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Spherical ␤-cyclodextrin-silica hybrid materials for multifunctional chiral stationary phases Litao Wang a , Shuqing Dong a , Feng Han a , Yingwei Zhao a , Xia Zhang a , Xiaoli Zhang a,b , Hongdeng Qiu a , Liang Zhao a,∗ a 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 Sciences, Lanzhou 730000, PR China b University of Chinese Academy of Sciences, Beijing 100039, PR China

a r t i c l e

i n f o

Article history: Received 20 August 2014 Received in revised form 9 January 2015 Accepted 12 January 2015 Available online xxx Keywords: Hybrid materials Cyclodextrins Multifunctional separations Chiral resolution Liquid chromatography

a b s t r a c t Spherical ␤-CD-silica hybrid materials have been prepared successfully by simple one-pot polymerization, which provide a new strategy to construct new type of HPLC chiral stationary phases. Various ␤-CD, ethane, triazinyl and 3,5-dimethylphenyl functional groups that can provide multiple interactions were introduced into the pore channels and pore wall framework of mesoporous materials, respectively. The materials towards some chiral, acidic, anilines and phenols compounds showed multiple chromatographic separation functions including racemic resolution, anion exchange and achiral separations with a typical feature of normal/reversed phase chromatography. Multi-tasking including racemic resolution and achiral separations for selected compounds were performed simultaneously on a chiral chromatographic column. The multifunctional character of materials arises from the multiple interactions including hydrophobic interaction, ␲–␲ interaction, anion exchange, inclusion interaction and hydrogen bonding interaction. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Chiral separation materials are the key and core parts of chromatographic techniques for the resolution of enantiomers in chiral separation science [1–6]. High performance liquid chromatography (HPLC) combining chiral stationary phases (CSPs) that affords a direct and simple approach for the enantiomer separation has become one of the most powerful tools not only on an analytical scale but also for production at a preparative level [7–12]. Considerable efforts have been dedicated to develop cyclodextrin (CD) bonded CSPs [13–20]. Although traditional preparation technologies of the CD-CSPs have made great progress, new type of CSPs develop slowly, and mostly is studies on CD derivatives bonded onto porous spherical silica gel surface by various different linkers. The bulky CDs may be cause non-uniform distribution and low bonded amount that is limited by the amount of Si–OH and incomplete reaction of Si–OH due to the steric hindrance of CD groups. Meanwhile, the preparation process has a certain degree of complexity. Moreover, thus CSPs are difficulty to achieve more separation functions because of the limited functional groups.

∗ Corresponding author. Tel.: +86 931 4968261; fax: +86 931 8277088. E-mail address: [email protected] (L. Zhao).

Multifunctional HPLC stationary phases that can separate multiple types of chiral and achiral analytes under the normal/reversed phase modes are the important research trend [21,22]. A desired goal for analysts is that chromatographic separation materials own more separation functions, which can provide a significant degree of separated efficiency, flexibility and cost savings. Recently, some multifunctional HPLC separation materials have been developed by chemical bonded methods [23–27]. However, these works did not involve chiral separation. The resolution of enantiomers and the separation of achiral analytes were performed employing multifunctional HPLC CSPs by multiple molecular interactions on a chromatographic column, which is a very valuable work to resolve optical natural products and large-scale production of single-isomer chiral drugs. Organic–inorganic hybrid materials that combine the advantages of the organic and inorganic units are receiving considerable attention for applications in separation, catalysis and electronics [28–34]. Such materials were endowed with new functions and features owing to the tunable functional organic groups in the pore walls or channels, which could achieve chromatographic separation not only on the surface of the materials but also at the inner part of the material so as to increase effective separation. Recently, Sanchez reviewed the hybrid materials science is a promised land for the integrative design of multifunctional materials [35]. To date,

http://dx.doi.org/10.1016/j.chroma.2015.01.023 0021-9673/© 2015 Elsevier B.V. All rights reserved.

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some small molecules such as phenyl [36,37], diaminocyclohexane [38,39] and binaphthyl group [40] hybrid organosilica spheres have been prepared and used for HPLC stationary phases, which showed relatively favorable chromatographic capability and significant enhanced column stability at high/low pH. It is known that CDs and derivatives are one of the commonly used chiral selectors and showed excellent chiral recognition potency, which are very important functional groups for the resolution of racemates. However, some efforts have been made for the ␤-CD-silica hybrid materials, but the spherical morphology has not obtained [41–45]. Therefore, the direct preparation of spherical ␤-CD-silica hybrid materials is still a challenging task by simple one-pot process. Moreover, various functional groups (such as ␤-CD, ethane, ethylene, phenyl, aminopropyl, triazinyl etc.) that can introduce multiple molecular interactions could be rationally designed into the pore wall framework and/or channel space of the hybrid materials, which could build the new pathways to prepare new type of chromatographic materials and achieve multifunctional chromatographic properties. Herein, we present the first results for a simple synthesis of ␤-CD functionalized and ethane-bridged mesoporous organosilica spheres (CD-EMOS) by one-pot polymerization. As illustrated in Fig. 1, ␤-CD and ethane groups were introduced into the pore channels and pore wall framework during the forming process of the hybrid mesoporous materials [41–46], and the triazinyl groups as linker were attached into the pore channels as well. The ␤-CD hydroxyls of the materials were further derivatized with 3,5-dimethylphenyl isocyanate after the template removal [47]. Therefore, the CD-EMOS materials own ␤-CD, ethane, triazinyl and 3,5-dimethylphenyl groups, which can provide multiple interactions including the inclusion interaction, hydrophobic interaction, ␲–␲ interaction, anion exchange and hydrogen bonding interaction to achieve a multifunctional chromatographic features. All kinds of the chiral analytes, polycyclic aromatic hydrocarbons, anilines, phenols and acidic compounds were used as the targets for separation under a normal and a reversed phase modes in HPLC. Multiple chromatographic separation functions including racemic resolution, anion exchange and achiral separation and a typical feature of normal/reversed phase chromatography have been evaluated. Thus far we know of no other such multifunctional CSPs have been reported, which make an important research work.

2. Experimental 2.1. Chemicals and materials (±)-1-(4-Chlorophenyl)-ethanol, (±)-1-phenylethanol, (−)1-phenylethanol, (±)-triadimenol, (±)-1-phenyl-2-propanol, (±)-albendazole sulfoxide, (±)-diclofop, (±)-chlorpheniramine, (±)-propranolol, (±)-metoprolol, (±)-atenolol, (±)-trazodone, (±)-promethazine, (±)-mexiletine, (±)-metalaxyl and HPLC-grade methanol, n-hexane and isopropanol (IPA) were purchased from Sigma–Aldrich (Shanghai, CHN). (±)-1-(2-Methoxyphenyl) ethanol, (±)-mandelonitrile and (±)-1,2,3,4-tetrahydronaphthalen -1-ol were purchased from J&K Chemicals (Beijing, CHN). ␤-cyclodextrin (MCT-␤-CD) was Monochlorotriazinyl purchased from Nanjing Duly Biotech Co., Ltd (CHN). 1,2Bis(triethoxysilyl)ethane (BTEE) and 3-aminopropyltriethoxysilane (APS) were purchased from Gelest, Inc (USA). Cetyltrimethylammonium bromide (CTAB), diethylamine (DEA), benzene, phenol and other aromatic compounds were purchased from Sinopharm Chemical Reagent Corporation (CHN). Stainless-steel tube (150 mm × 4.6 mm I.D.) was purchased from Hanbon Technologies (CHN). Ultrapure water was produced on a PureLab Classic (DI, UK) water purification system. The type of C18 column was

SymmetryShieldTM RP18 (5 ␮m, 3.9 mm × 150 mm, Waters, USA). Commercial CD column was purchased from Shiseido Investment Co., Ltd (JP), and the type of column was chiral CD-Ph (5 ␮m, 4.6 mm × 150 mm). Unless otherwise specified, all chemicals and reagents were analytical grade and used as received without further purification. 2.2. Apparatus The HPLC system consisted of a Waters 515 HPLC pump, a Rheodyne injector (model, 7725) equipped with 20 ␮L sample loop, and a Waters 2487 double ␭ absorbance detector (Waters, USA). Chromatographic data were acquired and processed by Millennium 32 chromatography manager software (Waters, USA). The HPLC slurry packing apparatus type is 95551U (Alltech, USA). Elemental analyses were determined on vario EL (Element, GER). The FTIR (KBr pellet) spectrum was recorded on an IFS120HR Fourier transform infrared spectrometer (Bruker, GER). The SEM images were recorded on a JSM-5600LV scanning electron microscope (JEOL, JP). The HRTEM images were obtained on a Tecnai-G2-F30 S-Twin operating at 300 kV (FEI, USA). The XPS measurements were performed with a VG ESCALAB 210 instrument provided with a dual Mg/Mg anode X-ray source (Thermo, USA). 2.3. Preparation of CD-EMOS As shown in Fig. 1, MCT-␤-CD (0.94 g, 0.60 mmol) and APS (0.40 g, 1.71 mmol) were dissolved in 5 mL ultrapure water, and sodium bicarbonate (0.05 g, 0.60 mmol) was added and stirred for 30 min at 40 ◦ C. This mixture was then added to a CTAB (0.55 g, 1.50 mmol) and NaOH (0.24 g, 6 mmol) solution (3.5 mL of ethanol and 11 mL of water) and stirred for 30 s, after which BTEE (0.8 mL, 2.25 mmol) with 1.5 mL ethanol was added. After stirring for 30 min at room temperature, the clear solution was transferred into a Teflon-lined autoclave and kept at 80 ◦ C for 18 h without agitation. The white precipitate was collected by filtration, and air-dried at room temperature. The powder (1 g) was treated with 200 mL ethanol–HCl (100/1, v/v) at 50 ◦ C for 10 h, then washed by Soxhlet extraction over ethanol for 48 h to provide the surfactant-extracted ␤-CD-silica hybrid materials. ␤-CD-silica hybrid mesoporous organosilica spheres (1 g) were added to 20 mL dry pyridine in a 100 mL round bottom flask. 3,5-Dimethylphenylcarbamate (3 mL) was added dropwise to this stirred mixture. Under the protection of nitrogen, the reaction mixture was heated to 80 ◦ C and stirred for 24 h. The faint yellow precipitate was collected by filtration and washed by Soxhlet extraction over ethanol for 24 h, dried under vacuum at 60 ◦ C for 24 h. The whole procedure was repeated three times, ␤-CD-silica hybrid stationary phase could be prepared well. 2.4. Column packing The prepared CD-EMOS materials were packed into previously polished stainless steel columns (150 mm × 4.6 mm i.d.) by a slurry packing method under a constant packing pressure of 50 MPa with methanol as the pushing solvent. 3. Results and discussion 3.1. Characterization of CD-EMOS The scanning electron microscopy (SEM) image of CD-EMOS materials was shown in Fig. 2. The materials possess well-defined spherical morphology and narrow distribution of particle size with average of 7.5 ␮m in Fig. S1. The results of the nitrogen

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Fig. 1. The preparation route of CD-EMOS and pore structure.

XPS spectrum (Fig. S4). Moreover, the elemental analysis of CDEMOS demonstrated that the hybrid materials with ␤-CD groups loading is 0.22 ␮mol m−2 [48], which is higher than that of the ␤CD-bonded CSPs [13,49]. These results confirmed that the CD-EMOS materials were successfully prepared as new type of ␤-CD-silica hybrid HPLC CSPs.

3.2. Racemic resolutions in normal phase mode

Fig. 2. The SEM image of CD-EMOS.

adsorption/desorption measurements (Fig. 3) indicated that the CD-EMOS materials possessed a type-IV isotherms characteristic, large BET surface area (760 m2 /g), pore volume (1.22 cm3 /g), pore size distribution of 2–4 nm, and average pore diameter of 3.2 nm (the inset in Fig. 3). High resolution transmission electron microscopy (HRTEM) image (see Fig. S2) well illustrates the obvious wormhole-like mesopores structure on the edge of CD-EMOS. In addition, the existence of ␤-CD, triazinyl and ethane groups on the CD-EMOS were proved by the Fourier transform infrared spectrometer (FTIR) and X-ray photoelectron spectroscope (XPS) which gave obvious signals, the ␤-CD groups (1040–1160 cm−1 ), triazinyl groups (1640–1415 cm−1 ) and carbonyl groups (1708 cm−1 ) were observed by characteristic IR peaks in Fig. S3, and the bands between 2975 and 2857 cm−1 are attributed to the C–H stretching of ethane and methyl groups. The distinct C, N, O and Si element signals are observed in the

Fig. 3. The nitrogen sorption–desorption isotherms of CD-EMOS. The inset is the pore size distribution.

The most essential advantage of CD-EMOS stationary phases is the enantiomeric recognition and resolution abilities. Chromatographic resolutions were first attempted using n-hexane/IPA as mobile phase in the normal phase mode, and tetrachloromethane was used for the determination of dead time. The comparative enantioseparations of 12 racemates were obtained on the CDEMOS column and commercial ␤-CD column. The retention factors (k), separation factors (a) and resolutions (Rs ) are summarized in Table 1, and representative chromatograms on CD-EMOS column were shown in Fig. 4. The Rs of compounds 1, 2, 3, 6 and 12 were 1.53, 0.58, 4.65, 2.42 and 1.87, respectively, which were higher than the values of commercial ␤-CD-bonded column. Compounds 4, 7, 9, 10 and 11 were not separated under the same chromatographic conditions on commercial ␤-CD column. Moreover, retention factors of most compounds on CD-EMOS column were greater than that of on commercial ␤-CD-bonded column, which is probably due to more hydrogen bond interactions arising from triazine ring in normal phase mode.

3.3. Racemic resolutions in reversed phase mode As shown in Table 2, 12 racemates were employed for resolution evaluation on CD-EMOS column and commercial ␤-CD column in reversed phase mode, and the acetone was used for the determination of dead time. The chromatograms were shown in Fig. 5. Most of racemates on CD-EMOS column have an effective resolution, and the Rs of 9 racemates are greater than 1.5. For compounds 2 and 17, the Rs value reached 2.84 and 2.77 which are much higher than that of 0.74 and 0.58 on conventional ␤-CD-bonded column, and the resolutions of compounds 3, 15 and 19 have not been observed. A plateau was observed during the separation of propranolol, which was the characteristic of interconversion of isomers. Moreover, new 12 racemates with the similar configuration in Table S1 were enantioseparated successfully with wonderful Rs for the first time. These results indicated that the CD-EMOS materials are an excellent chiral stationary phase for the resolution of the racemates that can be largely attributed to the inclusion interactions. Meanwhile, the synergistic effect of multiple interactions such as hydrophobic interaction, ␲–␲ interaction and hydrogen bonding interaction may help to enhance the enantioresolution. On the other hand, the results also showed that the CD-EMOS materials were highly functionalized with ␤-CD groups that contribute to favorable enantioseparation performance.

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1 2 3 4 5 6 7 8 9 10 11 12

␤-CD-EMOS column

Racemates

1-Phenylethanola 1-Phenyl-2-propanola Mandelonitrilea Diclofopa 1-(4-Chlorophenyl)ethanola 1,2,3,4-Tetrahydronaphthalen-1-ola 1,3-Diphenylpropane1,3-diola Triadimenol b Albendazole Sulfoxideb Trazodonec Metalaxylc Promethazined

Commercial ␤-CD column

k1

k2

a

Rs

a

Rs

1.63 1.92 0.49 0.24 0.53

1.91 2.26 0.73 0.39 0. 71

1.17 1.17 1.49 1.63 1.35

1.53 0.58 4.65 4.02 1.48

k1 1.49 1.22 3.41 1.45 1.47

k2 1.75 1.30 3.64 1.45 1.88

1.18 1.06 1.07 1.00 1.27

1.00 0.31 0.38 /f 1.56

0.60

0.91

1.52

2.42

1.32

2.05

1.55

2.39

5.04

5.75

1.14

1.15

1.10

1.10

1.00

/f

1.20 2.14

1.42 2.37

1.18 1.11

0.69 0.71

0.49 25.89e

1.00 25.89

2.03 1.00

0.63 /f

4.86 5.13 7.23

5.37 6.01 8.25

1.10 1.17 1.14

0.60 1.27 1.87

3.90 9.60 10.82

3.90 9.60 10.95

1.00 1.00 1.01

/f /f 0.51

Mobile phase, a Hexane/IPA (90/10, v/v). b Hexane/IPA (70/30, v/v). c Hexane/IPA/TFA (90:10:0.1, v/v/v). d Hexane/IPA/DEA (95:5:0.1, v/v/v). e Hexane/IPA (60/40, v/v). f Could not be separated. Flow rate at 0.5 mL/min, detection wavelength, 254 nm. Temperature, 25 ◦ C.

Table 2 Resolution of racemates in reversed phase mode. No.

1 2 3 6 8 13 14 15 16 17 18 19

␤-CD-EMOS column

Racemates

a

1-Phenylethanol 1-Phenyl-2-propanola Mandelonitrile a 1,2,3,4-Tetrahydronaph thalen-1-ola Triadimenolb 1-(2-Methoxyphenyl) ethanola 1,3-Diphenylprop2-en-1-olc Chlorpheniraminea Propranolold Metoprolold Atenolold Mexiletined

Commercial ␤-CD column

k1

k2

a

Rs

k1

1.27 0.97 1.13 2.23

1.54 1.19 1.37 2.65

1.21 1.23 1.22 1.19

3.70 2.84 2.66 3.54

2.92 5.02 5.89 3.50

8.91 2.02

9.99 2.39

1.12 1.18

0.72 0.97

3.84e 4.14

4.13

4.79

1.16

3.73

1.05 2.50 0.66 2.33 0.81

1.44 2.91 0.91 2.81 0.94

1.38 1.16 1.39 1.21 1.15

2.64 3.10 2.77 0.65 2.72

k2

a

Rs

6.92 5.77 5.89 5.79

2.37 1.15 1.00 1.65

6.22 0.74 /f 2.23

4.44 5.99

1.16 1.45

1.17 1.93

20.41

46.26

2.27

3.89

4.57 19.74 0.78 2.58 1.86

4.57 35.12 0.93 2.87 1.86

1.00 1.78 1.20 1.12 1.00

/f 3.01 0.58 0.46 /f

Mobile phase, a Methanol/1%TEAA, pH 4.0 (40/60, v/v). b Methanol/0.2% formic acid (35/65, v/v). c Methanol/H2 O (50/50, v/v). d Acetonitrile/0.1% TEAA, pH 5.2 (15/85, v/v). e Methanol/0.2% formic acid (60/40, v/v). f Could not be separated. Flow rate at 0.8 mL/min. Detection, UV 254 nm. Temperature, 25 ◦ C.

3.4. Achiral separations in reversed phase mode Besides the chiral resolution abilities of the CD-EMOS materials, the reversed chromatographic behaviors have been further evaluated using the Tanaka test mixture as shown in Fig. 6a, 7 kinds of the analytes were successfully separated. The hydrogen bonding capacity (˛C/P = kcaffeine /kphenol ) was determined as 2.8, which is much higher than LiChrosorb C18 column (5 ␮m, 4.6 mm × 150 mm) and other reported stationary phases [50]. These might be attributed to the stronger hydrogen bonding interaction from the N and O atoms on the triazinyl and ␤-CD groups with analytes. Moreover, the separation of geometric isomers was also observed using four isomers in Fig. 6b, the separation selectivity of CD-EMOS stationary phase was almost equal to commercial C18 stationary phase (Fig. S5), and the steric selectivity (˛T/O = ktriphenylene /ko-terphenyl ) was calculated to be 1.97. Trans/cis stilbene are also separated (Fig. S6), which is verified again the steric recognizing ability. As for the CD-EMOS stationary phase, the effective combination of ␤-CD and the multiple interactions would account for the enhanced performance of the columns for the separation of isomers and the polycyclic aromatic hydrocarbons. The retention behaviors of various polycyclic aromatic hydrocarbons on CD-EMOS column were also evaluated in reversed

phase mode using the mobile phase of methanol/H2 O (60/40, v/v) at 0.8 mL/min. The methylene selectivity tests were performed using a series of alkyl benzenes. The well separations with symmetrical peak shape can be obtained in Fig. 7a. The retention factors significantly increases with the increasing of aliphatic carbon chain as shown in Fig. S7. The value of the methylene selectivity (˛CH2 = kpentylbenzene /kbutylbenzene ) is 1.46, which is higher than mentioned above LiChrosorb C18 column (˛CH2 = 1.42) [27], indicating that the CD-EMOS stationary phase possessed excellent methylene selectivity probably due to the well coverage with ethane groups on the surface of pore walls, which can separate the compounds identifying only a methylene group. The planar selectivity were achieved as depicted in Fig. 7b, 5 kinds of the analytes with rigid plane structure were separated by baseline and showed enhanced chromatographic retention with the increasing phenyl rings, which demonstrated the good planar selectivity that can be mainly attributed to the ␲–␲ interaction provided by the phenyl groups. Moreover, the retention factors of biphenyl and pterphenyl are higher than those of naphthalene and pyrene (Figs. 7c and S7), which indicated the better linear selectivity compared to the planar selectivity. Moreover, these compounds were separated on commercial C18 column under the same chromatographic conditions in Fig. S8. The alkyl benzenes and polycyclic aromatic

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Fig. 4. Chiral separation of racemates in normal phase mode. Chromatographic condition, (a–g) Hexane/IPA (90/10, v/v). (h and i) Hexane/IPA (70/30, v/v). (j and k) Hexane/IPA/TFA (90:10:0.1, v/v/v). (l) Hexane/IPA/DEA (95:5:0.1, v/v/v). Flow rate at 0.5 mL/min, detection wavelength, 254 nm. Temperature, 25 ◦ C.

hydrocarbons were also well separated, and showed the strong retention for selected compounds with multiple phenyl rings and alkyl carbons. Next, some basic compounds were used for the separation evaluation of the CD-EMOS column. As shown in Fig. 8a, an excellent separation of 7 kinds of anilines mixtures with symmetrical peaks can be achieved in unbufferred mobile phase (methanol/water,

40/60, v/v). Strong retention of 1-naphthylamine was observed, which is ascribed to the strong hydrophobic and ␲–␲ interactions. The successful separations of the anilines including phenylenediamine on the CD-EMOS column are probably due to the presence of relatively few silanol groups on the surface of the ethane-bridged hybrid pore walls. Moreover, various phenols mixtures were also separated under the same chromatographic condition in Fig. 8b,

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Fig. 5. Chiral separation of racemates in reversed phase mode. Chromatographic condition, (a–f) Methanol/1%TEAA, pH 4.0 (40/60, v/v). (g) Methanol/0.2% formic acid (35/65, v/v). (h) Methanol/H2 O (50/50, v/v). (i–l) Acetonitrile/0.1% TEAA, pH 5.2 (15/85, v/v). Flow rate at 0.8 mL/min, detection wavelength, 254 nm. Temperature, 25 ◦ C.

and the order of separation is consistent with the polarity of the analytes from strong to weak which showed the typical feature of the reversed phase chromatography. More importantly, a mixture including chiral and achiral compounds was separated simultaneously accompanied by the enatioresolution as shown in Fig. 8c. The aniline and phenol compounds did not interfere with the chiral separation of the (±)-1-phenylethanol with the Rs at 3.54, which

demonstrates that CD-EMOS materials have good enantioselectivity under the existence of various compounds, mainly because of the inclusion interaction from ␤-CD. Correspondingly, the mixtures of anilines and phenols were also separated on commercial C18 column in Fig. S9. These results showed that the CD-EMOS column has an advantage for the separations of multiple types of compounds including racemates.

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Fig. 6. Separation of the mixtures of Tanata and geometric isomers including (a) uracil (1), phenol (2), caffeine (3), butylbenzene (4), o-terophenyl (5), pyrene (6) and triphenylene (7). (b) o-Terphenyl (1), m-terphenyl (2), p-terphenyl (3) and triphenylene (4). Mobile phase, methanol/H2 O (60/40, v/v) at 0.8 mL/min. Detection, UV 254 nm. Temperature, 25 ◦ C.

Fig. 8. Separation of the anilines, phenols and chiral mixtures including (a) maminophenol (1), o-phenylenediamine (2), phenylamine (3), 4-methylaniline (4), p-nitroaniline (5), 3-bromoaniline (6) and 1-naphthylamine (7). (b) 1,4-Benzenediol (1), phenol (2), 3,5-dimethylphenol (3), acetophenone (4) and p-tert-butylphenol (5). (c) 1,4-Benzenediol (1), o-phenylenediamine (2), (−)-1-phenylethanol (3), (+)-1phenylethanol (4) and p-tert-butylphenol (5). Mobile phase, methanol/H2 O (40/60, v/v) at 0.8 mL/min. Detection, UV 254 nm. Temperature, 25 ◦ C.

Fig. 7. Separation of the alkyl benzenes and polycyclic aromatic hydrocarbons mixtures including (a) benzene (1), toluene (2), ethylbenzene (3), propylbenzene (4), butylbenzene (5) and pentylbenzene (6). (b) Benzene (1), naphthalene (2), anthracene (3), pyrene (4) and benzanthracene (5). (c) Benzene (1), naphthalene (2), biphenyl (3), anthracene (4) and p-terphenyl (5). Mobile phase, methanol/H2 O (60/40, v/v) at 0.8 mL/min. Detection, UV 254 nm. Temperature, 25 ◦ C.

3.5. Anion exchange in reversed phase mode The triazinyl ring, the linker, is an important functional group which can provide the weak anion exchange interaction [27]. As can be seen from Fig. 9, five acidic compounds were separated by baseline according to the acidity from weak to strong follow the order of p-hydroxyphenylacetic acid, benzoic acid, terephthalic acid, 1-naphthalenesulfonic acid and 1,5-naphthalenedisulfonic acid under the methanol/0.05 M sodium dihydrogen phosphate, pH

Fig. 9. Separation of acidic mixtures including p-hydroxyphenylacetic acid (1), benzoic acid (2), terephthalic acid (3), 1-naphthalenesulfonic acid (4) and 1,5naphthalenedisulfonic acid (5). Mobile phase, methanol/0.05 M sodium dihydrogen phosphate, pH 3.5 (60/40, v/v) at 0.8 mL/min. Detection, UV 254 nm. Temperature, 25 ◦ C.

3.5 (60/40, v/v). The retention factors obviously increased as the enhanced acidity (Fig. S10). These results are mainly attributed to the anion exchange interaction. Moreover, the separation of above acidic compounds was tested on commercial C18 column under the methanol/0.05 M sodium dihydrogen phosphate, pH 3.5 (40/60, v/v) as shown in Fig. S11, almost inverse order with the separation results on CD-EMOS column was obtained according to the acidity from strong to weak, which is consistent with reversed-phase chromatographic rule by hydrophobic interaction force.

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Fig. 10. Separation of mixtures including 1,2-benzenediol (1), 1,3-benzenediol (2) and 1,4-benzenediol (3). Mobile phase, n-hexane/IPA (90/10, v/v) at 0.8 mL/min. Detection, UV 254 nm. Temperature, 25 ◦ C.

3.6. Achiral separations in normal phase mode To further explore the CD-EMOS stationary phase, the polycyclic aromatic hydrocarbons compounds are further studied under the normal phase condition. As shown in Fig. S12, these compounds were easy to achieve a good separation using n-hexane as mobile phase, and the analytes with planar structure have strong retention factors on the CD-EMOS stationary phase (Fig. S13), which is mainly due to the ␲–␲ interactions. Moreover, trans-cis stilbene are also separated by baseline and the peak shape was presented well in Fig. S14, which showed the favorable cis–trans recognition abilities at the normal phase condition. Various kinds of aniline and phenol compounds were separated under the n-hexane/IPA (90/10, v/v) as shown in Fig. S15a and b. The elution order of these compounds was in good agreement with the polarity from weak to strong, which showed the typical feature of the normal phase chromatography. More interestingly, the mixtures of aniline, phenol and chiral analytes have been successfully separated in Fig. S15c, and the enantioseparation of (±)-1-phenylethanol was not interfered with other aniline and phenol compounds. The acidic mixtures have been separated under the n-hexane/IPA/TFA (90/10/0.1, v/v/v) in Fig. S16. Moreover, the mixtures of o-, m-, p-benzenediol also have been separated at n-hexane/IPA (90/10, v/v) in Fig. 10, which indicated the good regioselectivity for the CD-EMOS stationary phases under normal phase mode. These separating results may be mainly attributed to the strong ␲–␲ and hydrogen bonding interactions. The CD-EMOS column were evaluated by various compounds under normal/reversed phase modes for more than a month, and the column keeps good separation selectivity for the same compound. The retention time is almost the same. Therefore, the CD-EMOS column prepared in this paper owns favorable separation selectivity and stability. 4. Conclusions In summary, we have succeeded in constructing an organic–inorganic hybrid way by simple one-pot polymerization reaction to prepare spherical ␤-CD-silica hybrid materials as new type of HPLC CSPs. Various functional groups including ␤-CD, ethane, triazinyl and 3,5-dimethylphenyl groups have been introduced into the pore channels and pore wall framework of mesoporous materials. These functional groups can introduce multiple molecular interactions (e.g. inclusion interaction, hydrophobic interaction, ␲-␲ interaction, ion exchange, hydrogen bonding interaction and stereochemical interaction),

which endow the materials with multifunctional chromatographic capabilities including racemic resolution, anion exchange and achiral separation and the typical features of normal and reversed phase chromatography for selected chiral analytes, polycyclic aromatic hydrocarbons, anilines, phenols and acidic compounds. Compared with the separation results of commercial C18 and ␤-CD columns, the CD-EMOS column prepared in this paper also showed excellent achiral and chiral separations for selected compounds. Organic–inorganic hybrid strategy represents a very efficient methodology for the preparation of CD-silica hybrid CSPs and thus opens a new way to access new type of HPLC CSPs (such as macrocyclic compounds, protein or polysaccharides-based CSPs). These materials could have important applications in HPLC CSPs, large-scale preparation of chiral drugs and environmental protection (e.g., the remove of the heavy metal or organic pollutants in waste water) [42,43,51]. Acknowledgements This work is supported by the National Natural Science Foundation of China (Nos. 21405162, 21405161, 21275150 and 20675085) and the Personnel Training Project of the West Light Foundation of the Chinese Academy of Sciences (2012, to Litao Wang). 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. 2015.01.023. References [1] S.-M. Xie, Z.-J. Zhang, Z.-Y. Wang, L.-M. Yuan, Chiral metal–organic frameworks for high-resolution gas chromatographic separations, J. Am. Chem. Soc. 133 (2011) 11892–11895. [2] A.A. Younes, D. Mangelings, Y. Vander Heyden, Chiral separations in reversedphase liquid chromatography: evaluation of several polysaccharide-based chiral stationary phases for a separation strategy update, J. Chromatogr. A 1269 (2012) 154–167. [3] J.P. Smuts, X.-Q. Hao, Z. Han, C. Parpia, M.J. Krische, D.W. Armstrong, Enantiomeric separations of chiral sulfonic and phosphoric acids with barium-doped cyclofructan selectors via an ion interaction mechanism, Anal. Chem. 86 (2014) 1282–1290. [4] X. Kuang, Y. Ma, H. Su, J. Zhang, Y.-B. Dong, B. Tang, High-performance liquid chromatographic enantioseparation of racemic drugs based on homochiral metal–organic framework, Anal. Chem. 86 (2014) 1277–1281. [5] P. Peluso, V. Mamane, S. Cossu, Homochiral metal–organic frameworks and their application in chromatography enantioseparations, J. Chromatogr. A 1363 (2014) 11–26. [6] T.J. Ward, K.D. Ward, Chiral separations: a review of current topics and trends, Anal. Chem. 84 (2012) 626–635. [7] R.M. Woods, D.C. Patel, Y. Lim, Z.S. Breitbach, H. Gao, C. Keene, G. Li, L. Kürti, D.W. Armstrong, Enantiomeric separation of biaryl atropisomers using cyclofructan based chiral stationary phases, J. Chromatogr. A 1357 (2014) 172–181. [8] M. Ahmed, A. Ghanem, Chiral ␤-cyclodextrin functionalized polymer monolith for the direct enantioselective reversed phase nano liquid chromatographic separation of racemic pharmaceuticals, J. Chromatogr. A 1345 (2014) 115–127. [9] Y.S. Nanayakkara, R.M. Woods, Z.S. Breitbach, S. Handa, L.M. Slaughter, D.W. Armstrong, Enantiomeric separation of isochromene derivatives by high-performance liquid chromatography using cyclodextrin based stationary phases and principal component analysis of the separation data, J. Chromatogr. A 1305 (2013) 94–101. [10] T. Ikai, Y. Okamoto, Structure control of polysaccharide derivatives for efficient separation of enantiomers by chromatography, Chem. Rev. 109 (2009) 6077–6101. [11] T.L. Chester, Recent developments in high-performance liquid chromatography stationary phases, Anal. Chem. 85 (2013) 579–589. [12] J. Shen, Y. Zhao, S. Inagaki, C. Yamamoto, Y. Shen, S. Liu, Y. Okamoto, Enantioseparation using ortho- or meta-substituted phenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography, J. Chromatogr. A 1286 (2013) 41–46. [13] C. Lin, W. Liu, J. Fan, Y. Wang, S. Zheng, R. Lin, H. Zhang, W. Zhang, Synthesis of a novel cyclodextrin-derived chiral stationary phase with multiple urea linkages and enantioseparation toward chiral osmabenzene complex, J. Chromatogr. A 1283 (2013) 68–74.

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