Evaluation of Poly(Glycidyl Methacrylate) Nanocoating for Chiral Separation with Glu-β-CD as Chiral Selector in Capillary Electrophoresis

Department of Neurology, Second Affiliated Hospital of Naval Medical University, Shanghai 200003, China Research and Development Center of Microelectronics, School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200444, China School of Medicine, Shanghai University, Shanghai 200444, China Department of Anesthesiology, Second Affiliated Hospital of Naval Medical University, Shanghai 200433, China


Introduction
The human body and the two enantiomers show different pharmacodynamics and pharmacokinetic properties, which means controlling the quality of the drug enantiomers is the key factor in the modern pharmaceutical industry. The development of effective and fast chiral drug analysis methods has attracted more and more attention [1][2][3]. Among various separation techniques, capillary electrophoresis (CE) has proven to be a useful method for chiral drugs due to its high efficiency, simplicity, low consumption, and flexibility of separation methods (e.g., micellar electrokinetic chromatography (MEKC), nonaqueous capillary electrophoresis (NACE), and capillary electrochromatography (CEC)) [4][5][6]. With further research, the traditional separated CE model has many limitations. Therefore, the induction of new type of materials to modify the inner wall of the capillary and enhance the selectivity and efficiency of CE has become a commonly used method [7,8].
After being modified by materials such as ionic liquids, nanoparticles, and even phospholipid vesicles, capillaries may have many characteristics, including inhibition of electroosmotic flow (EOF) and local enrichment of chiral selector [9,10]. In the past few decades, nanoparticles (NPs) have been used in many fields, including application as column coating for preparing capillary columns [11,12]. NPs can provide unique advantages, such as favorable surfaceto-volume ratio, significant mechanical strength, good chemical stability, and easy modification characteristics [13,14]. Due to specific physical and chemical properties, they can be easily connected to the capillary to improve the column capacity and efficiency of chiral CE [15][16][17][18][19][20]. Mayer and Schurig reported the first chiral open tubular CEC system based on Chirasil-Dex-(permethyl-β-cyclodextrin chemically linked to dimethylpolysiloxane-) modified column for enantioseparation. This method was developed in 1992 [21]. Our group explored an HKUST-1-functionalized CE column for the enantiomeric separation of several racemic drugs with CM-β-CD as the chiral selector in 2019. Compared with the uncoated bare column system, the HKUST-1 coating system has significantly improved separation for enantiomers [22].
In this study, poly(glycidyl methacrylate) nanoparticles (PGMA NPs) were immobilized on the inner wall of the capillary through a simple ring-opening reaction [23][24][25][26]. Then, Glu-β-CD was used as chiral selectors in the PGMA-coated column to construct a Glu-β-CD/PGMA column separation system. Two essential drugs were selected as models for evaluating enantioselectivity of PGMA nanocoating. Compared with bare column, the new system shows a satisfactory separation performance improvement. The main influencing factors such as chiral selector concentration and background electrolyte (BGE) pH are systematically studied. Glu-β-CD (purity > 99) and racemic drugs (nefopam (NEF) and chlorpheniramine (CHL)) are provided by Jiangsu Food and Drug Administration (Nanjing, China). The structure is shown in Figure 1. All experimental water is ultrapure water.     After removing the methanol with nitrogen, the capillary was heated in a vacuum oven at 100°C for about 1 hour. After cooling to room temperature, the APTES solution (50%, v/v) prepared in methanol was pumped through the capillary for 30 min. Then, the capillary column was sealed with rubber and reacted in a water bath at 55°C for 12 hours. In order to obtain the PGMA coating, the obtained capillary was rinsed with 0.5 mg/mL PGMA nanoparticles (pH 8.5, 50 mM phosphate buffer) for about 30 min at room temperature. The filled capillary was sealed and allowed to react at 55°C for 4 h. Wash the capillary with deionized water to remove excess PGMA nanoparticles. Repeat the above process 3 times.

Results and Discussion
3.1. Characterization of PGMA Nanoparticles and PGMA Nanocoating. The morphology of PGMA nanoparticles has been clearly characterized by SEM, as shown in Figure 2(a). All PGMA nanoparticles are spherical, with a uniform average diameter and an about 100 nm narrow particle size distribution. Figure 2(b) shows the SEM image of the inner surface of the PGMA-coated column. Very obvious spherical protrusions can be observed, which indicates that the PGMA nanoparticle coating has been successfully immobilized to the inner wall of the capillary.

Measurement of EOF.
EOF is a key influence due to the fact that it can reflect the ionic situation of capillary inner surface. Here, a range of pH 4.0-pH 9.0 with thiourea as electroosmotic flow marker in the uncoated capillary, APTSmodified capillary, and PGMA-coated column was investigated. As Figure 3 shows, EOF of the APTS-modified column and PGMA-coated column was negative in pH 4, which is due to the positive charge of the amino group (residual amino for the PGMA-coated column). After immobilization of PGMA NPs, the EOF was lower than that in the APTS column, which was due to the reaction of PGMA with the amino group. It is worth noting that mild EOF change with pH is favorable for obtaining high stability in migration time.
3.3. Performance of PGMA Nanocoating with Glu-β-CD as Chiral Selector. The capillary coating can suppress EOF and reduce the adsorption of analytes, thereby improving separation efficiency. NEF and CHL have been tested to evaluate the modification of PGMA coating. Using 20 mM phosphate buffer and a limited amount of Glu-β-CD, the drug is analyzed under optimized separation conditions in the PGMAcoated capillary. From Figure 4, the enantiomeric separation performance of NEF and CHL in PGMA coating system has been obtained.    Effect of Glu-β-CD Concentration for Enantioseparation in PGMA Nanocoating System. The concentration of the chiral selector is a key factor affecting the separation of enantiomers. In order to determine the optimal selection agent concentration for the system, a series of concentrations were studied using a 20 mM phosphate buffer solution. As shown in Figure 5, the migration time of racemic drugs all increase with the increase of Glu-β-CD concentration. The reason for this phenomenon may be the enhanced interaction between the chiral selector and the enantiomer led by local enrichment of Glu-β-CD in the PGMA nanocoating. And the increase in the viscosity of BGE may be the other reason. As the concentration of the chiral selector increases, Rs and α of the tested drug first increase and then decrease. The maximum value is obtained when the concentration of Glu-β-CD reaches 60 mM. Therefore, 60 mM Gluβ-CD was chosen for the next experiment.

Effect of Buffer pH for Enantioseparation in PGMA
Nanocoating System. The pH of the buffer is an important parameter of the separation system [28]. It determines the degree of ionization of the analyte and the chiral selector and the ion state of the capillary wall. Therefore, optimizing BGE pH is usually a key strategy for optimizing separation. A pH range of BGE was studied using a 20 mM phosphate buffer solution containing an optimized concentration of Glu-β-CD to isolate the selected drugs. As shown in Figure 6, Rs and α in the two PGMA-coated column systems have the same trend, increasing as the pH increases from 5.80 to 6.00. The change trend of resolution gradually decreased from pH 6.20 to 6.80, and the best separation effect was obtained at 6.20.

Conclusion
In this study, a PGMA nanocoating-based enantiomeric separation system with Glu-β-CD as the chiral selector was established to separate enantiomers. In this PGMA-coated column system, the enantiomeric separation results of the NEF and CHL have been obtained. PGMA nanocoating can achieve satisfactory stability of EOF. In addition, by evaluating influencing factors such as Glu-β-CD concentration and BGE pH, the best separation resolution can be obtained with pH 6.20 and 60 mM Glu-β-CD. This PGMA nanocoating can provide a support to the development of more new types of modification nanomaterials for enantioseparation.

Data Availability
Due to the confidentiality before public, this manuscript has no associate data.

Conflicts of Interest
The authors declare that they have no conflicts of interest.