20,21], we herein report per-4-chloro-3-methylphenylcarbamoylated -CD clicked CSP1 and per-5-chloro-2-methylphenylcarbamoylated -CD clicked CSP2. The enantioselectivities of these CSPs were evaluated with 29 racemates including aryl alcohols, flavanoids, adrenergic drugs and FMOC-amino acids in both normal-phase (NP), and reversedphase (RP) modes. The enantioseparation was tuned by optimizing the composition of mobile phases.
2. Experimental 2.1. Chemicals and materials Reagents including -CD, 4-chloro-3-methylaniline and 5chloro-2-methylphenyl isocyanate were purchased from Energy Chemical (Shanghai, China). Kromasil spherical silica gel (5 m, ˚ was obtained from Eka Chemicals (Bohus, Sweden). HPLC100 A) grade methanol (MeOH), acetonitrile (ACN), n-hexane (HEX), 2-propanol (IPA) and ethanol (EtOH) were purchased from Tedia (USA). HPLC-grade trifluoroacetic acid (TFA) and triethylamine (TEA) were purchased from J&K (Shanghai, China). The structures of racemates and CSPs are depicted in Fig. 1. Among of them, A6 and A9 were purchased from Sigma–Aldrich (St. Louis, MO), the other racemates were procured from J&K (Shanghai, China).
http://dx.doi.org/10.1016/j.chroma.2015.06.051 0021-9673/© 2015 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structure of 29 analytes and CSPs studied.
2.2. Instruments NMR spectra were collected on Bruker AVANCE 500 (500 MHz, Bruker Daltonics, Bremen, Germany). Fourier transform infrared spectra (FTIR) were recorded on Thermo Scientific Nicolet iS-10 FT-IR (Thermo Fisher Scientific, Waltham, MA). Elemental analysis was conducted on Vario EL-III CHONS record (Elementar Analysensysteme GmbH, Frankfurt, Germany). HPLC experiments were performed at Agilent 1260 system (Agilent Technologies, Palo Alto,
CA) equipped with G1315D diode array detection (DAD) system, G1329B quaternary pump, G1331 C automatic injector, G1316A temperature controller and Agilent ChemStation data manager software (Version No.C.01. 04). 2.3. HPLC procedures In HPLC, all analytes were prepared as 200 g/ml in MeOH for RP mode and 200 g/ml in IPA for NP mode. Each sample solution was
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Table 1 Optimized enantioseparations of selected racemates in RP-elution mode. Analytes
A1 A2a A3 A4 A5 A6b A7 A8 A9 A10 A11 B1 B2 B3 B4 B5 B6 B7 B8 B9 C1 C2 C3 C4 D1 D2 D3 D4
CSP1
CSP2
t1
t2
˛
Rs
Condition
– 4.7 6.1 6.8 8.0 7.0 12.9 – 3.0 16.0 16.2 6.5 10.5 9.9 9.7 5.3 6.2 5.9 5.7 9.0 4.2 11.7 7.5 4.7 8.3 31.8 – –
– 5.6 6.6 7.8 9.6 10.4 17.0 – 3.1 28.1 17.3 9.4 18.9 17.0 11.6 6.6 8.4 12.9 6.1 10.7 4.7 12.3 7.7 5.1 9.7 89.6 – –
– 1.55 1.17 1.26 1.31 1.88 1.41
– 2.50 1.50 2.50 3.17 5.16 0.79 – 0.45 5.68 1.01 4.45 4.92 5.85 2.45 3.82 3.68 7.62 0.95 2.01 1.28 0.71 0.56 0.65 2.11 4.83 – –
MeOH/H2 O(20/80;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(10/90;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(70/30;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(90/10;v/v) MeOH/TEAA(70/30;v/v) MeOH/TEAA(60/40;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(70/30;v/v) ACN/TEAA(80/20;v/v) MeOH/TEAA(70/30;v/v) ACN/TEAA(80/20;v/v) ACN/TEAA(80/20;v/v)
1.8 1.93 1.08 1.78 2.10 2.00 1.54 1.49 1.66 3.32 1.18 1.27 1.21 1.07 1.06 1.20 1.25 3.00 – –
␣
Rs
Condition
– 2.9 – – – –
– 3.1 – – – –
– 1.06 – – –
– – – – 11.5 23.6 14.3 – – 9.1 4.7 – – – – – – 4.5
– – – – 13.7 26.8 16.7 – 10.3 5.7 – – – – – – 7.3
– – – – 1.28 1.16 1.21 – – 1.22 1.52 – – – – – 3.00
– 0.79 – – – – – – – –
– –
– –
MeOH/H2 O(20/80;v/v) MeOH MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(20/80;v/v) MeOH/H2 O(10/90;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(50/50;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(70/30;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(60/40;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(90/10;v/v) MeOH/TEAA(70/30;v/v) MeOH/TEAA(60/40;v/v) MeOH/H2 O(80/20;v/v) MeOH/H2 O(80/20;v/v) MeOH/TEAA(90/10;v/v) ACN/TEAA(80/20;v/v) MeOH/TEAA(70/30;v/v) ACN/TEAA(80/20;v/v) ACN/TEAA(80/20;v/v)
t1
t2
– –
2.01 1.42 1.67 – 1.51 2.59 – – – – – 4.38 – –
Conditions: flow rate 1 ml/min, analytes’ concentration 200 g/ml unless specified, injection volume 10 L. a Analyte concentration 400 g/ml. b Analyte concentration 400 g/ml, 15 L injection volume.
injected 10 L otherwise specified. The detection wavelength range was 200–300 nm. All mobile phases were degassed with ultrasonication and all samples were filtered through a 0.22 m membrane before use. The CSPs were prepared and packed into columns in the lab (see Supplementary Information). 3. Results and discussion From elemental analysis, CSP1 exhibited a carbon content of 12.0% and nitrogen content of 0.364% while 12.3 and 0.35% for CSP2, which were greatly improved in comparison to 6.9% (C%) and 0.271% (N%) for alkynyl silica. The surface loading of CSP1 and CSPs was calculated to be 0.18 and 0.21 mol/ m2 according to literature [21]. The column efficiency was 10794 and 10130 plates/m for CSP1 and CSP2, respectively. 3.1. Evaluation of enantioselectivities of clicked CSPs in RP-elution mode In reversed-phase mode, CSP1 could baseline resolve 16 racemates among 28 selected analytes (Rs > 1.5), but CSP2 could only separate five compounds (See Table 1). For analytes A2-A4 containing electron-withdrawing halide groups (i.e.,–F, -Cl, and -Br) in aromatic moieties, CSP1 exhibited an enantioselectivity following the order: A2 > A4 > A3. B2 and B3 achieved better enantioseparation than B4 and B5 by CSPs, indicating the position of substituent on aromatic ring played an important role in enantioseparation. A5 was much better resolved than A3, attributed to the contribution of double bond to conjugation in CSP1. A7 was partially separated by CSP1 while A8 showed no separation since the larger steric hindrance of the ˛-substitute. A direct comparison study on the enantioselectivities of CSP1 with ACN/H2 O or MeOH/H2 O mobile phases was conducted by
using A3, A4, A5 and A10 as model analytes (See Fig. S2). For enantioseparations of analytes with both MeOH/H2 O and ACN/H2 O, all Rs were found to decrease with increased percentage of organic solvents (including both methanol and ACN) in the mobile phases. Faster elution was also observed with increased organic solvents percentage since the increased competition between the organic solvent and analytes in entering CD cavity when forming inclusion complexation. Enantioselectivities of CSP1 with MeOH/H2 O were found to be higher than those with ACN/H2 O, but with longer retention. The alkalescent -blockers (C1–C4) could not be resolved with both CSPs. Even by adding TEAA buffer into mobile phase, we could only obtain partial enantioseparation. However, enhanced enantioseparations were achieved for B8 and B9 on CSP1 with the addition of TEAA (1%) in mobile phase (see Fig. 2). D1 could be separated by both CSP1 and CSP2 at the presence of TEAA (1%, pH 4) in mobile phase. And Rs as high as 4.83 was obtained for D2 on CSP1 with MeOH/TEAA (1%, pH 4) (70/30, v/v). The rest FMOCamino acids could not be resolved with both CSPs under the mobile phases as explored. 3.2. Evaluation of the enantioselectivities of CD clicked CSPs in NP-elution mode In RP-elution mode, the inclusion complexation between CD and the analytes is considered as the main driving force for enantioseparations. But under NP-elution mode, such contribution of inclusion complexation is absent due to the prevailing inclusion of weak polar solvent inside CD’s cavity [22]. The enantioselectivities of our clicked CSPs at NP-elution mode were explored (see Table 2). As shown in Table 2, the retentions of enantiomers were shortened with the increased percentage of polar solvent in both
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Fig. 2. Enhanced resolution of B8 and B9 with CSP1 using 1% TEAA containing mobile phase.
ethanol/HEX and IPA/HEX mobile phases. In most cases, ethanol afforded both higher enantioselectivity and chiral resolution than IPA with the same percentage. In NP-elution mode, CD cavity is occupied by nonpolar HEX, the intermolecular interactions between CD functionalities and enantiomers are the dominant driving forces for enantioseparation. With ethanol used as organic modifier, all selected racemates were well resolved, with their best Rs obtained at 20% ethanol percentage. The highest ␣ values, however, were all obtained at 40% ethanol except A4 at 20% ethanol. Considering all aromatic alcohols could not be resolved by CSP2 and only two of them (A4 and A5) were separated by CSP1 in NPelution mode. In addition, Naringenin and Hesperetin were unable to be separated with IPA as organic modifier on CSP1, but they turned out to be easily resolved when ethanol was applied (see Fig. 3). Interestingly, Coumachlor could not be separated with both IPA and ethanol by CSP1. However, CSP2 exhibited great potential
to resolve it using ethanol as organic modifier. Moreover, the addition of TFA and TEA into the mobile phase could further improve the enantioseparations no matter what type of organic modifier was used. Based on the results discussed as above, our two chloromethylphenylcarbamoylated CD clicked CSPs exhibited quite different enantioselectivities for the model racemates. Similar substituents position-tuned enantioselectivities was earlier observed for chloromethylphenylcarbarmated cellulose CSPs [16], where the meta- and para-disubstituted derivatives were found to show higher chiral recognition than ortho- and metaor para-disubstituted ones. The reason was explained with more acidic N-H protons of meta- and para-disubstituted phenylcarbamate celluloses would interact more strongly with appropriate enantiomers via hydrogen-bonding. This may explain the higher enantioselectivities of CSP1 than CSP2 in our CD cases.
Table 2 Enantioseparations of aromatic alcohols and flavonoids with CSP1 or CSP2 using IPA/HEx or EtOH/HEX as mobile phases. Entry
CSP
Organic modifier/HEX 20/80;v/v
CSP1
A5
CSP1
B1
CSP1
B1
CSP2
B2
CSP1
B3
CSP1
B4
CSP2
B5
CSP2
B6
CSP1
B6
CSP2
B7
CSP1
B7
CSP2
40/60;v/v
50/50;v/v
Condi-tion
␣
Rs
t2
␣
Rs
t2
␣
Rs
␣
Rs
4.72 4.75 4.12 4.21 6.88 7.25
1.15 2.08 1.08 1.29 1.63 2.81
1.09 2.93 0.43 1.50 4.29 6.10
4.46 3.99 3.92 3.63 10.88 6.39
1.35 1.39 1.30 1.17 2.48 2.95
1.90 2.23 1.07 1.19 4.94 5.72
4.09 3.61 3.63 3.35 10.07 5.88
1.48 1.53 1.41 1.38 2.55 3.71
1.66 1.68 0.96 0.79 4.78 5.44
3.98 3.43 3.61 3.22 9.63 5.59
1.57 1.29 1.50 1.14 2.46 2.24
1.76 1.23 1.05 0.47 4.74 5.25
I II I II I II
5.05 4.72 13.56 16.86 9.56 10.61
1.17 2.50 1.83 2.66 1.88 2.84
1.20 1.38 6.23 7.63 4.39 7.42
4.83 4.42 28.04 13.50 19.11 9.16
1.21 1.50 2.58 3.14 2.87 3.60
1.20 1.33 4.55 7.35 5.34 7.18
4.61 4.16 23.82 11.60 17.49 8.30
1.25 1.89 2.64 3.47 2.96 4.78
1.14 1.22 4.48 7.12 5.18 6.84
4.53 4.03 21.7 10.5 16.7 7.84
1.22 1.30 2.67 3.17 3.01 3.34
1.11 1.14 4.36 7.01 5.21 6.83
I II I II I II
6.87 5.95 5.41 5.04 21.72 18.08
1.13 1.21 1.07 1.27 1.34 1.68
1.01 1.03 0.55 0.80 2.61 3.77
6.10 5.14 5.16 4.60 18.83 11.32
1.39 1.16 1.07 1.17 1.82 1.68
0.68 0.89 0.54 0.67 2.23 3.32
5.47 4.66 4.85 4.31 12.91 8.43
1.12 1.18 1.07 1.16 1.81 1.76
0.62 0.74 0.51 0.65 1.96 2.98
5.23 4.43 4.77 4.18 10.5 7.12
1.11 1.15 1.10 1.15 1.81 1.68
0.58 0.64 0.50 0.64 1.87 2.79
I II I II I II
12.02 9.80 28.50 28.95
1.10 1.20 2.46 3.97
0.74 1.68 6.96 7.49
8.09 6.78 45.40 18.09
1.09 1.22 6.16 3.97
0.55 1.41 4.01 6.71
6.25 5.42 31.19 13.23
1.09 1.22 6.17 4.39
0.47 1.18 3.89 6.22
5.52 4.03 25.0 10.9
1.05 1.22 3.75 4.08
0.32 0.98 3.97 5.93
I II I II
13.42 11.07
1.48 1.70
2.02 4.36
9.10 7.77
1.49 1.75
2.03 3.53
7.13 6.16
1.49 1.79
1.74 3.07
6.28 5.43
1.49 1.79
1.59 2.78
I II
t2 A4
30/70;v/v
t2
Note: condition I: IPA/HEX, condition II: EtOH/HEX.
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Fig. 3. Enantioseparations of naringenin, hesperetin and coumachlor with different organic modifiers.
4. Conclusion Two chloromethylphenylcarbamoylated CD clicked CSPs have been successfully developed and evaluated their enantioselectivities using 29 analytes in both RP- and NP-elution mode in HPLC. The 4-chloro-3-methylphenylcarbamoylated CD clicked CSP1 exhibited relatively better enantioseparation ability than 5-chloro-2-methylphenylcarbamoylated CD clicked CSP2 in both HPLC modes. Higher enantioselectivities were achieved in RPelution mode. The composition of mobile phases exerted significant impact on the enantioselectivities of the clicked CD CSPs. And the intermolecular interactions between CD and analytes played an important role for the chiral separation. This comparison study may provide some insight in molecular design of functionalized CD clicked CSPs for high-performance enantioseparation in HPLC. Acknowledgements 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 A Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.06. 051 References [1] M.E. Bosch, A.J.R. Sánchez, F.S. Rojas, C.B. Ojeda, Recent advances in analytical determination of thalidomide and its metabolites, J. Pharm. Biomed. Anal. 46 (2008) 9–17. [2] P. López-Ram-de-Víu, J.A. Gálvez, M.D. Díaz-de-Villegas, High-performance liquid chromatographic enantioseparation of unusual amino acid derivatives with axial chirality on polysaccharide-based chiral stationary phases, J. Chromatogr. A 1390 (2015) 78–85. [3] X. Lai, W. Tang, S.-C. Ng, Novel, -cyclodextrin chiral stationary phases with different length spacers for normal-phase high performance liquid chromatography enantioseparation, J. Chromatogr. A 1218 (2011) 3496–3501. [4] S. Mao, Y. Zhang, S. Rohani, A.K. Ray, Chromatographic resolution and isotherm determination of (R,S)-mandelic acid on Chiralcel-OD column, J. Sep. Sci. 35 (2012) 2273–2281.
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