Grafting (S)-2-Phenylpropionic Acid on Coordinatively Unsaturated Metal Centers of MIL−101(Al) Metal–Organic Frameworks for Improved Enantioseparation

Chiral metal–organic frameworks (cMOFs) are emerging chiral stationary phases for enantioseparation owing to their porosity and designability. However, a great number of cMOF materials show poor separation performance for chiral drugs in high-performance liquid chromatography (HPLC). The possible reasons might be the irregular shapes of MOFs and the low grafting degree of chiral ligands. Herein, MIL−101−Ppa@SiO2 was synthesized by a simple coordination post-synthetic modification method using (S)-(+)-2-Phenylpropionic acid and applied as the chiral stationary phase to separate chiral compounds by HPLC. NH2−MIL−101−Ppa@SiO2 prepared via covalent post-synthetic modification was used for comparison. The results showed that the chiral ligand density of MIL−101−Ppa@SiO2 was higher than that of NH2−MIL−101−Ppa@SiO2, and the MIL−101−Ppa@SiO2 column exhibited better chiral separation performance and structural stability. The binding affinities between MIL−101−Ppa@SiO2 and chiral compounds were simulated to prove the mechanism of the molecular interactions during HPLC. These results revealed that cMOFs prepared by coordination post-synthetic modification could increase the grafting degree and enhance the separation performance. This method can provide ideas for the synthesis of cMOFs.


Introduction
Chiral compounds, which exist in two forms, the R-enantiomer and S-enantiomer, exhibit identical physical and chemical properties. In the chiral microenvironment, one enantiomer may be active, but the other may show negative or toxic effects [1,2]. For example, S-hydrochloroquine and S-chloroquine have a higher response to SARS-CoV-2 when studying COVID-19 [3]. With the increasing research and development of chiral compounds, they have been widely applied in the fields of pharmacology, agriculture, flavors and life science [4][5][6]. In order to obtain a high efficacy of pure optical chiral compounds, the discrimination of enantiomers appears particularly necessary [7,8].
To date, many technologies for enantiomeric separation have been developed, such as enantioselective crystallization [9], membrane resolution [10,11], biokinetic resolution [12] and chromatography. Among them, chromatography technology attracts more attention owing to its simple operation and wide usage [13]. Among various chromatographic methods, HPLC serves as the most applicable enantioseparation strategy [14,15] due to its high efficiency and low cost [16]. It is well known that the enantiomeric recognition of chiral stationary phases (CSPs) is the key parameter that significantly influences the performance of enantioseparation. Therefore, many researchers pay more attention to developing novel CSPs. In recent decades, a variety of chiral stationary phases have emerged. Cellulose, cyclodextrin and other traditional materials were first applied as CSPs in the 1990s [17][18][19].

Preparation of Chiral Stationary Phases 2.2.1. Synthesis of SiO 2 -NH 2
The original silica microspheres were activated first. Briefly, spherical silica gel (5.0 g) was placed in 100 mL of HCl solution (20%, v/v) and subjected to ultrasound treatments. The mixture was then stirred in a three-necked round-bottom flask at 90 • C for 3 h. The product was washed with ultrapure water to a neutral pH and vacuum-dried at 110 • C overnight.
Amino-functionalized SiO 2 was obtained according to the method of Chen et al. [46]. Briefly, activated silica microspheres (5.0 g) were mixed in 200 mL of anhydrous ethanol and stirred at room temperature for 30 min. Then, 2.0 mL of 3-Aminopropyltriethoxysilane (APTES) was introduced dropwise into the mixture and stirred at 70 • C for 24 h. The obtained SiO 2 −NH 2 microspheres were finally washed with anhydrous ethanol more than 3 times and vacuum dried at 70 • C for 10 h.

Synthesis of MIL−101@SiO 2
The SiO 2 microspheres were first modified with −COOH before preparing MIL−101@SiO 2 [47]. Succinic anhydride (25.0 g) was dissolved in 200 mL of DMF, and nitrogen was blown onto the mixture for 10 min to remove oxygen. The obtained SiO 2 −NH 2 microspheres (5.0 g) were then dispersed into the above solution and reacted at room temperature for 24 h. The product (SiO 2 −COOH) was treated with DMF and anhydrous ethanol more than 3 times and dried overnight.
The construction of MIL−101@SiO 2 was carried out according to the method reported by Bromberg et al. [48]. Briefly, 1.0 g of carboxylic silica spheres (SiO 2 −COOH) and aluminum chloride hexahydrate (AlCl 3 ·6H 2 O) (1.2 mmol, 0.2897 g) were mixed in 300 mL of DMF and stirred at room temperature for 3 h. Subsequently, 299.034 mg of terephthalic acid (BDC) dissolved in 300 mL DMF was added dropwise to the mixture and heated to 130 • C for 48 h. The products were first centrifuged using DMF at 700 rpm to remove the MIL−101 crystal impurities and then washed with dichloromethane more than 3 times.
2.2.3. Synthesis of NH 2 −MIL−101@SiO 2 NH 2 −MIL−101 (Al) microspheres were prepared using the same synthesis method as that used for MIL−101 (Al) with slight alterations [48]. In brief, the organic linker was changed from BDC to 2-amino-terephthalic acid (amino-BDC), and the other experimental conditions were kept the same.

Synthesis of MIL−101−Ppa@SiO 2
First, 0.5 g of MIL−101@SiO 2 and 18 mmol (2500 µL) S-2-Ppa were added to 50 mL of DMF and agitated at 100 • C for 9 h. The product was then cleaned using the same treatment procedure as that used for MIL−101@SiO 2 .  [49]. First, 2500 µL of S-2-Ppa was added to 20 mL of a dichloromethane solution of PyBrOP (18 mmol, 8.3914 g) and stirred at room temperature for 1 h. Then, NH 2 −MIL−101@SiO 2 (0.5 g) and DMAP (36 mmol, 4.3981 g) dissolved in dichloromethane (30 mL) were added. The resulting mixture was stirred at room temperature for 4 days. These materials were washed with dichloromethane and vacuum dried.

Characterization
Scanning electron microscopy (SEM) was conducted on a JSM-7401F, 20 kV instrument (Jeol, Tokyo, Japan). Before observation, the samples were covered with gold to increase their conductivity. The infrared absorption spectra were taken on an Avatar 370 infrared Fourier transform spectrometer (Nicolet, Oshkosh, WI, USA). The powder X-ray diffraction (PXRD) data were collected on a Bruker D8 Focus diffractometer (Bruker, Karlsruhe, Germany). The surface area and porosity were recorded with the Quantachrome Autosorb IQ3 (Quantachrome, Boynton Beach, FL, USA) using N 2 adsorption at 77 K. NMR samples were prepared in 2.5 mm NMR tubes, and liquid 1 H NMR data were recorded on a Bruker AV 300 spectrometer (Bruker, Germany). Before 1 H-NMR testing, MIL−101−Ppa@SiO 2 was dissolved in a mixture of deuterated dimethyl sulfoxide (d6-DMSO) and 20% deuterated hydrochloric acid (DCl) in D 2 O (molar ratio 7:1) [50].

Column Packing
The packed column was prepared using a high-pressure slurry packing method. Before packing them into the column, the obtained materials were activated in dichloromethane for three days. The materials (0.5 g) were then dispersed in 50 mL of n-hexane/isopropanol (v:v = 95:5) under ultrasonication for 5 min and packed into a stainless-steel column (100 mm long × 2.1 mm i.d, IDEX CORPORATION, Lake Forest, IL, USA) using n-hexane/isopropanol (v:v = 95:5) under a pressure of 50 MPa for 50 min. The MIL−101−Ppa@SiO 2 and NH 2 −MIL−101−Ppa@SiO 2 packed columns were prepared following the same procedure. The packed column was conditioned/equilibrated with isopropanol at a flow rate of 0.1 mL·min −1 for 8 h before chromatographic experiments [51].

HPLC
HPLC analysis was performed on the Shimadzu series system with a Shimadzu LC-20AT pump and a Shimadzu SPD-20A UV detector (Shimadzu, Kyoto, Japan). In order to choose an appropriate eluent, various compositions of mobile phase systems were compared and optimized, such as methanol-water, n-hexane-isopropanol and nhexane-dichloromethane. The working solutions of chiral compounds were prepared at a concentration of 1 mg·mL −1 . All HPLC separations were carried out at a 40 • C temperature. The flow rate was 0.5 mL·min −1 , and the injection volume was 2 µL.

Molecular Docking
The interactions between modified MOFs (MIL−101−Ppa and NH 2 −MIL−101−Ppa) and racemates were investigated by molecular docking using the Autodock Vina 1.1.2 software [52]. Structural models of the modified MOFs were built on the basis of the MIL−101(Cr) crystal structure [53] via a modification of the metal-coordination network (MIL−101−Ppa) or the linker (NH 2 −MIL−101−Ppa), as illustrated in Scheme 1. MIL−101 has two types of mesoporous cages with diameters of ∼29 and 34 Å, formed by 20 and 28 hybrid super-tetrahedra (ST) building blocks, respectively. We chose two adjacent cages (denoted as ST20 and ST28) for modification, which were immersed in a cubic box of isopropanol solvent (one of the mobile phases in HPCL experiments) and then optimized via energy minimization using the GROMACS 2018 software [54] for subsequent docking calculations. The force-field parameters of the modified MOFs were generated by the OBGMX toolkit [55], and atomic charges of S-2-Ppa and linker groups were computed with the AM1-BCC charge model [56,57] using the "antechamber" tool [58]. The parameters of isopropanol were taken from previous work [59]. During docking, the search space was defined by the interior of the ST20 and ST28 cages and the channel between the two cages, and potential binding to the exterior of MIL−101 MOFs was blocked by isopropanol molecules. Such a task can be completed with the Visual Molecular Dynamics (VMD) software [60] by removing the solvent molecules in the search space from the energyminimized structures. Note that there is no built-in parameter for the Al ion in the Autodock Vina software, and we used the Fe ion instead for a rough estimate. For each compound, docking was run 100 times with random seeds, and the best binding model with the lowest binding energy for each run was used for data collection. and potential binding to the exterior of MIL−101 MOFs was blocked by isopropanol molecules. Such a task can be completed with the Visual Molecular Dynamics (VMD) software [60] by removing the solvent molecules in the search space from the energy-minimized structures. Note that there is no built-in parameter for the Al ion in the Autodock Vina software, and we used the Fe ion instead for a rough estimate. For each compound, docking was run 100 times with random seeds, and the best binding model with the lowest binding energy for each run was used for data collection.

Results and Discussion
In order to evaluate the performance of cMOFs@SiO2 prepared by coordination PSM, the MIL−101 crystal was deposited onto the surface of SiO2−COOH by solvothermal synthesis.

Results and Discussion
In order to evaluate the performance of cMOFs@SiO 2 prepared by coordination PSM, the MIL−101 crystal was deposited onto the surface of SiO 2 −COOH by solvothermal synthesis. (S)-(+)-2-Phenylpropionic acid (S-Ppa) was then grafted onto the unsaturated metal sites of MIL−101@SiO 2 frameworks through a coordination reaction. This method provided a facile and short-time synthesis route to obtain chiral MIL−101−Ppa@SiO 2 . Moreover, NH 2 −MIL−101−Ppa@SiO 2 was prepared by covalent PSM for comparison. The structures of MOF crystals and the general procedures for separation are illustrated in Scheme 1.

Characterization
The morphologies of SiO 2 −COOH, MIL−101@SiO 2 , NH 2 −MIL−101@SiO 2 , MIL−101 −Ppa@SiO 2 and NH 2 −MIL−101−Ppa@SiO 2 materials were characterized by Energy-Dispersive Spectroscopy (EDS) mapping and scanning electron microscopy (SEM). In the EDS mapping images of MIL−101@SiO 2 and NH 2 −MIL−101@SiO 2 , the element Al was homogeneously dispersed in silica, which proved the formation of both MOFs@SiO 2 composites ( Figure S1). In addition, SEM was used to further observe the morphology of MOFs@SiO 2 . As shown in Figure 1a-e, all of the materials showed good dispersity and a regular shape. As shown in Figure 1a, the SiO 2 −COOH microspheres had a smooth surface with an average diameter of about 5 µm. After the immobilization of MIL−101 on the surface of SiO 2 microspheres, MIL−101@SiO 2 presented a rough surface. The average diameter of MIL−101@SiO 2 increased to 5.2 µm (Figure 1b), which indicated the successful synthesis of MIL−101@SiO 2 , and the shell thickness was about 200 nm. Though a similar rough surface of NH 2 −MIL−101@SiO 2 could be observed, the shell thickness of NH 2 −MIL−101 (140 nm) was a little thinner than that of MIL−101@SiO 2 ( Figure 1d). It is presumed that the growth of the MIL−101@SiO 2 crystal was better than that of NH 2 −MIL−101@SiO 2 . Figure 1c,e shows the images of MIL−101−Ppa@SiO 2 and NH 2 −MIL−101−Ppa@SiO 2 morphologies. Few changes were observed after modification, which suggested that the post-synthetic modification with Ppa might not affect the morphologies of MOFs. Figure 1a-e, all of the materials showed good dispersity and a regular shape. As shown in Figure 1a, the SiO2−COOH microspheres had a smooth surface with an average diameter of about 5 μm. After the immobilization of MIL−101 on the surface of SiO2 microspheres, MIL−101@SiO2 presented a rough surface. The average diameter of MIL−101@SiO2 increased to 5.2 μm (Figure 1b), which indicated the successful synthesis of MIL−101@SiO2, and the shell thickness was about 200 nm. Though a similar rough surface of NH2−MIL−101@SiO2 could be observed, the shell thickness of NH2−MIL−101 (140 nm) was a little thinner than that of MIL−101@SiO2 (Figure 1d). It is presumed that the growth of the MIL−101@SiO2 crystal was better than that of NH2−MIL−101@SiO2. Figure 1c, e shows the images of MIL−101−Ppa@SiO2 and NH2−MIL−101−Ppa@SiO2 morphologies. Few changes were observed after modification, which suggested that the post-synthetic modification with Ppa might not affect the morphologies of MOFs.  Figure 1f. In the spectrum of SiO2−COOH, the peak intensity at 1093 cm −1 was ascribed to the vibration of Si-O-Si [32]. An adsorption peak at 1510 cm −1 in the spectrum of MIL−101 was attributable to the asymmetric and symmetric vibrations of the benzene group in MIL−101 [61]. These characteristic peaks of SiO2−COOH and MIL−101 could be observed in the spectrum of MIL−101@SiO2, which indicated that the MIL−101@SiO2 composite was formed [32]. Comparing MIL−101@SiO2 with MIL−101−Ppa@SiO2, the peak of the Al-O stretch slightly Fourier-transform infrared spectroscopy (FT-IR) was used to further confirm the preparation of MIL−101@SiO 2 , NH 2 −MIL−101@SiO 2 , MIL−101−Ppa@SiO 2 and NH 2 −MIL− 101−Ppa@SiO 2 materials, as shown in Figure 1f. In the spectrum of SiO 2 −COOH, the peak intensity at 1093 cm −1 was ascribed to the vibration of Si-O-Si [32]. An adsorption peak at 1510 cm −1 in the spectrum of MIL−101 was attributable to the asymmetric and symmetric vibrations of the benzene group in MIL−101 [61]. These characteristic peaks of SiO 2 −COOH and MIL−101 could be observed in the spectrum of MIL−101@SiO 2 , which indicated that the MIL−101@SiO 2 composite was formed [32]. Comparing MIL−101@SiO 2 with MIL−101−Ppa@SiO 2 , the peak of the Al-O stretch slightly shifted from 594 cm −1 to 596 cm −1 , which resulted from the introduction of Ppa to MIL−101 via coordination coupling [62]. In the spectrum of NH 2 −MIL−101@SiO 2 , the appearance of the characteristic band at 1666 cm −1 was due to the -NH 2 linkage, which proved the successful synthesis of NH 2 −MIL−101 [63]. Evidence that NH 2 −MIL−101 was deposited onto the SiO 2 microspheres could also be obtained from the spectrum of NH 2 −MIL−101@SiO 2 in the same way as MIL−101@SiO 2 . Comparing the spectrum of NH 2 −MIL−101@SiO 2 with that of NH 2 −MIL−101−Ppa@SiO 2 , the characteristic peak at 2975 cm −1 originating from the N-H bond of NH 2 −MIL−101 was increased, suggesting that the amide condensation reaction occurred between Ppa and NH 2 −MIL−101@SiO 2 . A characteristic peak appeared at 1655 cm −1 (NH 2 −MIL−101−Ppa@SiO 2 ), which was attributed to symmetric stretching vibrations of −C=O in amide groups [64].

MOFs@SiO2. As shown in
The powder X-ray diffraction (PXRD) patterns of the materials are shown in Figure 1g. As can be seen, a pronounced peak in the range of 20-25 • corresponding to the spectrum of SiO 2 appeared in the pattern of MIL−101@SiO 2 [32], which proved the successful growth of MIL−101 on the SiO 2 microspheres. According to the pattern of NH 2 −MIL−101@SiO 2 , the same evidence could be obtained to demonstrate the successful preparation of NH 2 −MIL−101@SiO 2 composites. In addition, characteristic peaks at 2θ = 9.23 • and 18 • with high intensity belonged to MIL−101 crystals [65]. The same signal could be observed in the PXRD pattern of MIL−101@SiO 2 and NH 2 −MIL−101@SiO 2 , which also indicated the successful synthesis of MOFs@SiO 2 composites. However, the characteristic peaks of NH 2 −MIL−101@SiO 2 were wider and weaker than those of MIL−101@SiO 2 , which indicated the poorer crystallinity of NH 2 −MIL−101@SiO 2 . This broad Bragg reflection of NH 2 −MIL−101@SiO 2 might result from the small size effect [66]. Comparing the crystal peaks before and after modification, MIL−101−Ppa@SiO 2 and NH 2 −MIL− 101−Ppa@SiO 2 had no new diffraction peaks, so it was assumed that Ppa did not influence the crystal structure of MOFs.
The . Meanwhile, the average pore size was reduced from 1.25 nm to 1.14 nm, which indicated that the chiral ligand occupied the channel space but might not destroy the crystal structure. This is in good agreement with the results of FTIR and XRD, which further confirmed that MIL−101@SiO 2 had better stability.
In order to obtain the grafting degree of MIL−101−Ppa@SiO 2 , 1 H NMR analysis was performed. The NMR spectrum shows obvious peaks of BDC and Ppa in Figure S2. According to Yan's and Ma's reports, the peaks around 7.95 and 7.18 ppm were assigned to the hydrogen signals of the BDC and Ppa ligands, respectively [68,69]. The grafting ratio was defined as the ratio of the weights of grafted chiral ligands to the weights before grafting [70]. According to this definition, the grafting degree of MIL−101−Ppa@SiO 2 was about 22.92%, which proved that Ppa was successfully modified. Furthermore, this grafting degree was higher than that of NH 2 −MIL−101−Ppa reported by Yan's synthesis [69]. In order to obtain the grafting degree of MIL−101−Ppa@SiO2, 1 H NMR analysis was performed. The NMR spectrum shows obvious peaks of BDC and Ppa in Figure S2. According to Yan's and Ma's reports, the peaks around 7.95 and 7.18 ppm were assigned to the hydrogen signals of the BDC and Ppa ligands, respectively [68,69]. The grafting ratio was defined as the ratio of the weights of grafted chiral ligands to the weights before grafting [70]. According to this definition, the grafting degree of MIL−101−Ppa@SiO2 was about 22.92%, which proved that Ppa was successfully modified. Furthermore, this grafting degree was higher than that of NH2−MIL−101−Ppa reported by Yan's synthesis [69].

HPLC Separation of Racemic Compounds
In order to investigate the chiral separation ability of the MIL−101−Ppa@SiO2 CSPs, various types of racemic compounds were employed as targets. Herein, nine racemic compounds were used: rac-ketoprofen, (±)-naproxen, rac-ibuprofen, R, S-phenylethanol, R, S-1-Phenyl-1,2-ethanediol, (±)-mandelic acid, DL-alpha-methylbenzylamine, DL-phenylglycinol and 2-amino-1,2-diphenylethanol racemates, which were derived from non-steroidal drugs, phenylethanol and its derivatives and are widely used in the pharmaceutical industry, the cosmetic industry, protein engineering and biological chemistry [71][72][73][74]. The structures of these chiral compounds are shown in Figure 3. The separation results of all racemic compounds after optimizing the chromatographic mobile phase conditions are shown in Figure 4 and Table 1. The baseline separation of naproxen and ibuprofen with corresponding symmetric peak shapes was achieved, which exhibited the high enantioselectivity and good separation performance of the MIL−101−Ppa@SiO2 packed column. The common structural feature of these enantiomers was that they all possessed hydroxyl groups, which might be capable of hydrogen-bonding interactions with the carboxyl of MIL−101−Ppa@SiO2 [75]. Additionally, other possible interactions, including van der Waals forces, hydrophobic interactions and π-π interactions, might also contribute to the chiral recognition among the chiral target compounds and the MIL−101−Ppa@SiO2 column. For the other enantiomers, including ketoprofen, mandelic acid, alpha-methylbenzylamine, DL-phenylglycinol and 2-amino-1,2-diphenylethanol, complete separation could not be achieved. According to the structures of these racemic compounds, it was assumed that hydrogen-bonding interactions, π-π interactions and hydrophobic

HPLC Separation of Racemic Compounds
In order to investigate the chiral separation ability of the MIL−101−Ppa@SiO 2 CSPs, various types of racemic compounds were employed as targets. Herein, nine racemic compounds were used: rac-ketoprofen, (±)-naproxen, rac-ibuprofen, R, S-phenylethanol, R, S-1-Phenyl-1,2-ethanediol, (±)-mandelic acid, DL-alpha-methylbenzylamine, DL-phenylglycinol and 2-amino-1,2-diphenylethanol racemates, which were derived from non-steroidal drugs, phenylethanol and its derivatives and are widely used in the pharmaceutical industry, the cosmetic industry, protein engineering and biological chemistry [71][72][73][74]. The structures of these chiral compounds are shown in Figure 3. The separation results of all racemic compounds after optimizing the chromatographic mobile phase conditions are shown in Figure 4 and Table 1. The baseline separation of naproxen and ibuprofen with corresponding symmetric peak shapes was achieved, which exhibited the high enantioselectivity and good separation performance of the MIL−101−Ppa@SiO 2 packed column. The common structural feature of these enantiomers was that they all possessed hydroxyl groups, which might be capable of hydrogen-bonding interactions with the carboxyl of MIL−101−Ppa@SiO 2 [75]. Additionally, other possible interactions, including van der Waals forces, hydrophobic interactions and π-π interactions, might also contribute to the chiral recognition among the chiral target compounds and the MIL−101−Ppa@SiO 2 column. For the other enantiomers, including ketoprofen, mandelic acid, alpha-methylbenzylamine, DL-phenylglycinol and 2-amino-1,2-diphenylethanol, complete separation could not be achieved. According to the structures of these racemic compounds, it was assumed that hydrogen-bonding interactions, π-π interactions and hydrophobic interactions might occur between chiral compounds and CSPs. Among them, carboxyl groups or amido groups supplied such significant hydrogen-bonding interaction sites in their structures that both the R-enantiomer and S-enantiomer showed strong retention behavior on the MIL−101−Ppa@SiO 2 column. The peaks of two enantiomers were more likely to overlap, which led to low separation factors (Rs). Additionally, although R, S-phenylethanol and R, S-1-Phenyl-1,2-ethanediol had benzene ring and hydroxyl groups, they could not be separated due to their strong binding. In addition to the nine racemates, attempts were made to separate some other chiral compounds using the MIL−101−Ppa@SiO 2 column, but they could not be isolated. For example, in Figure S3a, the chromatogram of 1,1 -bi-2-naphthol only has one peak owing to the steric effect. The molecular dimensions of 1,1 -bi-2-naphthol were larger than the pore size of MIL−101−Ppa@SiO 2 , so 1,1 -bi-2-naphthol could not enter the pores [28]. This illustrated that the size exclusion of MIL−101−Ppa@SiO 2 played an important part, as well. DL-Ethyl-3-hydroxybutyrate does not have benzene rings and could not serve as an active hydrogen-bond donor ( Figure S3b). Therefore, it was unable to be completely separated. According to the above results, the main mechanism of separation may be hydrogen-bonding interactions and π-π interactions.
behavior on the MIL−101−Ppa@SiO2 column. The peaks of two enantiomers were more likely to overlap, which led to low separation factors (Rs). Additionally, although R, Sphenylethanol and R, S-1-Phenyl-1,2-ethanediol had benzene ring and hydroxyl groups, they could not be separated due to their strong binding. In addition to the nine racemates, attempts were made to separate some other chiral compounds using the MIL−101−Ppa@SiO2 column, but they could not be isolated. For example, in Figure S3a, the chromatogram of 1,1′-bi-2-naphthol only has one peak owing to the steric effect. The molecular dimensions of 1,1′-bi-2-naphthol were larger than the pore size of MIL−101−Ppa@SiO2, so 1,1′-bi-2-naphthol could not enter the pores [28]. This illustrated that the size exclusion of MIL−101−Ppa@SiO2 played an important part, as well. DL-Ethyl-3-hydroxybutyrate does not have benzene rings and could not serve as an active hydrogen-bond donor ( Figure S3b). Therefore, it was unable to be completely separated. According to the above results, the main mechanism of separation may be hydrogen-bonding interactions and π-π interactions.      c Separation factor α = k 2 , where t 0 is the column void time determined by 1,3,5-tri-tert-butyl-benzene.
d Binding affinities between the modified MIL−101 MOFs and chiral compounds (R/S isomers) from 100 replicates of docking calculations; the lowest binding affinities are given in parentheses.
In order to further evaluate the separation capacity of cMOFs, NH 2 −MIL−101−Ppa@SiO 2 prepared by the covalent PSM method was applied to separate chiral compounds. As seen in Figure S4, only three compounds could be isolated on the NH 2 −MIL−101−Ppa@SiO 2 column. Their factors (Rs) were similar to those of MIL−101−Ppa@SiO 2 . However, the others could not be separated using the NH 2 −MIL−101−Ppa@SiO 2 column. The separation performance of the NH 2 −MIL−101−Ppa@SiO 2 column was unsatisfactory. One of the possible reasons is that the exposure of the metal sites led to high electronegativity [76]. The other reasons were that the crystal forms of the original MOFs were better than those of NH 2 −MOFs.

Evaluation of Separation Performance
In order to evaluate the separation performance of the MIL−101−Ppa@SiO 2 column, a comparison between the MIL−101−Ppa@SiO 2 column and commercial column (Chiralpak OD column) was carried out. For the Chiralpark OD column, the separation factors (α) of naproxen, ibuprofen and ketoprofen were 1.03, 1.13 and 1.03, respectively [77], while the factors of the three above compounds on the MIL−101−Ppa@SiO 2 column were 35.71, 21.98 and 10.02, respectively. The separation results showed that MIL−101−Ppa@SiO 2 had better resolution and required less time. This confirmed that the MIL−101−Ppa@SiO 2 column has the potential to be applied in the separation of these chiral compounds.
In addition, the reproducibility was tested by repeatedly separating alphamethylbenzylamine on the MIL−101−Ppa@SiO 2 column at 40 • C using n-hexane/isopropanol (10:90, v:v) as the mobile phase. There were no obvious changes in the 50th, 100th, 150th, 200th or 250th injections, which demonstrates the good reproducibility of MIL−101−Ppa@SiO 2 after 250 times (Figure 5a). The relative standard deviations (RSDs, n = 5) of the retention time and peak area for replicate separations were 0.52% and 2.68%, respectively, which indicated the good reproducibility of the MIL−101−Ppa@SiO 2 column, as well. After one month, alpha-methylbenzylamine could still be completely separated, with little change in the separation factor (Figure 5b). nol (10:90, v:v) as the mobile phase. There were no obvious changes in the 50th, 100th, 150th, 200th or 250th injections, which demonstrates the good reproducibility of MIL−101−Ppa@SiO2 after 250 times (Figure 5a). The relative standard deviations (RSDs, n = 5) of the retention time and peak area for replicate separations were 0.52% and 2.68%, respectively, which indicated the good reproducibility of the MIL−101−Ppa@SiO2 column, as well. After one month, alpha-methylbenzylamine could still be completely separated, with little change in the separation factor (Figure 5b). The stability of the MIL−101−Ppa@SiO2 column was characterized by SEM and PXRD, as well. Compared with the XRD spectrum of MIL−101−Ppa@SiO2 before packing the column, MIL−101−Ppa@SiO2 after separation still maintained its original crystal structure, as shown in Figure S5. The morphology of MIL−101−Ppa@SiO2 did not significantly change

Docking Predictions
The interiors of two mesoporous cages (ST20 and ST28) and the channels between these two cages in MIL−101 MOFs were chosen as possible binding sites for the racemates, as indicated by the light-gray beads in Figure 7a, b. The binding affinities averaged over 100 independent docking runs for the R and S isomers are tabulated in Table 1. Due to the availability of reference standards, we could only determine the elution order of three racemates in HPLC experiments with MIL−101−Ppa and NH2−MIL−101−Ppa as the stationary phases. The R isomer of (±)-mandelic acid had a longer retention time than its S isomer, whereas the R isomer was eluted first with a shorter retention time compared to the S isomer of DL-phenylglycinol and 2-amino-1,2-diphenylethanol. A longer retention time indicates a stronger interaction (or binding affinity) with the stationary phase. Our docking calculations predicted binding affinities of ca. −8.0 and −8.4 kcal/mol for the binding of the R and S isomers of 2-amino-1,2-diphenylethanol to MIL−101−Ppa, in good agreement with the experimental elution order (Figure 4i and Table 1). Similarly, good agreement was obtained for the binding of 2-amino-1,2-diphenylethanol isomers to NH2−MIL−101−Ppa ( Figure S4 and Table 1). For (±)-mandelic acid and DL-phenylglycinol, large errors for the predicted binding affinities precluded a clear-cut measurement, although the average values and/or the lowest binding affinities showed good agreement with the experiment ( Table 1).
The racemates preferred to interact with the channel between the two adjacent cages of MIL−101 MOFs over the interiors of the cages, as shown in Figure 7c-h. This may be ascribed to the fact that the cage interior is surrounded only by aromatic groups, and additional interactions, such as hydrogen bonds, can be provided by the metal-coordination network and/or the modified MOF linkers. Hydrogen bonds, Pi-Pi stacking between aromatic rings, and Pi-alkyl hydrophobic interactions with the Ppa alkyl group are the driv-

Docking Predictions
The interiors of two mesoporous cages (ST20 and ST28) and the channels between these two cages in MIL−101 MOFs were chosen as possible binding sites for the racemates, as indicated by the light-gray beads in Figure 7a,b. The binding affinities averaged over 100 independent docking runs for the R and S isomers are tabulated in Table 1. Due to the availability of reference standards, we could only determine the elution order of three racemates in HPLC experiments with MIL−101−Ppa and NH 2 −MIL−101−Ppa as the stationary phases. The R isomer of (±)-mandelic acid had a longer retention time than its S isomer, whereas the R isomer was eluted first with a shorter retention time compared to the S isomer of DL-phenylglycinol and 2-amino-1,2-diphenylethanol. A longer retention time indicates a stronger interaction (or binding affinity) with the stationary phase. Our docking calculations predicted binding affinities of ca. −8.0 and −8.4 kcal/mol for the binding of the R and S isomers of 2-amino-1,2-diphenylethanol to MIL−101−Ppa, in good agreement with the experimental elution order (Figure 4i and Table 1). Similarly, good agreement was obtained for the binding of 2-amino-1,2-diphenylethanol isomers to NH 2 −MIL−101−Ppa ( Figure S4 and Table 1). For (±)-mandelic acid and DL-phenylglycinol, large errors for the predicted binding affinities precluded a clear-cut measurement, although the average values and/or the lowest binding affinities showed good agreement with the experiment (Table 1).

Conclusions
In this study, the enantioseparation performance of cMOFs prepared by two different post-synthetic modifications and the roles of MOFs and chiral ligands were investigated. Therein, MIL−101−Ppa@SiO2 was prepared via coordination coupling, and NH2−MIL−101−Ppa@SiO2 composites were synthesized by covalent bonding. According to the characterization results, MIL−101−Ppa@SiO2 had a larger surface area and higher grafting density than NH2−MIL−101−Ppa@SiO2. Moreover, as the chiral stationary phase, MIL−101−Ppa@SiO2 could separate more stereoselective drugs and intermediates within a shorter time and exhibited good chiral separation performance. According to the good The racemates preferred to interact with the channel between the two adjacent cages of MIL−101 MOFs over the interiors of the cages, as shown in Figure 7c-h. This may be ascribed to the fact that the cage interior is surrounded only by aromatic groups, and additional interactions, such as hydrogen bonds, can be provided by the metal-coordination network and/or the modified MOF linkers. Hydrogen bonds, Pi-Pi stacking between aromatic rings, and Pi-alkyl hydrophobic interactions with the Ppa alkyl group are the driving forces responsible for binding (Figure 7d,e,g,h). Both the hydroxyl and amino groups of 2-amino-1,2-diphenylethanol were capable of hydrogen bonding with the carboxyl group of MOF linkers and the Ppa hydroxyl group. The R isomer of 2-amino-1,2-diphenylethanol used its hydroxyl group as a hydrogen-bond donor (Figure 7d), while its S isomer used the amino group. The amino group (Figure 7e,h) appears to form more hydrogen bonds with MIL−101 MOFs than the hydroxyl group (Figure 7d,g). Moreover, the R isomer had two Pi-Pi stacking and three Pi-Alkyl contacts with MIL−101−Ppa (Figure 7d), while the S isomer formed three Pi-Pi stacking and two Pi-Alkyl contacts. These findings led to a difference in the binding affinity between two isomers and MIL−101 MOFs. For NH 2 −MIL−101−Ppa, the modified linker was likely to produce steric hindrance, thereby preventing close contact with the metal-coordination networks (Figure 7g-h).

Conclusions
In this study, the enantioseparation performance of cMOFs prepared by two different post-synthetic modifications and the roles of MOFs and chiral ligands were investigated. Therein, MIL−101−Ppa@SiO 2 was prepared via coordination coupling, and NH 2 −MIL−101−Ppa@SiO 2 composites were synthesized by covalent bonding. According to the characterization results, MIL−101−Ppa@SiO 2 had a larger surface area and higher grafting density than NH 2 −MIL−101−Ppa@SiO 2 . Moreover, as the chiral stationary phase, MIL−101−Ppa@SiO 2 could separate more stereoselective drugs and intermediates within a shorter time and exhibited good chiral separation performance. According to the good reproducibility and stability, MIL−101−Ppa@SiO 2 could be used for enantioseparation by HPLC. The molecular docking calculations also supported the experimental data and provide insights into the separation capacity of MIL−101−Ppa@SiO 2 . This work supplies a simple method to synthesize cMOFs and apply them as CSPs. Thus, this synthetic method may have more promising applications in the future.