Graphene quantum dots functionalized β-cyclodextrin and cellulose chiral stationary phases with enhanced enantioseparation performance

Journal of Chromatography A, 1600 (2019) 209–218

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Graphene quantum dots functionalized ␤-cyclodextrin and cellulose chiral stationary phases with enhanced enantioseparation performance Qi Wu a,b , Jie Gao a,b , Lixiao Chen a,b , Shuqing Dong a , Hui Li a , 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, China b University of Chinese Academy of Sciences, Beijing, China

a r t i c l e

i n f o

Article history: Received 13 March 2019 Received in revised form 18 April 2019 Accepted 19 April 2019 Available online 22 April 2019 Keywords: Graphene quantum dots Cyclodextrin Cellulose Molecular modeling Enantioseparation

a b s t r a c t Graphene quantum dots (GQD) functionalized ␤-cyclodextrin (␤-CD) and cellulose silica composites were first prepared and applied in HPLC as chiral stationary phases (CSP) to investigate the effect of GQDs on chiral separation. Through comparing the enantioseparation performance of GQDs functionalized ␤-CD or cellulose CSPs and unmodified ␤-CD or cellulose CSPs, we found GQDs enhanced the enantioseparation performance of nature ␤-CD, ␤-CD-3,5-dimethylphenylcarbamate derivative and cellulose-3,5-dimethylphenylcarbamate derivative. Molecular modeling was applied to understand and theoretically study the enhancement mechanism of GQDs for enantioseparation. According to molecular simulation results, GQDs provide extra interactions such as hydrophobic, hydrogen bond and ␲-␲ interaction when chiral selector interacts with enantiomers, which enhances the chiral recognition ability indirectly. The molecular simulation results showed a good agreement with the experimental results. Our work reveals the enhancement performance of GQDs for chiral separation, it can be expected that GQDsbased chiral composites and chiral GQDs have great prospect in chiral separation and other research fields such as asymmetric synthesis, chiral catalysis, chiral recognition and drug delivery. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Chirality is an essential attribute of nature. The separation of enantiomers of chiral compounds continues to be of great interest due to their prevalence in the pharmaceutical industry, agrochemicals and food additives [1]. The task of enantioseparation is extremely challenging, because enantiomers are identical in most regards: they have the same densities and solubilities, boiling and melting points, electronic and vibrational frequencies, reactivities and refractivities, etc [2]. Chiral separation materials are the core to achieve enantioseparation. Cyclodextrins (CD) and their derivatives as well as cellulose derivatives are the most commonly used chiral selectors at present [3–6]. With the rapid development of pharmaceutical industry and more and more attention to life safety, higher demands will be put forward for chiral separation. It can be expected that developing novel and effective chiral separation materials has great significance.

∗ Corresponding authors at: No. 18, Tianshui Middle Road, Lanzhou, China. E-mail addresses: [email protected] (H. Qiu), [email protected] (L. Zhao). https://doi.org/10.1016/j.chroma.2019.04.053 0021-9673/© 2019 Elsevier B.V. All rights reserved.

Carbon nanomaterials (CNM) with good physical and chemical characteristics have been widely used in various disciplines [7–12]. With the development of CNMs in separation science, various CNMs have been applied to enantioseparation in recent years. Chiral single-walled carbon nanotubes and multi-walled carbon nanotubes have been directly applied for the enantioseparation of pharmaceuticals and biologicals by using them as stationary or pseudostationary phases in chromatographic separation techniques including high performance liquid chromatography (HPLC), capillary electrochromatography (CEC) and gas chromatography (GC) [13]. Collectively, functionalized carbon nanotubes have been indirectly applied in separation science by enhancing the enantioseparation of different chiral selectors [13,14]. Chiral selectors functionalized graphene oxide (GO) composites have been applied in CEC enantioseparation [15–19], however, their application in HPLC enantioseparation was relatively scarce [20,21]. In CEC, Qiu and coworkers prepared a series of chiral selectors functionalized GO nanocomposites [15–17] as chiral stationary phases (CSP) for chip-based open-tubular CEC. Li, Ji and coworkers prepared three types of GO-functionalized chiral affinity capillary silica monoliths [18]. Du and coworkers established a GO-modification CEC system

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for enantioseparation with methyl-␤-CD as chiral mobile phase additives [19]. Research results demonstrated that GO possessed large surface area and abundant functional groups which were necessary for chiral selectors immobilization and could provide various interactions for enantioseparation with improved performance. In HPLC, Li and coworkers prepared cellulose derivative coated rGObonded silica CSP [21]. Due to the existence of rGO on the CSP, this CSP obtained stronger interaction with the analytes leading to better enantioseparation performance compared with cellulose derivative modified silica CSP. According to previous research, we may find that CNMs have great prospect in chiral separation. Graphene quantum dots (GQD), as a new type of CNM, have been widely used in bioimaging and fluorescent sensing because of good water solubility, biocompatibility and unique fluorescence stability [22]. Recently, great efforts have been devoted to explore potential applications of GQDs in separation science, GQDs are gradually applied in GC [23], CEC [24] and HPLC [25–29] showing satisfactory separation performance. As chromatographic material, GQDs have some unique characteristics such as small dimension, high thermostability and good dispersibility, which make them easier to be immobilized on the silica support. Moreover, GQDs possess highly delocalized conjugate system of ␲-electron and abundant oxygen-containing groups, which enables them to provide various interactions including ␲-␲, hydrophobic, hydrogen-bonding and hydrophilic interactions and thus to be used as multifunctional separation material. It is reasonable to believe that GQDs have great prospect in separation science. Though the separation performance of GQDs for all kinds of achiral compounds has been investigated systematically, their application in chiral separation has not been explored. We wonder if GQDs, as good chromatographic material and biocompatible nanomaterial, could be the chiral enhancement material for nature chiral selectors. Hence, GQDs functionalized ␤-CD and cellulose silica composites were first prepared and applied in HPLC as CSPs to investigate the effect of GQDs on chiral separation. Ten chiral compounds were chosen as probes to evaluate the enantioseparation performance of the prepared CSPs. ␤-CD CSP and cellulose CSP were prepared and used as references. Generally, 3,5-dimethylphenyl isocyanate is always used as hydroxyls derivatization reagent of ␤-CD and cellulose to enhance their enantioseparation ability [3–5,30,31]. Especially for cellulose CSPs, the available cellulose CSPs are cellulose derivatives-based CSPs since the poor solubility of cellulose [32,33]. To investigate the difference between GQDs and 3,5-dimethylphenyl isocyanate in abilities to enhance chiral separation performance, the enantioseparation performance of GQDs functionalized ␤-CD CSP was also compared with that of 3,5-dimethylphenylcarbamate derived ␤-CD CSP. In addition, ␤CD-3,5-dimethylphenylcarbamate CSP was also functionalized by GQDs to further explore the chiral enhancement effect of GQDs to ␤-CD-3,5-dimethylphenylcarbamate derivative.

2. Materials and methods 2.1. Materials and instruments Silica gel (70 Å, 800 Å, 5 ␮m in diameter) was supplied by Fuji Silysia Chemical Ltd (Japan). 3-aminopropyltriethoxysilane (APTES), 3-isocyanatopropyltriethoxysilane, 3,5-dimethylphenyl isocyanate, ␤-CD and microcrystalline cellulose were obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Meryer Chemical Technology Co. Ltd (Shanghai, China). All chiral chemicals used for chromatographic separation were purchased from Sigma-Aldrich (Shanghai, China).

Elemental analysis was performed on vario EL (Elementar, Germany). The Fourier transform infrared spectroscopy (FTIR) spectra was acquired on an IFS120HR Fourier transform infrared spectrometer (Bruker, Germany). Laser scanning confocal microscope (LSCM) images were obtained on a FLUOVIEW FV1200 biological confocal laser scanning microscope (OLYMPUS, Japan). 2.2. Preparation of Si-GQD-CD and Si-GQD-CDD GQDs used in this work were prepared by previously reported methods [27,28,34]. The particle size of the prepared GQDs can be identified as around 20 nm and the main oxygen-containing functional groups of the prepared GQDs were hydroxy, carboxyl and epoxy/ether according to our previously characterization [27,28]. Silica with small pore size (70 Å) was used for the preparation of ␤-CD-based CSPs according to previous research [35]. GQDs bonded silica composite (Si-GQD) was obtained by covalently coupling the silanized GQDs and silica. First, GQDs were reacted with APTES to form silanized GQDs. 0.4 g GQDs were dispersed in N,Ndimethylformamide (DMF) and 0.4 g EDC/NHS were added into above dispersion to activate the carboxyl groups of GQDs. After half an hour, 1.0 mL APTES was added into above dispersion and the dispersion was stirred at 25 ◦ for 12 h. Second, 5.0 g silica was added into the dispersion of silanized GQDs and the mixture was stirred at 90 ◦ for 6 h. The products were washed with DMF for several times to obtain Si-GQD. GQDs functionalized ␤-CD CSP (Si-GQD-CD) was prepared as follows. The schematic diagram of the preparation process was shown in Fig. 1. First, 2.0 g ␤-CD and 2.0 mL 3-isocyanatopropyltrimethoxysilane were added into 50 mL dry pyridine and the solution was stirred for 8 h at 80 ◦ . Second, 5.0 g Si-GQD were added into above solution and the mixture was stirred at 90 ◦ for 12 h. After reaction, the products were washed with pyridine, ethanol and methanol for several times and dried under vacuum at 60 ◦ C for 12 h. The blank column ␤-CD CSP (Si-CD) was prepared using the same method except for that silica was used in the second step. The Si-GQD-CD was further derived with 3,5-dimethylphenyl to obtain GQDs functionalized and 3,5isocyanate dimethylphenylcarbamate derived ␤-CD CSP (Si-GQD-CDD). 2.5 g Si-GQD-CD was dispersed in pyridine and 3.0 mL 3,5dimethylphenyl isocyanate was added, then the dispersion was stirred at 80 ◦ for 24 h. After that, the products were washed with pyridine, ethanol and methanol for several times and dried under vacuum at 60 ◦ C for 12 h. The 3,5-dimethylphenylcarbamate derived ␤-CD CSP (Si-CDD) was prepared using the same method except for that Si-CD was used for derivation reaction. 2.3. Preparation of Si-GQD-Cellulose Silica with large pore size (800 Å) was used for the preparation of cellulose-based CSPs according to previous experience [31,36]. The GQDs functionalized cellulose CSP (Si-GQD-Cellulose) was prepared by coating cellulose-3,5-dimethylphenylcarbamate derivatives onto the surface of Si-NH2 -GQD which was prepared by covalently coupling the carboxyl groups of GQDs and the amino groups of aminosilica as previously reported method [27–29]. First, cellulose-3,5-dimethylphenylcarbamate derivatives were prepared according to reported methods [30,36]. 1.0 g microcrystalline cellulose was dispersed in pyridine and heated at 90 ◦ C for 24 h, then, 5.0 mL 3,5-dimethylphenyl isocyanate was added to the mixture, the mixture was continued to heat for 24 h. The obtained product was isolated as a methanol-insoluble fraction. The white solid was filtered and washed thoroughly with methanol and then dried under vacuum. Second, 0.15 g cellulose derivatives and 1.5 g Si-NH2 -GQD were dispersed in tetrahydrofuran, the mixture

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211

Fig. 1. Synthetic process of GQDs functionalized ␤-CD and cellulose CSPs.

was heated at 40 ◦ C to vaporize the tetrahydrofuran solvent to form the Si-GQD-Cellulose CSP. The blank column Si-Cellulose CSP was prepared using the same method except for that Si-NH2 was used in the second step. The preparation process was shown in Fig. 1. 2.4. Column packing The Si-CD, Si-GQD-CD, Si-CDD and Si-GQD-CDD composites were packed into stainless steel tubes (150 mm × 4.6 mm i.d.) by a slurry packing method under a constant packing pressure of 40 MPa using 1,4-dioxane and carbon tetrachloride (1/1, v/v) as homogenate solvents and methanol as propulsion solvent. The Si-GQD-Cellulose and Si-Cellulose composites were packed into stainless steel tubes (100 mm × 4.6 mm i.d.) by a slurry packing method under a constant packing pressure of 40 MPa using n-hexane and isopropanol (1/1, v/v) as homogenate solvents and n-hexane as propulsion solvent.

Fig. 2. FTIR spectra of Si-CD, Si-GQD-CD, Si-CDD, Si-GQD-CDD, Si-Cellulose and SiGQD-Cellulose.

2.5. Chromatographic separation All chromatographic separation was performed on a Waters HPLC system (Waters, USA), consisted of a Waters 515 HPLC pump, a Rheodyne 7725i injector equipped with 20 ␮L sample loop and a Waters 996 Photodiode Array detector. The detection wavelength was at 254 nm. The preparation of two components mobile phases (MeOH/H2 O, ACN/H2 O and n-hexane/isopropanol) was according to their volume ratio (v/v). Acetone was used as test analyte to determine the dead time of ␤-CD-based columns under reversed phase chromatography mode using methanol as mobile phase, the flow rate was 1.0 mL/min [6]. The void volume of the four ␤CD-based columns was about 2.1 cm3 . 1,3,5-tri-tert-butylbenzene was used as test analyte to determine the dead time of cellulosebased columns under normal phase chromatography mode using n-hexane/isopropanol (50/50) as mobile phase, the flow rate was 1.0 mL/min [31]. The void volume of the cellulose-based columns was about 1.6 cm3 . 3. Results and discussion 3.1. Characterization Fig. 2 showed the FTIR spectra of Si-CD, Si-GQD-CD, Si-CDD, SiGQD-CDD, Si-Cellulose and Si-GQD-Cellulose. Compared to Si-CD,

the enhanced peak at 1731 cm−1 (C O in O C O) of Si-GQD-CD indicated the existence of GQDs since GQDs had COOH. In Si-CDD and Si-GQD-CDD, the enhanced peaks at 1731 cm−1 , 1627 cm−1 and 1561/1442 cm−1 which was assigned to the characteristic absorption peak of O C O, N H and benzene ring demonstrated that ␤-CDs were derived with 3,5-dimethylphenyl isocyanate successfully. Si-Cellulose and Si-GQD-Cellulose showed same characteristic adsorption peaks, but Si-GQD-Cellulose had stronger stretching vibration peaks at 1731 cm−1 , which indicated the presence of GQDs. All of these showed that Si-CD, Si-GQD-CD, Si-CDD, Si-GQD-CDD, Si-Cellulose and Si-GQD-Cellulose CSPs had been prepared successfully. From the LSCM images (Fig. 3), compared with unmodified CSPs, homogeneous fluorescence was observed on the surface of Si-GQDCD, Si-GQD-CDD and Si-GQD-Cellulose, which indicated successful functionalization of Si-CD, Si-CDD and Si-Cellulose with GQDs. Elemental analysis data of Si-CD, Si-GQD, Si-GQD-CD, Si-CDD, SiGQD-CDD, Si-Cellulose and Si-GQD-Cellulose were listed in Table 1. The C content of Si-CD which was come from grafting of ␤-CD was 7.52%. According to the C content data of Si-GQD (1.81%) and SiGQD-CD (7.64%), the increased C content which come from grafting of ␤-CD was calculated to be 5.83%, which indicated that the content of chiral selector ␤-CD in Si-GQD-CD was lower than that in Si-CD. This was because that GQDs had limited carboxy groups

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Fig. 3. The LSCM images of (a) Si-CD, (b) Si-GQD-CD, (c) Si-CDD, (d) Si-GQD-CDD, (e) Si-Cellulose and (f) Si-GQD-Cellulose under 405 nm excitation.

Table 1 Elemental analysis data. Elemental analysis (%)

Si-CD Si-GQD Si-GQD-CD Si-CDD Si-GQD-CDD Si-NH2 Si-NH2 -GQD Si-Cellulose Si-GQD-Cellulose

N

C

H

1.00 0.50 1.05 2.67 2.15 0.68 0.85 1.01 1.19

7.52 1.81 7.64 22.63 17.92 2.71 3.20 9.12 9.61

1.46 0.73 1.55 2.44 2.17 0.61 0.64 1.12 1.13

amount compared to hydroxy amount in silica gel. After the chiral selector ␤-CDs in Si-CD and Si-GQD-CD were derived with 3,5-dimethylphenyl isocyanate, their C and N content increased obviously. Since the content of chiral selector ␤-CD in Si-GQD-CD was lower than that in Si-CD, the C content of Si-GQD-CDD was lower than that of Si-CDD after derivation with 3,5-dimethylphenyl isocyanate. The C content of Si-Cellulose and Si-GQD-Cellulose was similar. Since the presence of GQDs in CSP, the C and N content of Si-GQD-Cellulose was higher than that in Si-Cellulose. Because the coating amount of cellulose derivatives on Si-NH2 -GQD and Si-NH2 was same, thus the amount of chiral selectors in Si-GQD-Cellulose and Si-Cellulose was same. 3.2. Enantioseparation Ten chiral racemates benzoin (1), benzoin methyl ether (2), benzoin ethyl ether (3), 6,6 -dibromo-1,1 -bi-2-naphthol (4), transstilbene oxide (5), flavanone (6), 6-hydroxyflavanone (7), naphthyl ethanol (8), diclofop (9), metalaxyl (10) (Fig. 4) which could be recognized on ␤-CD-based or cellulose-based CSPs were chosen as model analytes to evaluate the prepared CSPs.

3.2.1. Enantioseparation on ˇ-CD-based CSPs Seven chiral compounds including benzoin (1), benzoin methyl ether (2), benzoin ethyl ether (3), 6,6 -dibromo-1,1 -bi-2-naphthol (4), trans-stilbene oxide (5), flavanone (6) and 6-hydroxyflavanone (7) were used as probes to evaluate the enantioseparation performance of Si-CD, Si-GQD-CD, Si-CDD and Si-GQD-CDD columns. Their chromatograms were shown in Fig. 5. Fig. 5(1) showed the chromatograms of benzoin racemates separated on the four chiral columns, from which we saw benzoin enantiomers were not recognized on Si-CD column in proper analysis time but were identified on Si-GQD-CD column. This indicated that GQDs played a positive role on the enantiomeric recognition ability of ␤-CD CSP. It must be noted that the ␤-CD content in Si-GQD-CD column was lower than that in Si-CD column as we discussed in characterization part, which demonstrated the enhancement effect of GQDs on enantioseparation more strongly. The benzoin enantiomers were also recognized on Si-CDD column, which demonstrated that derivatization of ␤-CD with 3,5-dimethylphenylcarbamate could improve the enantioseparation performance of ␤-CD CSP. Compared the enantioseparation performance of Si-GQD-CD and Si-CDD, Si-CDD showed higher selectivity for benzoin racemates, it seemed that 3,5-dimethylphenyl isocyanate derivatization reagent could improve the enantioseparation performance of ␤CD more effectively. This may be due to that small molecules can modify ␤-CD more adequately. Interestingly, the benzoin enantiomers obtained highest selectivity on Si-GQD-CDD column within the same analysis time, which indicated that GQDs not only enhanced the enantioseparation performance of ␤-CD, but also improved the enantioseparation performance of ␤-CD-3,5dimethylphenylcarbamate derivative. Fig. 5(2) and (3) showed the chromatograms of benzoin methyl ether and benzoin ethyl ether racemates separated on the four columns, respectively. From the chromatograms we saw benzoin methyl ether and benzoin ethyl ether enantiomers could be recognized on Si-CD column but with poor selectivity, however, their enantiomers could be separated effectively on Si-GQD-CD column. GQDs improved the enantioseparation performance of ␤-CD distinctly. However, the circumstance was different from that of benzoin, the enantioseparation performance for benzoin methyl ether and benzoin ethyl ether were not improved on Si-CDD and Si-GQD-CDD columns. After derivatization with 3,5-dimethylphenylcarbamate, the enantiomeric recognition performance of ␤-CD for benzoin methyl ether and benzoin ethyl ether decreased and GQDs could not turn the tide. Fig. 5(4) showed the chromatograms of 6,6 -dibromo-1,1 bi-2-naphthol enantiomers separated on the four columns, we saw 6,6 -dibromo-1,1 -bi-2-naphthol enantiomers achieved higher selectivity on Si-GQD-CD column than on Si-CD column, which was in accord with above circumstances that the enantioseparation performance of ␤-CD was improved by functionalization with GQDs. Si-CDD showed worse enantiomeric recognition performance for 6,6 -dibromo-1,1 -bi-2-naphthol compared with Si-CD column. After derivatization with 3,5-dimethylphenylcarbamate, the enantiomeric recognition ability of ␤-CD for 6,6 -dibromo1,1 -bi-2-naphthol decreased and GQDs could not turn the tide. It seemed that the chiral enhancement effect of 3,5dimethylphenylcarbamate depended on the style of chiral compounds. Fig. 5(5) showed the chromatograms of trans-stilbene oxide enantiomers separated on the four columns. Compared the separation performance of Si-CD and Si-GQD-CD, Si-GQD-CD showed higher enantiomeric selectivity, which indicated GQDs improved the enantiomeric recognition ability of ␤-CD. Compared the separation performance of Si-CDD and Si-GQD-CD, Si-CDD showed higher enantiomeric recognition ability, which indicated that 3,5dimethylphenylcarbamate could improve the enantioseparation

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213

Fig. 4. Chiral compounds used for enantioseparation.

performance of ␤-CD more effectively than GQDs for trans-stilbene oxide. Among the four columns, Si-GQD-CDD presented the highest enantioseparation performance, this demonstrated that GQDs also played a positive role on the enantioseparation performance of ␤-CD-3,5-dimethylphenylcarbamate derivative, on the other hand, this indicated that GQDs and 3,5-dimethylphenyl isocyanate had certain synergistic effect on the improvement of chiral separation. As shown in Fig. 5(6), flavanone enantiomers could be separated effectively on the four columns, even so, the enhancement effect of GQDs and 3,5-dimethylphenyl isocyanate could be still observed. It was found that 3,5-dimethylphenyl isocyanate could improve the enantioseparation performance of ␤-CD more effectively than GQDs for flavanone. GQDs and 3,5-dimethylphenyl isocyanate had certain synergistic effect, thus Si-GQD-CDD showed the highest enantioseparation performance. 6-Hydroxyflavanone obtained similar resolution on Si-CD and Si-GQD-CD columns, GQDs showed slight enhancement effect for 6-hydroxyflavanone (Fig. S1). However, 6-hydroxyflavanone were completely separated on Si-CDD and Si-GQD-CDD, and higher resolution was obtained on Si-GQD-CDD. This further demonstrated the synergistic effect of GQDs and 3,5-dimethylphenyl isocyanate for chiral separation. Chromatographic parameters of the seven chiral compounds on the four columns were listed in Table 2. Compared the enantioseparation performance of Si-CD, Si-GQD-CD, Si-CDD and Si-GQD-CDD columns, Si-GQD-CD column showed the highest enantioseparation performance for chiral compounds 2,3,4 and Si-GQD-CDD column showed the highest enantioseparation performance for chiral compounds 1,5,6,7. The columns which were functionalized with GQDs had the best enantioseparation performance, which indicated the enhancement effect of GQDs on chiral separation. Compared the enhancement effect of GQDs and 3,5-dimethylphenyl isocyanate on ␤-CD’s enantioseparation performance, it was found that GQDs had enhancement effect for all the tested chiral compounds in different degree, while 3,5-dimethylphenyl isocyanate had enhancement effect for chiral compounds (1,5,6,7) and negative effect for chiral compounds (2,3,4). We also note that 3,5-dimethylphenyl isocyanate had higher enhancement effect than GQDs for chiral compounds 1,5,6,7. From the separation results of the selected seven chiral compounds on Si-CD and Si-CDD columns, we saw ␤-CD and

␤-CD-3,5-dimethylphenylcarbamate derivative showed certain complementary effect on enantiomeric recognition ability. ␤-CD had higher enantiomeric recognition ability for chiral compounds 2,3,4, while ␤-CD-3,5-dimethylphenylcarbamate derivative had higher enantiomeric recognition ability for chiral compounds 1,5,6,7. After Si-CD and Si-CDD were functionalized with GQDs, their enantioseparation performance were both improved in different agree, the complementary effect could also be seen on Si-GQD-CD and Si-GQD-CDD columns. These phenomena give us some inspiration that excellent enantioseparation performance may achieve through adjusting the 3,5-dimethylphenyl isocyanate derivatization degree and GQDs content in ␤-CD CSPs. 3.2.2. Effect of organic modifiers on enantioseparation Organic modifier in mobile phase plays an important role in the enantioseparation. Seven chiral compounds were chosen as model analytes to evaluate the influence of organic modifier on ␤CD-based CSPs. Generally, ACN have stronger elution power than MeOH as the organic modifier in reversed-phase LC due to the more hydrophobility of ACN. To make the comparison more reliable, the ACN and MeOH proportion in mobile phase was adjusted to make analytes have similar retention time. In the same analysis time, the resolutions of the tested racemates using ACN and MeOH as organic modifier were calculated and listed in Tables S1 and S2. As shown in Table S1, for Si-CD CSP, ACN could offer better resolution than MeOH for chiral compounds 1,2,3,6 especially for flavanone (6); MeOH could offer better resolution than ACN for chiral compounds 4,5,7. MeOH and ACN showed similar ability as organic modifier for enantioseparation. While for Si-GQD-CD CSP, ACN offered similar resolution with MeOH for chiral compounds 1,2,3 and better resolution than MeOH for flavanone (6); MeOH offered better resolution than ACN for chiral compounds 4,5,7. Take a comprehensive consideration, MeOH was more suitable as organic modifier for SiGQD-CD CSP. It seems that GQDs functionalization enhances the applicability of MeOH as organic modifier for enantioseparation. As shown in Table S2, MeOH offered better resolution than ACN for all the model analytes, this phenomenon appeared on both Si-CDD and Si-GQD-CDD CSPs. We may draw a conclusion that MeOH was more suitable used as organic modifier in 3,5-dimethylphenylcarbamate derived and GQDs functionalized ␤-CD CSPs.

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Fig. 5. Chromatograms of benzoin (1), benzoin methyl ether (2), benzoin ethyl ether (3), 6,6 -dibromo-1,1 -bi-2-naphthol (4), trans-stilbene oxide (5) and flavanone (6) racemates separated on Si-CD (a), Si-GQD-CD (b), Si-CDD (c) and Si-GQD-CDD (d) CSPs. Mobile phase: (1a, 1b, 1c, 1d), MeOH/H2 O (30/70, 35/65, 40/60, 45/55); (2a, 2b, 2c, 2d), MeOH/H2 O (40/60, 45/55, 50/50, 50/50); (3a, 3b, 3c, 3d), MeOH/H2 O (40/60, 40/60, 50/50, 50/50); (4a, 4b, 4c, 4d), MeOH/H2 O (50/50, 50/50, 75/25, 75/25); (5a, 5b, 5c, 5d), MeOH/H2 O (45/55, 50/50, 60/40, 65/35); (6a, 6b), ACN/H2 O (30/70), (6c, 6d), MeOH/H2 O (80/20). Flow rate: 1.0 mL min−1 .

3.2.3. Enantioseparation on cellulose-based CSPs To further verify the enhancement effect of GQDs on chiral separation, GQDs functionalized cellulose CSP was prepared and its enantioseparation performance was compared with that of cellulose CSP. Chiral compounds (1,2,3,5,6,8,9,10) were chosen as model analytes to evaluate Si-GQD-Cellulose and Si-Cellulose CSPs. Comparable chromatograms were shown in Fig. 6. To compare their chiral separation performance fairly, analysis time was stipulated as the common conditions. It was found that Si-GQD-Cellulose showed better chiral separation performance than Si-Cellulose

in the same analysis time for all the tested chiral compounds. The chromatographic parameters of the tested chiral compounds on Si-Cellulose and Si-GQD-Cellulose CSPs were calculated and listed in Table 3. Through comparing the mobile phase conditions, we found Si-GQD-Cellulose needed stronger eluent to achieve the same retention time with Si-Cellulose, which indicated SiGQD-Cellulose had stronger retention ability than Si-Cellulose. The stronger retention ability helped the enantioseparation to some extent. For example, benzoin ethyl ether (3) and trans-stilbene oxide (5) racemates had very weak retention on Si-Cellulose thus

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Table 2 Chromatographic parameters of chiral compounds on ␤-CD-based CSPs. Chiral sample number 1 2 3 4 5 6 7

Si-GQD-CD

Si-CD

Si-CDD

Si-GQD-CDD

k1

˛

Rs

k1

˛

Rs

k1

˛

Rs

k1

˛

Rs

19.09 9.64 10.33 7.81 14.86 4.46 14.84

1.00 1.03 1.08 1.08 1.09 1.13 1.06

0.00 0.10 0.60 0.50 0.55 1.28 0.50

16.52 7.76 9.46 5.58 16.34 3.97 12.25

1.05 1.11 1.20 1.22 1.14 1.23 1.05

0.50 0.89 1.27 0.94 1.00 1.92 0.50

14.96 8.74 11.63 7.06 12.74 3.21 11.13

1.07 1.00 1.00 1.00 1.20 1.63 1.18

0.78 0.00 0.00 0.00 1.22 4.16 1.58

12.87 11.2 13.93 5.96 8.28 3.15 11.04

1.12 1.00 1.00 1.00 1.22 1.71 1.44

1.03 0.00 0.00 0.00 1.92 4.26 2.93

k: retention factor, k = (tR -t0 )/t0 , tR : retention time, t0 : dead time; k1 : k of the first eluted enantiomer. ˛: selectivity factor, ˛ = k2 /k1 . Rs : Resolution, Rs = 2(tR2 -tR1 )/(W1 + W2 ), W: width of peak base. The chromatographic conditions were identical to Fig. 5, Fig S1.

Fig. 6. Chromatograms of benzoin (1), benzoin methyl ether (2), benzoin ethyl ether (3), trans-stilbene oxide (5), flavanone (6), naphthyl ethanol (8), diclofop (9), metalaxyl (10) separated on Si-Cellulose (a) and Si-GQD-Cellulose (b) CSPs. Mobile phase: (1a, 1b), hexane/isopropanol (90/10, 90/20); (2a, 2b), hexane/isopropanol (99/1, 95/5); (3a, 3b), hexane/isopropanol (99.5/0.5); (5a, 5b), hexane/isopropanol (99.5/0.5, 99/1); (6a, 6b), hexane/isopropanol (99.5/0.5, 99/1); (8a, 8b), hexane/isopropanol (90/10, 90/20); (9a, 9b), hexane/isopropanol (95/5, 90/10); (10a, 10b), hexane/isopropanol (90/10, 90/20). Flow rate: 1.0 mL min−1 .

they could not be separated efficiently, while they had longer retention time on Si-GQD-Cellulose thus they were better separated on Si-GQD-Cellulose. The existence of GQDs in CSPs could provide extra interactions including ␲-␲ and hydrogen-bond interaction with analytes, thus Si-GQD-Cellulose had stronger retention ability than Si-Cellulose.

3.3. Exploration of chiral enhancement mechanism Take inspiration from previous reports [19,21], a hypothesis was proposed that GQDs which possess large ␲ electron system and abundant oxygen-containing groups, can provide ␲-␲ and hydrogen-bond interaction with ␤-CD or cellulose and ana-

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Table 3 Chromatographic parameters of chiral compounds on cellulose-based CSPs. Chiral sample number 1 2 3 5 6 8 9 10

Table 4 Data from molecular modeling.

Si-GQD-Cellulose

Si-Cellulose k1

˛

Rs

k1

˛

Rs

2.88 1.87 1.80 1.02 4.72 1.74 1.69 4.40

1.31 1.37 1.00 1.31 1.20 1.45 1.82 2.23

1.15 1.17 0.00 0.60 0.68 1.45 1.79 2.38

2.72 1.78 2.37 2.14 4.90 1.64 1.64 3.21

1.33 1.72 1.18 2.18 1.41 1.51 2.08 3.29

1.47 1.74 0.70 5.50 2.12 1.75 2.89 3.20

The chromatographic conditions were identical to Fig. 6.

lytes, these extra interactions may prolong the interaction time between enantiomers and chiral selectors, and thus enhance the enantioseparation ability [37,38]. According to experimental results, stronger elution was used for GQDs-functionalized ␤-CD and cellulose columns, which indicated GQDs-functionalized ␤-CD and cellulose columns had stronger retention ability than ␤-CD and cellulose columns. These experimental phenomena were in accord with our hypothesis that GQDs provided some interactions with analytes during the separation process. Derivatization of ␤CD with 3,5-dimethylphenylcarbamate also introduced ␲-␲ and hydrogen-bond interactions, however, their contribution to enantioseparation ability was different from GQDs’. It was found that GQDs had enhancement effect for all the texted chiral compounds, while 3,5-dimethylphenyl isocyanate had enhancement effect for four chiral compounds (1,5,6,7) and negative effect for another three chiral compounds (2,3,4). This might be due to that derivatization of ␤-CD edges with 3,5-dimethylphenylcarbamate introduced steric-hindrance and thus influenced inclusion interaction between ␤-CD and analytes. Through comparison we realized that different substances had different effect on enantioseparation ability though they introduced the same interaction force types. There are two reasons to explain the positive role of GQDs on chiral separation performance: one reason was that GQDs introduced extra interactions including ␲-␲ and hydrogen-bond interactions which helped enantioseparation; another reason was that nanoscale GQDs had certain space structure which influenced surrounding environment of chiral selectors. In recent years, molecular modeling has been considered as a good tool to explain the enantiomer recognition mechanism [39,40]. Du and coworkers used molecular modeling to investigate the chiral recognition mechanism of GO-modification enantioseparation CEC system [19]. Wang, Xiao and coworkers used molecular dynamics simulation to reveal the essential factors for CD’s chiral discrimination behaviors [41]. In this work, molecular modeling was applied to understand and theoretically study the enhancement mechanism of GQD for enantioseparation. Here we take the ␤-CD system as an example to explain the enhancement mechanism. The starting geometries of GQD and ␤-CD were constructed by Discovery Studio software according to the previous related literatures [19,41]. The possible structures of GQD/␤-CD complex were designed and energetically minimized, from which a configuration with lowest-energy was chosen as GQD/␤-CD complex model. The structures of ␤-CD and GQD/␤-CD complex were optimized by molecular dynamics simulation, the simulation process was based on CHARMm force field. Each simulation of enantiomer/␤-CD inclusion complex was performed by molecular docking (CDOCKER). The whole molecular docking studies were divided into two parts. In the first part, enantiomers were docked to ␤-CD. In the second part, enantiomers were docked to GQD/␤CD complex. The interaction energy (E) between enantiomers and ␤-CD or GQD/␤-CD receptor was calculated from the dominant

Chiral sample number 1 2 3 4 5 6 7

Si-GQD-CD

Si-CD ER

ES

E

ER

ES

E

−10.78 −10.42 −9.73 −18.33 −17.77 −21.79 −53.86

−12.15 −11.98 −11.72 −20.61 −19.90 −24.71 −55.43

1.37 1.56 1.99 2.28 2.13 2.92 1.57

−20.91 −17.58 −22.15 −27.62 −19.45 −40.46 −60.02

−23.30 −20.50 −26.70 −31.02 −22.57 −44.62 −62.15

2.39 2.92 4.55 3.40 3.12 4.16 2.13

E: Interaction energy, units are in kcal mol−1 . E: Value of difference of interaction energies between the (R)- and (S)-enantiomer.

Fig. 7. Molecular docking configurations for benzoin ethyl ether enantiorecognition in ␤-CD system (a, b) and GQD/␤-CD complex system (c, d). The left is R-enantiomer and the right is S-enantiomer. The interaction is indicated by color line.

docking conformations using “calculate interaction energy” function of Discovery Studio software. The data from molecular simulation was summarized in Table 4. It was generally observed that the magnitude of E of all studied analytes in GQD/␤-CD complex was significantly enlarged compared with the single ␤-CD system, which indicated that the existence of GQD could enhance the affinity between analytes and chiral selectors. On the other hand, the GQD/␤-CD complex obviously increased the E between enantiomers, which demonstrated the enhancement of chiral discrimination and consequently the improvement of chiral selectivity. The molecular simulation results showed a good agreement with experimental results. The molecular docking configurations for benzoin ethyl ether enantiorecognition on both Si-CD CSP and Si-GQD-CD CSP were shown in Fig. 7. According to the previous literature [40], drug enantiomers were preferentially inserted into the ␤-CD from the wider rim, which was in accordance with the molecular docking results. As observed, (R)- and (S)-benzoin ethyl ether were both inserted into the cavity of ␤-CD, (R)-benzoin ethyl ether had carbon hydrogen bond interaction, pi-donor hydrogen bond interaction and pi-sigma interaction with ␤-CD, while (S)-benzoin ethyl ether had carbon hydrogen bond interaction and pi-sigma interaction with ␤-CD. The difference of interactions between enantiomers and ␤CD results in the discrimination of enantiomers. In the GQD/␤-CD complex system, (R)-benzoin ethyl ether had carbon hydrogen bond interaction and pi-donor hydrogen bond interaction with ␤-CD, it also formed pi-alkyl interaction with GQDs meanwhile; (S)-benzoin ethyl ether had carbon hydrogen bond interaction and pi-sigma interaction with ␤-CD, it also had pi-alkyl interaction with GQDs. GQDs can also provide ␲-␲ and hydrogen bond interactions in other molecular docking configurations. Synthetically considering, the existence of GQDs provides interactions with enantiomers during the inclusion process and changes the types of interactions between enantiomers and ␤-CD, which helps to discriminate the enantiomers and improves the enantioseparation performance.

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3.4. Stability The stability of Si-GQD-CD, Si-GQD-CDD and Si-GQD-Cellulose columns were estimated by calculating the RSD of retention factor (k) and resolution (Rs ) obtained from replicated analyses of enantiomers. The run-to-run, day-to-day and column-to-column max RSD values were listed in Table S3. Finally, it can be concluded that these CSPs can provide satisfactory repeatability and reproducibility. The useful temperature range of Si-GQD-CD, Si-GQD-CDD and Si-GQD-Cellulose columns were estimated by adjusting the column temperature from 20 ◦ to 40 ◦ considering the volatilization of organic solvent. With the increase of column temperature, the retention time decreased, no significant column efficiency loss was found, which indicated the GQDs-functionalized CSPs could be used in the temperature range of 20 ◦ –40 ◦ . Considering that silica is stable in the pH range of 2.0–8.0, we examined the stability of GQDs-functionalized ␤-CD CSPs in pH 2.5 and 7.6 under the reversed phase chromatography condition. 50:50 MeOH/20 mM ammonium formate (pH 2.5 and 7.6) was passed through the column for fixed periods and chromatography separation was carried out and the process repeated. Both acid and base exposure experiments were carried out until 25 h passed. Test results showed the columns were stable in the pH range of 2.5–7.6. 3.5. Column efficiency Chiral compounds (1–7) were used as probes to test the column efficiency of the four ␤-CD-based columns. The column efficiency of Si-CD, Si-GQD-CD, Si-CDD and Si-GQD-CDD columns is 6700 plates/m, 8214 plates/m, 8976 plates/m, 9368 plates/m, respectively. Chiral compounds (1,2,3,5,6,8,9,10) were used as probes to test the column efficiency of the cellulose-based columns. The column efficiency of Si-Cellulose and Si-GQD-Cellulose columns is 2230 plates/m and 5923 plates/m, respectively. GQDs functionalization increased the column efficiency of ␤-CD and cellulose chiral columns. 4. Conclusions To investigate the effect of GQDs on chiral separation, GQDs functionalized ␤-CD and cellulose silica composites were first prepared and applied in HPLC enantioseparation. Through comparing the enantioseparation performance of GQDs functionalized ␤-CD or cellulose CSPs and unmodified ␤-CD or cellulose CSPs, it was found GQDs could enhance the enantioseparation performance of nature ␤-CD, ␤-CD-3,5-dimethylphenylcarbamate derivative and cellulose-3,5-dimethylphenylcarbamate derivative. GQDs had unique advantages in chiral separation compared with conventional chiral enhancement reagent 3,5-dimethylphenyl isocyanate. GQDs had a general enhancement effect for all the tested chiral compounds, while 3,5-dimethylphenyl isocyanate showed negative effect for certain chiral compounds. According to experimental results, GQDs functionalized CSPs have stronger retention ability, this was because GQDs provided extra interactions including ␲-␲ and hydrogen-bonding interactions with analytes. Molecular modeling was applied to get an in-depth understanding of the enhancement mechanism of GQDs for enantioseparation, which showed a good agreement with the experimental results. Our work demonstrates that GQDs have enhancement effect on chiral separation, which gives a guidance for the fabrication of new-style chiral separation materials and has inspiration significance for asymmetric synthesis, chiral recognition, drug delivery and other research fields.

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Conflicts of interest There are no conflicts to declare.

Acknowledgement This work was supported by the National Natural Science Foundation of China (Nos. 21405162, 21405161, 21675163 and 21702210).

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 04.053.

References [1] T.J. Ward, K.D. Ward, Chiral separations: a review of current topics and trends, Anal. Chem. 84 (2012) 626–635. [2] I.D. Rukhlenko, N.V. Tepliakov, A.S. Baimuratov, S.A. Andronaki, Y.K. Gun’ko, A.V. Baranov, A.V. Fedorov, Completely chiral optical force for enantioseparation, Sci. Rep. 6 (2016), 36884. [3] T. Ikai, C. Yamamoto, M. Kamigaito, Y. Okamoto, Enantioseparation by HPLC using phenylcarbonate, benzoylformate, p-toluenesulfonylcarbamate, and benzoylcarbamates of cellulose and amylose as chiral stationary phases, Chirality 17 (2005) 299–304. [4] Y. Okamoto, T. Ikai, Chiral HPLC for efficient resolution of enantiomers, Chem. Soc. Rev. 37 (2008) 2593–2608. [5] T. Ikai, Y. Okamoto, Structure control of polysaccharide derivatives for efficient separation of enantiomers by chromatography, Chem. Rev. 109 (2009) 6077–6101. [6] L. Wang, S. Dong, F. Han, Y. Zhao, X. Zhang, X. Zhang, H. Qiu, L. Zhao, Spherical beta-cyclodextrin-silica hybrid materials for multifunctional chiral stationary phases, J. Chromatogr. A 1383 (2015) 70–78. [7] H. Zhang, J. Chen, Y. Yang, L. Wang, Z. Li, H. Qiu, Discriminative detection of glutathione in cell lysates based on oxidase-like activity of magnetic nanoporous graphene, Anal. Chem. 91 (2019) 5004–5010. [8] L. Wang, Z. Li, J. Chen, Y. Huang, H. Zhang, H. Qiu, Enhanced photocatalytic degradation of methyl orange by porous graphene/ZnO nanocomposite, Environ. Pollut. 249 (2019) 801–811. [9] L. Song, H. Zhang, T. Cai, J. Chen, Z. Li, M. Guan, H. Qiu, Porous graphene decorated silica as a new stationary phase for separation of sulfanilamide compounds in hydrophilic interaction chromatography, Chin. Chem. Lett. https://doi.org/10.1016/j.cclet.2018.10.040. [10] Z. Li, X. Zhang, H. Tan, W. Qi, L. Wang, M.C. Ali, H. Zhang, J. Chen, P. Hu, C. Fan, H. Qiu, Combustion fabrication of nanoporous graphene for ionic separation membranes, Adv. Funct. Mater. 28 (2018), 1805026. [11] H. Zhang, B. Zhang, C. Di, M.C. Ali, J. Chen, Z. Li, J. Si, H. Zhang, H. Qiu, Label-free fluorescence imaging of cytochrome c in living systems and anti-cancer drug screening with nitrogen doped carbon quantum dots, Nanoscale 10 (2018) 5342–5349. [12] X. Liang, X. Hou, J.H.M. Chan, Y. Guo, E.F. Hilder, The application of graphene-based materials as chromatographic stationary phases, TrAC, Trends Anal. Chem. 98 (2018) 149–160. [13] A.L. Hemasa, N. Naumovski, W.A. Maher, A. Ghanem, Application of carbon nanotubes in chiral and achiral separations of pharmaceuticals, biologics and chemicals, Nanomaterials 7 (2017) 186. [14] Y. Ji, J. Ke, F. Duan, J. Chen, Preparation and application of novel multi-walled carbon nanotubes/polysulfone nanocomposite membrane for chiral separation, Desalin. Water Treat. 87 (2017) 179–187. [15] R.P. Liang, C.M. Liu, X.Y. Meng, J.W. Wang, J.D. Qiu, A novel open-tubular capillary electrochromatography using beta-cyclodextrin functionalized graphene oxide-magnetic nanocomposites as tunable stationary phase, J. Chromatogr. A 1266 (2012) 95–102. [16] R.P. Liang, X.N. Wang, C.M. Liu, X.Y. Meng, J.D. Qiu, Facile preparation of protein stationary phase based on polydopamine/graphene oxide platform for chip-based open tubular capillary electrochromatography enantioseparation, J. Chromatogr. A 1323 (2014) 135–142. [17] R.P. Liang, X.Y. Meng, C.M. Liu, J.W. Wang, J.D. Qiu, Enantiomeric separation by open-tubular capillary electrochromatography using bovine-serum-albumin-conjugated graphene oxide-magnetic nanocomposites as stationary phase, Microfluid. Nanofluid. 16 (2014) 195–206. [18] T. Hong, X. Chen, Y. Xu, X. Cui, R. Bai, C. Jin, R. Li, Y. Ji, Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis, J. Chromatogr. A 1456 (2016) 249–256.

218

Q. Wu et al. / J. Chromatogr. A 1600 (2019) 209–218

[19] Z.R. Liu, Y.X. Du, Z.J. Feng, Enantioseparation of drugs by capillary electrochromatography using a stationary phase covalently modified with graphene oxide, Microchim. Acta 184 (2017) 583–593. [20] L. Candelaria, L.V. Frolova, B.M. Kowalski, K. Artyushkova, A. Serov, N.G. Kalugin, Surface-modified three-dimensional graphene nanosheets as a stationary phase for chromatographic separation of chiral drugs, Sci. Rep. 8 (2018), 14747. [21] Y. Li, Q. Li, N. Zhu, Z. Gao, Y. Ma, Cellulose type chiral stationary phase based on reduced graphene oxide@silica gel for the enantiomer separation of chiral compounds, Chirality 30 (2018) 996–1004. [22] P. Zheng, N. Wu, Fluorescence and sensing applications of graphene oxide and graphene quantum dots: a review, Chem.-Asian J. 12 (2017) 2343–2353. [23] X. Zhang, H. Ji, X. Zhang, Z. Wang, D. Xiao, Capillary column coated with graphene quantum dots for gas chromatographic separation of alkanes and aromatic isomers, Anal. Methods 7 (2015) 3229–3237. [24] Y. Sun, Q. Bi, X. Zhang, L. Wang, X. Zhang, S. Dong, L. Zhao, Graphene quantum dots as additives in capillary electrophoresis for separation cinnamic acid and its derivatives, Anal. Biochem. 500 (2016) 38–44. [25] T.P. Cai, H.J. Zhang, A. Rahman, Y.P. Shi, H.D. Qiu, Silica grafted with silanized carbon dots as a nano-on-micro packing material with enhanced hydrophilic selectivity, Microchim. Acta 184 (2017) 2629–2636. [26] H. Zhang, X. Qiao, T. Cai, J. Chen, Z. Li, H. Qiu, Preparation and characterization of carbon dot-decorated silica stationary phase in deep eutectic solvents for hydrophilic interaction chromatography, Anal. Bioanal. Chem. 409 (2017) 2401–2410. [27] Q. Wu, Y. Sun, X. Zhang, X. Zhang, S. Dong, H. Qiu, L. Wang, L. Zhao, Multi-mode application of graphene quantum dots bonded silica stationary phase for high performance liquid chromatography, J. Chromatogr. A 1492 (2017) 61–69. [28] Q. Wu, Y. Sun, J. Gao, L. Chen, S. Dong, G. Luo, H. Li, L. Wang, L. Zhao, Ionic liquid-functionalized graphene quantum dot-bonded silica as multi-mode HPLC stationary phase with enhanced selectivity for acid compounds, New J. Chem. 42 (2018) 8672–8680. [29] Q. Wu, L. Chen, J. Gao, S. Dong, H. Li, D. Di, L. Zhao, Graphene quantum dots-functionalized C18 hydrophobic/hydrophilic stationary phase for high performance liquid chromatography, Talanta 194 (2019) 105–113. [30] X. Zhang, L. Wang, S. Dong, X. Zhang, Q. Wu, L. Zhao, Y. Shi, Nanocellulose 3, 5-dimethylphenylcarbamate derivative coated chiral stationary phase: preparation and enantioseparation performance, Chirality 28 (2016) 376–381.

[31] E. Yashima, P. Sahavattanapong, Y. Okamoto, HPLC enantioseparation on cellulose tris(3,5-dimethylphenylcarbamate) as a chiral stationary phase: influences of pore size of silica gel, coating amount, coating solvent, and column temperature on chiral discrimination, Chirality 8 (1996) 446–451. [32] B. Chankvetadze, Recent developments on polysaccharide-based chiral stationary phases for liquid-phase separation of enantiomers, J. Chromatogr. A 1269 (2012) 26–51. [33] X. Zhang, L. Wang, S. Dong, X. Zhang, Q. Wu, L. Zhao, Y. Shi, Nanocellulose derivative/silica hybrid core-shell chiral stationary phase: preparation and enantioseparation performance, Molecules 21 (2016) 561. [34] Y. Liu, R. Wang, J. Lang, X. Yan, Insight into the formation mechanism of graphene quantum dots and the size effect on their electrochemical behaviors, Phys. Chem. Chem. Phys. 17 (2015) 14028–14035. [35] Q. Qin, S. Zhang, W.G. Zhang, Z.B. Zhang, Y.J. Xiong, Z.Y. Guo, J. Fan, S. Run-Zheng, D. Finlow, Y. Yin, The impact of silica gel pore and particle sizes on HPLC column efficiency and resolution for an immobilized, cyclodextrin-based, chiral stationary phase, J. Sep. Sci. 33 (2010) 2582–2589. [36] C. Yamamoto, S. Inagaki, Y. Okamoto, Enantioseparation using alkoxyphenylcarbamates of cellulose and amylose as chiral stationary phase for high-performance liquid chromatography, J. Sep. Sci. 29 (2006) 915–923. [37] L. Wang, M. Lv, D. Pei, Y. Wang, Q. Wang, S. Sun, H. Wang, Wide pH range enantioseparation of cyclodextrin silica-based hybrid spheres for high performance liquid chromatography, J. Chromatogr. A https://doi.org/10. 1016/j.chroma.2019.02.040. [38] X. Yao, H. Zheng, Y. Zhang, X. Ma, Y. Xiao, Y. Wang, Engineering thiol-ene click chemistry for the fabrication of novel structurally well-defined multifunctional cyclodextrin separation materials for enhanced enantioseparation, Anal. Chem. 88 (2016) 4955–4964. [39] P. Peluso, A. Dessi, R. Dallocchio, V. Mamane, S. Cossu, Recent studies of docking and molecular dynamics simulation for liquid-phase enantioseparations, Electrophoresis (2019) 1–16. [40] C.S. Nascimento, J.F. Lopes, L. Guimaraes, K.B. Borges, Molecular modeling study of the recognition mechanism and enantioseparation of 4-hydroxypropranolol by capillary electrophoresis using carboxymethyl-beta-cyclodextrin as the chiral selector, Analyst 139 (2014) 3901–3910. [41] X. Li, X. Yao, Y. Xiao, Y. Wang, Enantioseparation of single layer native cyclodextrin chiral stationary phases: effect of cyclodextrin orientation and a modeling study, Anal. Chim. Acta 990 (2017) 174–184.