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Qifeng Fu Fengqing Yang Hua Chen Zhining Xia School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, P. R. China
Received March 9, 2014 Revised May 27, 2014 Accepted June 19, 2014
Research Article
Enhancement of enantioselectivity in chiral capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as chiral selector under molecular crowding conditions induced by dextran or dextrin Molecular crowding is a new approach to enhance the retention properties and selectivity of molecularly imprinted polymers. In this work, this concept was first applied to chiral CE to enhance its enantioselectivity. A model system, enantioseparation of salbutamol using hydroxypropyl-beta-cyclodextrin as chiral selector in the presence of dextran or dextrin as crowding-inducing agents, was chosen to demonstrate its potency. Some parameters, especially the concentration of crowding-inducing agents and cyclodextrins were investigated intensively. Moreover, based on fluorescence spectroscopy and affinity CE, it was found that the presence of crowding-inducing agents could promote the association of enantiomers with cyclodextrins and intensify the interacting differences of two enantiomers with cyclodextrins. As a result, the essential concentration of cyclodextrins to make the enantiomers reach baseline separation was significantly decreased with the aid of molecular crowding. This study shows that molecular crowding is an effective strategy to enhance the enantioselectivity of cyclodextrin in chiral CE. Keywords: CE / Chiral separation / Molecular crowding / Salbutamol enantiomers DOI 10.1002/elps.201400116
1 Introduction CE is a popular and evolving separation technique for chiral compounds. Up to now, a variety of chiral selectors have been used for the enantioseparation with CE, such as cyclodextrins [1, 2], macrocyclic antibiotics [3], chiral crown ethers [4], proteins [5], and bacteria [6]. Among them, cyclodextrins and their derivatives are the most frequently used ones. However, good enantioseparation in chiral CE cannot be obtained by using one kind of cyclodextrin alone in some cases [7–9]. Vescina et al. [8] investigated the effectiveness of many commercially available cyclodextrin derivatives for chiral CE and the highest success rate was only 68% in the 35 tested model compounds. To improve the enantioselectivity of cyclodextrins in chiral CE, some approaches have been presented. For example, various new cyclodextrin derivatives possessing novel functional groups have been synthesized for achieving better enantiomeric separation [10]. Nevertheless, these novel cy-
Correspondence: Professor Zhining Xia, School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400030, P. R. China E-mail: [email protected] Fax: +86-23-65106615
Abbreviations: HP--CD, hydroxypropyl-beta-cyclodextrin; MIP, molecularly imprinted polymer C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
clodextrin derivatives are not easily obtained commercially. Na et al. [11] proposed an alternative protocol related to polystyrene nanoparticles to enhance the enantioselectivity of cyclodextrins. In addition, cyclodextrin-modified gold nanoparticles also can be used in the chiral CE as pseudostationary phase [12] or stationary phase [13] to obtain better enantioselectivity. However, the synthesis of nanoparticles is not easy and it is costly . Therefore, it is essential to develop new applicable approaches to improve the enantioselectivity of cyclodextrins in chiral CE. Molecular crowding, which is an important feature of the molecular environments in biological cells, can enhance the stability of higher-order structures of biopolymers and promote the association reactions of biomolecules [14–16]. Inspired by the unique environmental characteristic of biomolecules, Matsui et al. [17] found that molecular crowding can shift the equilibrium of template molecules reacting with functional monomers in the direction of complex formation side. As a result, crowding-assisted molecularly imprinted polymers (MIPs) can result in higher retention and better selectivity than noncrowding assisted MIPs. On the basis of this principle, imprinted monolithic column for HPLC and imprinted microparticles used in CEC have also been prepared successfully under molecular crowding conditions [18–21]. In chiral CE, the chiral discrimination of cyclodextrins is based on the differences in stability between the complexes generated by the complexation between the cyclodextrins and the enantiomers [10, 22].
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Considering the similarity between inclusion reaction and the association reaction in MIPs, it is reasonable to envision that molecular crowding can also enhance the stability of the complexes and intensify the interacting differences of enantiomers with cyclodextrins, leading to higher enantioselectivity of cyclodextrins in chiral CE. In this study, the concept of molecular crowding was first used to enhance the enantioselectivity of cyclodextrins in chiral CE. Hydroxypropyl-beta-cyclodextrin (HP--CD) is a very commonly used cyclodextrin in chiral CE. However, good enantioseparation cannot be achieved by using only HP--CD in some cases. Therefore, HP--CD was chosen as the model cyclodextrin. On the other hand, dextran, a common hydrophilic polysaccharide, had been successfully used as crowding-inducing agent in the macromolecular reactions [14, 16]. Therefore, dextran and its short chain analog dextrin were used as the crowding-inducing agents in the present study. Salbutamol is a selective 2-adrenoreceptor agonist extensively used in the treatment of pulmonary disease [23], and its therapeutic activity was attributed to the R-(-)-enantiomer [24]. However, it was difficult to separate salbutamol enantiomers using HP--CD as chiral selector alone by chiral CE [9, 25]. Herein, with the aid of crowdinginducing agents dextran or dextrin, the chiral separation of salbutamol using HP--CD as chiral selector was intensively investigated. Additionally, both fluorescence spectroscopy and ACE technique were utilized to evaluate the association constants between enantiomers and cyclodextrins with and without crowding-inducing agents.
2 Materials and methods
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tion software. Separations were done on an uncoated fused silica capillary of 50 m id, 375 m od, 48.5 cm total length and 40.0 cm to the detector (Yongnian Optical Fiber Factory, Hebei, China). The capillary was sequentially flushed with 0.1 M NaOH for 20 min at the beginning of every day, ultrapure water for 10 min, and running buffer for 15 min at 940 mbar. Between each runs, the capillary was flushed with 0.1 M NaOH, ultrapure water and the running buffer for 3 min, respectively. All the experiments were performed at 16 kV and 25°C in positive mode. The samples were detected at 200 nm and injected using a pressure of 35 mbar. To keep the injection amount consistent, the time of injection was adjusted from 5 to 25 s by the viscosity of different running buffer, which can affect the migration rate of samples. A series of phosphate buffer with different concentrations were prepared in ultrapure water and the pH was adjusted with phosphoric acid by a Delta 320 pH meter (MettlerToledo Instruments, Shanghai, China). The running buffer was prepared by dissolving appropriate amounts of HP--CD and neutral polysaccharides or PEG as crowding-inducing agents into the corresponding phosphate buffer. In consideration that the analysis time should not be too long and the solubility of some additives was limited, the concentration ranges tested were 10–80 mM for HP--CD, 1–15% w/v for dextran, 1–9% for dextrin, PEG-20000, and PEG-6000. A stock solution containing 1 mg/mL racemic salbutamol sulfate was prepared with ultrapure water. Before analysis, the solution was diluted with phosphate buffer to 100 g/mL. All solutions were filtered through a 0.45 m membrane filter (Auto science instrument Co., Tianjin, China) and degassed by sonication prior to experiments.
2.1 Materials and reagents Racemic salbutamol sulfate was purchased from Adamas Reagent Co. (Shanghai, China). R-salbutamol hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). HP--CD with degree of substitution 0.97 and molecular weight of 1530 was from Xinda Biotechnology Co. (Shandong, China). Dextran with an average molecular weight of 40 000, and dextrin were all brought from Sinopharm Chemical Reagent Co. (Shanghai, China). PEG with the molecular weight of 20 000 (PEG-20000) and 6000 (PEG-6000) were supplied by KeLong Chemical Reagent Co. (Chengdu, China). Sodium hydroxide, sodium dihydrogen phosphate, and phosphoric acid were all analytical grade and from Beibei Chemical Reagent Factory (Chongqing, China). The ultrapure water used throughout was produced by AK’s laboratory water purification system (Tang’s Kangning Science and Technology Development Co., Chengdu, China).
2.3 Fluorescence spectroscopy measurement Fluorescence spectroscopy was used to determine the association constants between salbutamol and HP--CD. It was determined on a RF-5301 (Shimadzu, Japan) spectrofluorimeter and the emission spectra of salbutamol were monitored between 285 and 500 nm (3 nm slit width) with excitation at 280 nm (3 nm slit width) in 2.5 mL cuvettes. To simulate the optimized conditions of CE, ionic strength and pH of all the solutions used in fluorescence measurement were identical to the optimal CE buffer, i.e. 60 mM phosphate buffer at pH 2.5. The tested solutions were prepared by dissolving different amounts of HP--CD and dextran as crowdinginducing agents into the 4 × 10−5 M salbutamol solutions.
2.2 CE procedures
2.4 Calculations
All the CE experiments were performed with an Agilent 7100 3D CE system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector and an Agilent ChemSta-
The association constants between two salbutamol enantiomers and HP--CD were obtained by ACE and fluorescence spectroscopy measurement. For the ACE
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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technique, the calculation was based on the Scatchard equation [26]:
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3 Results and discussion 3.1 Selection of crowding-inducing agents
1 1 1 1 + = ep − ep, f (c − ep, f )K [C D] c − ep, f
(1)
where ep denotes the experimentally determined effective mobility of salbutamol in the running buffer containing different concentrations of HP--CD and constant concentrations of polysaccharides, ep,f and c are the effective mobilities of the free and completely complexed enantiomer, respectively, K represents the association constant, and [CD] is the concentration of HP--CD. When HP--CD and crowding-inducing agents coexist in the CE system, the migration behavior of salbutamol would be affected by the possible side interaction and it can be described by the following equation [27]: Aep =
ep,A +KAC [C]ep,AC +KAD [D]ep,AD 1 + KAC [C] + KAD [D]
(2)
A where ep is the net effective mobility of salbutamol, ep,A denotes the mobility of the free salbutamol, C and D represent HP--CD and crowding-inducing agents, respectively, KAC and KAD are the association constants for the complexes AC and AD, and ep,AC and ep,AD represent the effective mobility of the complexes AC and AD, respectively. Because electrophoretic mobility was inversely proportional to the viscosity of running buffer, the viscosity correction was applied for the evaluation of the ACE measurement by means of monitoring the change of current as follows [27, 28]:
corr = uncorr ×
I[add]=0 I[add]⬎0
(3)
where corr and uncorr are the viscosity corrected and uncorrected mobility, respectively, I[add] = 0 is the current of the buffer without any additive, I[add]⬎0 denotes the current value of the buffer it consist of different concentrations of HP--CD or polysaccharides. In addition, fluorescence assay was carried out by the Benesi–Hildebrand equation and Lineweaver–Burk equation as follows [29, 30]: 1 1 1 + = F − F0 (F∞ − F0 )K [C D] F∞ − F0
(4)
1 1 1 = + F0 − F F0 K F0 [Q]
(5)
where F and F0 are the fluorescence intensity of salbutamol in the presence and absence of HP--CD/dextran, respectively, F is the fluorescence intensity when all of the salbutamol molecules are completely complexed with HP--CD, K is the association constant, [CD] represents the concentration of HP--CD, and [Q] is the concentration of dextran. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Initially, we chose PEG and neutral polysaccharides, including PEG-20000, PEG-6000, dextran, and dextrin as the candidate crowding-inducing agents. These agents were chosen because (i) all of them have good solubility in aqueous solution and good UV transmittance; (ii) PEG and dextran have been successfully used as crowding-inducing agents in the research on macromolecular reactions [14,16]; (iii) these polymers are hydrophilic and do not interfere with the inclusion of salbutamol into the cavity of HP--CD. In order to further ascertain the most appropriate crowding-inducing agents, we made a comparison on the effect of these agents by preliminary CE experiments. For the PEG-6000 and PEG-20000, unfortunately, both of them caused severe instabilities of the UV detector signal. Although this problem could be overcome when a low pressure was applied to the inlet of the capillary as previously reported [31], the resolution of sample will be inevitably decreased because of the introduction of additional laminar flow. Therefore, PEG6000 and PEG-20000 were not suitable for further studies. Then the separation effectiveness using the same amount of HP--CD incorporated with different amounts of neutral polysaccharides were compared, respectively. It was found that the addition of dextran or dextrin greatly enhanced the enantioselectivity of HP--CD as expected. In addition, to ensure the observed enhanced enantioselectivity was not due to the inherent chiral recognition abilities of dextran and dextrin [32,33], the CE experiments using only dextran or dextrin as buffer additives were carried out as control. The results indicated that dextrin and dextran had no enantioselectivity to salbutamol without HP--CD (data not shown). Thus, dextran and dextrin were selected for the further studies.
3.2 Enhanced chiral CE under molecular crowding conditions 3.2.1 Effect of buffer concentration The concentration of the buffer can affect the buffering capacity, the peak shape, and the interaction intensity between cyclodextrins and enantiomers. The chiral separation of salbutamol was studied when the buffer concentration ranged from 20 to 140 mM and the pH was fixed at 2.50 (the other conditions were as follows: phosphate buffer containing 50 mM HP--CD and/or simultaneously containing 9% dextran or 6% dextrin, 16 kV of applied voltage and 25°C of cassette temperature). As shown in Fig. 1A, with the phosphate buffer at a concentration of 60 mM, the optimal resolution can be obtained in the presence of these polysaccharides and the resolution without polysaccharides was also close to the optimum concentration of 80 mM. Hence, 60 mM of phosphate buffer was selected for further study. www.electrophoresis-journal.com
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centration. However, if the concentration of dextran was too high, the buffer viscosity would be too high to get rapid separations. So the system with higher concentration of dextran (15%) was not investigated. In the case of dextrin, the greatest improvement in resolution occurred at the moderate concentration (6% w/v), and the resolution began to decrease at higher concentration ranges. The different effects on chiral separation induced by dextran and dextrin deserved attention and the potential mechanism would be discussed in Section 3.3 below.
3.2.4 Effect of the concentration of HP--CD
Figure 1. Effect of buffer concentration (A), buffer pH (B), polysaccharides concentration (C), and HP--CD concentration (D) on the resolution of salbutamol enantiomers without polysaccharides (•), in presence of dextran ()and dextrin ().
3.2.2 Effect of pH The pH of running buffer has significant influences on the enantioseparation. For example, both electroosmotic flow and the degree of ionization of samples are affected by the buffer pH. The alteration of these parameters may further affect the interaction between cyclodextrins and enantiomers. In the study, the effect of pH was investigated intensively (the other conditions were as follows: 60 mM phosphate buffer containing 50 mM HP--CD and/or simultaneously containing 9% dextran or 6% dextrin, 16 kV of operation voltage and 25°C of cassette temperature). The results shown in Fig. 1B revealed that in the presence of dextran or dextrin, the resolution of salbutamol decreased as pH increased from 2.5 to 4.5. To the buffer solution without dextran or dextrin, the resolution increased slightly at low pH and began to decrease intensely with a pH of higher than 3.0. Taken together, pH 2.5 was adequate for all the three kinds of systems.
The concentration of cyclodextrin is an essential parameter for optimizing chiral separation. The influence of HP--CD concentration (10–80 mM) on the resolution of salbutamol in the presence of dextrin or dextran was studied when the other conditions were as follows: 60 mM phosphate buffer at pH 2.50 containing 15% dextran or 6% dextrin, 16 kV of operation voltage, and 25°C of cassette temperature. The results shown in Fig. 1D indicated that the enantioselectivity of HP--CD to salbutamol was so poor that the essential concentration of HP--CD to reach baseline separation was about 70 mM. Using the buffer incorporated with 15% dextran or 6% dextrin, the minimal concentration of HP--CD for baseline separation decreased greatly. Especially for dextran, the concentration of HP--CD to reach baseline separation was only about 40 mM. These results demonstrated the positive effect of molecular crowding on the enhancement of enantioseparation in chiral CE. Based on the above investigation, the optimal CE buffers for the analysis were selected: 60 mM phosphate buffer at pH 2.5 with appropriate amounts of HP--CD in the presence of 15% dextran or 6% dextrin. Under these optimized conditions, the separation effectiveness of HP--CD was enhanced significantly by the crowding-inducing agents and the typical electrophorograms were shown in Fig. 2. The first peak was identified as R-salbutamol by comparing the retention time to the individual standard of R-salbutamol under the identical conditions.
3.2.3 Effect of the concentrations of dextran and dextrin The addition of dextran or dextrin provided a molecular crowding surrounding in the process of electrophoresis, which was expected to play a positive role in the chiral separation. Different concentrations of dextran and dextrin were investigated when the other conditions were as follows: 60 mM phosphate buffer at pH 2.50 containing 50 mM HP--CD, 16 kV of operation voltage and 25°C of cassette temperature. The results in Fig. 1C demonstrated the influence of the amounts of dextran and dextrin in the running buffer upon the resolution of salbutamol enantiomers, and as expected, it was enhanced gradually with the increase in dextran con C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.3 The mechanism of enhanced chiral separation under molecular crowding The above results demonstrated that dextran and dextrin can significantly enhance the enantioselectivity of HP--CD to salbutamol as crowding-inducing agents, though they had no inherent enantioselectivity to salbutamol. More interestingly, they had different effects on chiral separation with changes in concentration (Fig. 1C). To better clarify these phenomena, the interaction between enantiomers and HP--CD was evaluated by means of fluorescence spectroscopy and ACE technique. www.electrophoresis-journal.com
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Figure 2. Electropherograms of salbutamol enantiomers with the 60 mM phosphate buffer (pH 2.5) containing 50 mM HP--CD (A), buffer containing 50 mM HP--CD and 6% dextrin (B), buffer containing 50 mM HP--CD and 15% dextran (C). 1, R-Sabutamol; 2, S-Sabutamol. Other conditions: fused silica capillary 50 m (48.5 cm × 40 cm), UV detected at 200 nm, 16 kV of operation voltage and 25°C of cassette temperature.
Figure 3. Fluorescence enhancement of salbutamol by HP--CD without dextran (A), with 5% dextran (B), with 10% dextran (C) with 15% dextran (D), and fluorescence quenching of salbutamol by dextran (E). From 1 to 7, the concentration of HP--CD were 0, 4, 8, 12, 16, 20, 24 mM, and the concentration of dextran were 0, 1, 2, 3, 4, 5, 6%, respectively. Operation conditions: excitation and emission slit width, 3 nm; excitation wavelength, 280 nm; phosphate buffer solution, 60 mM, pH 2.5.
3.3.1 Fluorescence spectroscopy studies The interaction between salbutamol and HP--CD in the presence of dextran with different concentrations was initially investigated using fluorescence spectroscopy. It was found that within the concentration range of dextran studied, the fluorescence intensity increased along with the increase of HP--CD concentration. The corresponding spectra were shown in Fig. 3 and the results revealed that salbutamol was transferred from the polar water solution into the apolar cavity of HP--CD, which can protect the guest molecular from quenching or other processes occurring in the bulk solvent. To quantitatively compare the interacting intensity of inclusion reaction at different concentrations of dextran, we C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
fitted the spectrum data to the Benesi–Hildebrand expression. The calculated association constants were summarized in Table 1, with a good linearity. Thus the 1:1 binding ratio between HP--CD and salbutamol can be confirmed. By comparing the association constants listed in Table 1, it can be seen that the intensity of combination was enhanced dramatically along with the increase of dextran. Accordingly, the result of fluorescence studies was in line with the expectation that molecular crowding can facilitate the association reaction between HP--CD and salbutamol. On the other hand, as shown in Fig. 3E, the fluorescence intensities of salbutamol decreased with the increase of dextran concentration and the quenching effect indicated the analyte can interact with dextran. The quenching www.electrophoresis-journal.com
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Table 1. The association constants between salbutamol enantiomers and HP--CD calculated by the Benesi–Hildebrand equation
Buffer components
Linear equation
R2
Kb (×10−3 L/g)
HP--CD HP--CD and 5% dextran HP--CD and 10% dextran HP--CD and 15% dextran Dextran
y = 1.18x + 4.71 × 10−3 y = 6.29 × 10−1 x + 5.40 × 10−3 y = 7.39 × 10−1 x + 9.58 × 10−3 y = 7.51×10−1 x + 1.25 × 10−2 y = 1.50x + 7.21 × 10−4
0.9960 0.9990 0.9963 0.9985 0.9948
3.99 8.58 12.96 16.64 0.48
Table 2. The association constants between salbutamol enantiomers and HP--CD determined by Scatchard analysis
Kb (×10−3 L/g)
Buffer containing HP--CD
Buffer containing 15% dextran and HP--CD
Buffer containing 6% dextrin and HP--CD
K1 (correlation coefficient) K2 (correlation coefficient) K2 /K1
4.52 (0.9923) 4.98 (0.9947) 1.102
8.46 (0.9866) 9.71 (0.9889) 1.148
7.83 (0.9890) 8.89 (0.9871) 1.135
mechanism was confirmed as static quenching based on the results of fluorescence analysis at different temperatures (data not shown). Hence, the related association constant can be calculated based on the Lineweaver–Burk equation. The result listed in Table 1 showed that the interaction intensity of salbutamol with dextran was weak as compared with HP-CD. Therefore, the side interaction introduced by dextran may have no significant impact on the chiral separation. Besides, to confirm the reliability of fluorescence studies, we further carried out the ACE analysis.
Table 3. The experimentally determined parameters for the calculation based on Eq. (2)
Types of additives
HP--CD Dextran
Kb (×10−3 L/g)
(×10−9 m2 V−1 s−1 )
K1
K2
ep,AC /ep,AD
A ep
4.52 0.36
4.98 0.36
8.20 1.58
18.32 18.32
3.3.2 ACE analysis The ACE technique and Scatchard analysis were utilized to evaluate the differences of two salbutamol enantiomers that interacted with HP--CD. By preparing a series of running buffers containing different concentrations of HP--CD in the presence or absence of dextran and dextrin, we determined the change in apparent mobility of each salbutamol enantiomers and calculated respective association constants using Scatchard equation. As shown in Table 2, both the association constants and their ratio were increased significantly after adding dextran or dextrin to the buffer containing HP-CD. The magnitude of association constants determined by ACE was in good accordance with the results of fluorescence studies. Moreover, the higher ratio of association constants also corresponded with the anticipation that molecular crowding can enhance the interacting differences of two enantiomers with cyclodextrins. Hence, the ACE analysis not only confirmed the reliability of fluorescence studies, but also offered more evidences that molecular crowding was an effective strategy to enhance the enantioselectivity of cyclodextrin in chiral CE. Not only interaction of salbutamol with HP--CD, the ACE technique also can be used to investigate the potential side interaction introduced by the crowding-inducing agents [27, 34, 35]. Since salbutamol interacted not only with cyclodextrins but also with crowding-inducing agents, the side interaction would shift the association constants of salbu C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 4. Theoretical and experimental dependence of mobility ratios on the concentration of HP--CD in the system containing different concentrations of HP--CD with or without 15% dextran.
tamol with HP--CD measured by ACE and may bring about better resolution [34,35]. Therefore, it is necessary to evaluate the side interaction based on Eq. (2) listed in Section 2.4, and the related parameters were determined by ACE and listed in Table 3. The small association constants indicated the weak interaction of salbutamol with dextran, which was consistent with the result of fluorescence analysis. Beyond that, based on these parameters and Eq. (2), the theoretical mobility ratios of the enantiomers were calculated in the presence of different concentrations of HP--CD with or without 15% dextran. As shown in Fig. 4, the side interaction introduced by dextran www.electrophoresis-journal.com
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can lead to a slight decrease of the calculated mobility ratios, which was unfavorable to the enhancement of enantioseparation. Conversely, the experimentally determined ratios were significantly higher than the calculated values. Thus, the observed enhanced enantioselectivity can be attributed to the effect of molecular crowding rather than the weak interaction of salbutamol with dextran compared with HP--CD. Molecular crowding not only can explain why the enantioselectivity was enhanced significantly, but also can illustrate the different effects of dextran and dextrin on chiral separation as the change of concentration. Molecular crowding, which retards diffusional motion, also can decrease the inclusion reaction rate on the condition that the overall rate of the inclusion reaction is controlled by the diffusion mobility of reactant molecules [14]. Thus, considering the two opposite effects and the different constitution of dextran and dextrin, it was reasonable that they had different impact on the chiral separation of salbutamol with the change of concentration. For the dextran, the positive effect of molecular crowding occupied a dominant position in the whole concentration range studied. Consequently, the stronger stability of formed complexes enlarged the interacting differences of two enantiomers with HP--CD and enhanced the enantioselectivity of HP--CD. As for dextrin, the aforementioned two opposite effects achieved a balance at the moderate concentration, i.e. 6% w/v, and the separation effectiveness reached optimum simultaneously.
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tioseparation of salbutamol, the model drug, was enhanced by using HP--CD as chiral selector and dextran or dextrin as the crowding-inducing agents. Compared with the results obtained without crowding-inducing agents, the presence of dextrin and dextran, especially the dextran, can significantly improve the enantioselectivity of HP--CD. The mechanism of enhanced enantioselectivity was also investigated intensively through fluorescence spectroscopy and ACE technique. It can be attributed to the effect of molecular crowding which can promote the interaction of enantiomers with cyclodextrins and intensify the interacting differences of two enantiomers with cyclodextrins. It should be noted that the analysis time was long in the presence of dextran. Hence, further work should be considered in the future, for example, searching for more available crowding-inducing agents in chiral CE. In conclusion, molecular crowding was an effective strategy to enhance the enantioselectivity of cyclodextrin in chiral CE and has potential for more extensive use such as applications in greater number of analytes. This work was supported by the National Natural Science Foundation of China (21175159, 21275169 and 81202886). The authors have declared no conflict of interest.
5 References 3.3.3 The viscous effects in chiral CE Apart from molecular crowding, the change of buffer viscosity caused by the addition of crowding-inducing agents also deserved attention. There were two opposing effects of increased viscosity on the resolution of enantioseparation. Higher viscosity will increase the analysis time and decrease the diffusion coefficient of solutes, and it seemingly contributed to the improvement of resolution. But on the other hand, the mobility differences of the enantiomers decreased with the growing viscosity, which would be expected to lower the resolution. To have an insight into the viscous effects on separation in CE, Schure et al. [36] synthesized the two aspects and developed a theory to describe the viscous effect of buffer additives on the resolution of small molecules. They found that the resolution was independent of the viscosity of the running buffer. Moreover, Shimizu et al. [37] investigated the relation between mobility, , diffusion coefficient, D, of a small molecule and the viscosity of the buffer containing PEG. The results also showed that the ratio /D related to the resolution was nearly independent of viscosity. Therefore, we can further confirm the predominant role of molecular crowding in the enhanced chiral separation of salbutamol.
4 Concluding remarks In the present study, we revealed the application of molecular crowding for enantioseparation in chiral CE. The enan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Qifeng Fu Fengqing Yang Hua Chen Zhining Xia School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, P. R. China
Received March 9, 2014 Revised May 27, 2014 Accepted June 19, 2014
Research Article
Enhancement of enantioselectivity in chiral capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as chiral selector under molecular crowding conditions induced by dextran or dextrin Molecular crowding is a new approach to enhance the retention properties and selectivity of molecularly imprinted polymers. In this work, this concept was first applied to chiral CE to enhance its enantioselectivity. A model system, enantioseparation of salbutamol using hydroxypropyl-beta-cyclodextrin as chiral selector in the presence of dextran or dextrin as crowding-inducing agents, was chosen to demonstrate its potency. Some parameters, especially the concentration of crowding-inducing agents and cyclodextrins were investigated intensively. Moreover, based on fluorescence spectroscopy and affinity CE, it was found that the presence of crowding-inducing agents could promote the association of enantiomers with cyclodextrins and intensify the interacting differences of two enantiomers with cyclodextrins. As a result, the essential concentration of cyclodextrins to make the enantiomers reach baseline separation was significantly decreased with the aid of molecular crowding. This study shows that molecular crowding is an effective strategy to enhance the enantioselectivity of cyclodextrin in chiral CE. Keywords: CE / Chiral separation / Molecular crowding / Salbutamol enantiomers DOI 10.1002/elps.201400116
1 Introduction CE is a popular and evolving separation technique for chiral compounds. Up to now, a variety of chiral selectors have been used for the enantioseparation with CE, such as cyclodextrins [1, 2], macrocyclic antibiotics [3], chiral crown ethers [4], proteins [5], and bacteria [6]. Among them, cyclodextrins and their derivatives are the most frequently used ones. However, good enantioseparation in chiral CE cannot be obtained by using one kind of cyclodextrin alone in some cases [7–9]. Vescina et al. [8] investigated the effectiveness of many commercially available cyclodextrin derivatives for chiral CE and the highest success rate was only 68% in the 35 tested model compounds. To improve the enantioselectivity of cyclodextrins in chiral CE, some approaches have been presented. For example, various new cyclodextrin derivatives possessing novel functional groups have been synthesized for achieving better enantiomeric separation [10]. Nevertheless, these novel cy-
Correspondence: Professor Zhining Xia, School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400030, P. R. China E-mail: [email protected] Fax: +86-23-65106615
Abbreviations: HP--CD, hydroxypropyl-beta-cyclodextrin; MIP, molecularly imprinted polymer C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
clodextrin derivatives are not easily obtained commercially. Na et al. [11] proposed an alternative protocol related to polystyrene nanoparticles to enhance the enantioselectivity of cyclodextrins. In addition, cyclodextrin-modified gold nanoparticles also can be used in the chiral CE as pseudostationary phase [12] or stationary phase [13] to obtain better enantioselectivity. However, the synthesis of nanoparticles is not easy and it is costly . Therefore, it is essential to develop new applicable approaches to improve the enantioselectivity of cyclodextrins in chiral CE. Molecular crowding, which is an important feature of the molecular environments in biological cells, can enhance the stability of higher-order structures of biopolymers and promote the association reactions of biomolecules [14–16]. Inspired by the unique environmental characteristic of biomolecules, Matsui et al. [17] found that molecular crowding can shift the equilibrium of template molecules reacting with functional monomers in the direction of complex formation side. As a result, crowding-assisted molecularly imprinted polymers (MIPs) can result in higher retention and better selectivity than noncrowding assisted MIPs. On the basis of this principle, imprinted monolithic column for HPLC and imprinted microparticles used in CEC have also been prepared successfully under molecular crowding conditions [18–21]. In chiral CE, the chiral discrimination of cyclodextrins is based on the differences in stability between the complexes generated by the complexation between the cyclodextrins and the enantiomers [10, 22].
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Considering the similarity between inclusion reaction and the association reaction in MIPs, it is reasonable to envision that molecular crowding can also enhance the stability of the complexes and intensify the interacting differences of enantiomers with cyclodextrins, leading to higher enantioselectivity of cyclodextrins in chiral CE. In this study, the concept of molecular crowding was first used to enhance the enantioselectivity of cyclodextrins in chiral CE. Hydroxypropyl-beta-cyclodextrin (HP--CD) is a very commonly used cyclodextrin in chiral CE. However, good enantioseparation cannot be achieved by using only HP--CD in some cases. Therefore, HP--CD was chosen as the model cyclodextrin. On the other hand, dextran, a common hydrophilic polysaccharide, had been successfully used as crowding-inducing agent in the macromolecular reactions [14, 16]. Therefore, dextran and its short chain analog dextrin were used as the crowding-inducing agents in the present study. Salbutamol is a selective 2-adrenoreceptor agonist extensively used in the treatment of pulmonary disease [23], and its therapeutic activity was attributed to the R-(-)-enantiomer [24]. However, it was difficult to separate salbutamol enantiomers using HP--CD as chiral selector alone by chiral CE [9, 25]. Herein, with the aid of crowdinginducing agents dextran or dextrin, the chiral separation of salbutamol using HP--CD as chiral selector was intensively investigated. Additionally, both fluorescence spectroscopy and ACE technique were utilized to evaluate the association constants between enantiomers and cyclodextrins with and without crowding-inducing agents.
2 Materials and methods
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tion software. Separations were done on an uncoated fused silica capillary of 50 m id, 375 m od, 48.5 cm total length and 40.0 cm to the detector (Yongnian Optical Fiber Factory, Hebei, China). The capillary was sequentially flushed with 0.1 M NaOH for 20 min at the beginning of every day, ultrapure water for 10 min, and running buffer for 15 min at 940 mbar. Between each runs, the capillary was flushed with 0.1 M NaOH, ultrapure water and the running buffer for 3 min, respectively. All the experiments were performed at 16 kV and 25°C in positive mode. The samples were detected at 200 nm and injected using a pressure of 35 mbar. To keep the injection amount consistent, the time of injection was adjusted from 5 to 25 s by the viscosity of different running buffer, which can affect the migration rate of samples. A series of phosphate buffer with different concentrations were prepared in ultrapure water and the pH was adjusted with phosphoric acid by a Delta 320 pH meter (MettlerToledo Instruments, Shanghai, China). The running buffer was prepared by dissolving appropriate amounts of HP--CD and neutral polysaccharides or PEG as crowding-inducing agents into the corresponding phosphate buffer. In consideration that the analysis time should not be too long and the solubility of some additives was limited, the concentration ranges tested were 10–80 mM for HP--CD, 1–15% w/v for dextran, 1–9% for dextrin, PEG-20000, and PEG-6000. A stock solution containing 1 mg/mL racemic salbutamol sulfate was prepared with ultrapure water. Before analysis, the solution was diluted with phosphate buffer to 100 g/mL. All solutions were filtered through a 0.45 m membrane filter (Auto science instrument Co., Tianjin, China) and degassed by sonication prior to experiments.
2.1 Materials and reagents Racemic salbutamol sulfate was purchased from Adamas Reagent Co. (Shanghai, China). R-salbutamol hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). HP--CD with degree of substitution 0.97 and molecular weight of 1530 was from Xinda Biotechnology Co. (Shandong, China). Dextran with an average molecular weight of 40 000, and dextrin were all brought from Sinopharm Chemical Reagent Co. (Shanghai, China). PEG with the molecular weight of 20 000 (PEG-20000) and 6000 (PEG-6000) were supplied by KeLong Chemical Reagent Co. (Chengdu, China). Sodium hydroxide, sodium dihydrogen phosphate, and phosphoric acid were all analytical grade and from Beibei Chemical Reagent Factory (Chongqing, China). The ultrapure water used throughout was produced by AK’s laboratory water purification system (Tang’s Kangning Science and Technology Development Co., Chengdu, China).
2.3 Fluorescence spectroscopy measurement Fluorescence spectroscopy was used to determine the association constants between salbutamol and HP--CD. It was determined on a RF-5301 (Shimadzu, Japan) spectrofluorimeter and the emission spectra of salbutamol were monitored between 285 and 500 nm (3 nm slit width) with excitation at 280 nm (3 nm slit width) in 2.5 mL cuvettes. To simulate the optimized conditions of CE, ionic strength and pH of all the solutions used in fluorescence measurement were identical to the optimal CE buffer, i.e. 60 mM phosphate buffer at pH 2.5. The tested solutions were prepared by dissolving different amounts of HP--CD and dextran as crowdinginducing agents into the 4 × 10−5 M salbutamol solutions.
2.2 CE procedures
2.4 Calculations
All the CE experiments were performed with an Agilent 7100 3D CE system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector and an Agilent ChemSta-
The association constants between two salbutamol enantiomers and HP--CD were obtained by ACE and fluorescence spectroscopy measurement. For the ACE
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technique, the calculation was based on the Scatchard equation [26]:
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3 Results and discussion 3.1 Selection of crowding-inducing agents
1 1 1 1 + = ep − ep, f (c − ep, f )K [C D] c − ep, f
(1)
where ep denotes the experimentally determined effective mobility of salbutamol in the running buffer containing different concentrations of HP--CD and constant concentrations of polysaccharides, ep,f and c are the effective mobilities of the free and completely complexed enantiomer, respectively, K represents the association constant, and [CD] is the concentration of HP--CD. When HP--CD and crowding-inducing agents coexist in the CE system, the migration behavior of salbutamol would be affected by the possible side interaction and it can be described by the following equation [27]: Aep =
ep,A +KAC [C]ep,AC +KAD [D]ep,AD 1 + KAC [C] + KAD [D]
(2)
A where ep is the net effective mobility of salbutamol, ep,A denotes the mobility of the free salbutamol, C and D represent HP--CD and crowding-inducing agents, respectively, KAC and KAD are the association constants for the complexes AC and AD, and ep,AC and ep,AD represent the effective mobility of the complexes AC and AD, respectively. Because electrophoretic mobility was inversely proportional to the viscosity of running buffer, the viscosity correction was applied for the evaluation of the ACE measurement by means of monitoring the change of current as follows [27, 28]:
corr = uncorr ×
I[add]=0 I[add]⬎0
(3)
where corr and uncorr are the viscosity corrected and uncorrected mobility, respectively, I[add] = 0 is the current of the buffer without any additive, I[add]⬎0 denotes the current value of the buffer it consist of different concentrations of HP--CD or polysaccharides. In addition, fluorescence assay was carried out by the Benesi–Hildebrand equation and Lineweaver–Burk equation as follows [29, 30]: 1 1 1 + = F − F0 (F∞ − F0 )K [C D] F∞ − F0
(4)
1 1 1 = + F0 − F F0 K F0 [Q]
(5)
where F and F0 are the fluorescence intensity of salbutamol in the presence and absence of HP--CD/dextran, respectively, F is the fluorescence intensity when all of the salbutamol molecules are completely complexed with HP--CD, K is the association constant, [CD] represents the concentration of HP--CD, and [Q] is the concentration of dextran. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Initially, we chose PEG and neutral polysaccharides, including PEG-20000, PEG-6000, dextran, and dextrin as the candidate crowding-inducing agents. These agents were chosen because (i) all of them have good solubility in aqueous solution and good UV transmittance; (ii) PEG and dextran have been successfully used as crowding-inducing agents in the research on macromolecular reactions [14,16]; (iii) these polymers are hydrophilic and do not interfere with the inclusion of salbutamol into the cavity of HP--CD. In order to further ascertain the most appropriate crowding-inducing agents, we made a comparison on the effect of these agents by preliminary CE experiments. For the PEG-6000 and PEG-20000, unfortunately, both of them caused severe instabilities of the UV detector signal. Although this problem could be overcome when a low pressure was applied to the inlet of the capillary as previously reported [31], the resolution of sample will be inevitably decreased because of the introduction of additional laminar flow. Therefore, PEG6000 and PEG-20000 were not suitable for further studies. Then the separation effectiveness using the same amount of HP--CD incorporated with different amounts of neutral polysaccharides were compared, respectively. It was found that the addition of dextran or dextrin greatly enhanced the enantioselectivity of HP--CD as expected. In addition, to ensure the observed enhanced enantioselectivity was not due to the inherent chiral recognition abilities of dextran and dextrin [32,33], the CE experiments using only dextran or dextrin as buffer additives were carried out as control. The results indicated that dextrin and dextran had no enantioselectivity to salbutamol without HP--CD (data not shown). Thus, dextran and dextrin were selected for the further studies.
3.2 Enhanced chiral CE under molecular crowding conditions 3.2.1 Effect of buffer concentration The concentration of the buffer can affect the buffering capacity, the peak shape, and the interaction intensity between cyclodextrins and enantiomers. The chiral separation of salbutamol was studied when the buffer concentration ranged from 20 to 140 mM and the pH was fixed at 2.50 (the other conditions were as follows: phosphate buffer containing 50 mM HP--CD and/or simultaneously containing 9% dextran or 6% dextrin, 16 kV of applied voltage and 25°C of cassette temperature). As shown in Fig. 1A, with the phosphate buffer at a concentration of 60 mM, the optimal resolution can be obtained in the presence of these polysaccharides and the resolution without polysaccharides was also close to the optimum concentration of 80 mM. Hence, 60 mM of phosphate buffer was selected for further study. www.electrophoresis-journal.com
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centration. However, if the concentration of dextran was too high, the buffer viscosity would be too high to get rapid separations. So the system with higher concentration of dextran (15%) was not investigated. In the case of dextrin, the greatest improvement in resolution occurred at the moderate concentration (6% w/v), and the resolution began to decrease at higher concentration ranges. The different effects on chiral separation induced by dextran and dextrin deserved attention and the potential mechanism would be discussed in Section 3.3 below.
3.2.4 Effect of the concentration of HP--CD
Figure 1. Effect of buffer concentration (A), buffer pH (B), polysaccharides concentration (C), and HP--CD concentration (D) on the resolution of salbutamol enantiomers without polysaccharides (•), in presence of dextran ()and dextrin ().
3.2.2 Effect of pH The pH of running buffer has significant influences on the enantioseparation. For example, both electroosmotic flow and the degree of ionization of samples are affected by the buffer pH. The alteration of these parameters may further affect the interaction between cyclodextrins and enantiomers. In the study, the effect of pH was investigated intensively (the other conditions were as follows: 60 mM phosphate buffer containing 50 mM HP--CD and/or simultaneously containing 9% dextran or 6% dextrin, 16 kV of operation voltage and 25°C of cassette temperature). The results shown in Fig. 1B revealed that in the presence of dextran or dextrin, the resolution of salbutamol decreased as pH increased from 2.5 to 4.5. To the buffer solution without dextran or dextrin, the resolution increased slightly at low pH and began to decrease intensely with a pH of higher than 3.0. Taken together, pH 2.5 was adequate for all the three kinds of systems.
The concentration of cyclodextrin is an essential parameter for optimizing chiral separation. The influence of HP--CD concentration (10–80 mM) on the resolution of salbutamol in the presence of dextrin or dextran was studied when the other conditions were as follows: 60 mM phosphate buffer at pH 2.50 containing 15% dextran or 6% dextrin, 16 kV of operation voltage, and 25°C of cassette temperature. The results shown in Fig. 1D indicated that the enantioselectivity of HP--CD to salbutamol was so poor that the essential concentration of HP--CD to reach baseline separation was about 70 mM. Using the buffer incorporated with 15% dextran or 6% dextrin, the minimal concentration of HP--CD for baseline separation decreased greatly. Especially for dextran, the concentration of HP--CD to reach baseline separation was only about 40 mM. These results demonstrated the positive effect of molecular crowding on the enhancement of enantioseparation in chiral CE. Based on the above investigation, the optimal CE buffers for the analysis were selected: 60 mM phosphate buffer at pH 2.5 with appropriate amounts of HP--CD in the presence of 15% dextran or 6% dextrin. Under these optimized conditions, the separation effectiveness of HP--CD was enhanced significantly by the crowding-inducing agents and the typical electrophorograms were shown in Fig. 2. The first peak was identified as R-salbutamol by comparing the retention time to the individual standard of R-salbutamol under the identical conditions.
3.2.3 Effect of the concentrations of dextran and dextrin The addition of dextran or dextrin provided a molecular crowding surrounding in the process of electrophoresis, which was expected to play a positive role in the chiral separation. Different concentrations of dextran and dextrin were investigated when the other conditions were as follows: 60 mM phosphate buffer at pH 2.50 containing 50 mM HP--CD, 16 kV of operation voltage and 25°C of cassette temperature. The results in Fig. 1C demonstrated the influence of the amounts of dextran and dextrin in the running buffer upon the resolution of salbutamol enantiomers, and as expected, it was enhanced gradually with the increase in dextran con C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.3 The mechanism of enhanced chiral separation under molecular crowding The above results demonstrated that dextran and dextrin can significantly enhance the enantioselectivity of HP--CD to salbutamol as crowding-inducing agents, though they had no inherent enantioselectivity to salbutamol. More interestingly, they had different effects on chiral separation with changes in concentration (Fig. 1C). To better clarify these phenomena, the interaction between enantiomers and HP--CD was evaluated by means of fluorescence spectroscopy and ACE technique. www.electrophoresis-journal.com
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Figure 2. Electropherograms of salbutamol enantiomers with the 60 mM phosphate buffer (pH 2.5) containing 50 mM HP--CD (A), buffer containing 50 mM HP--CD and 6% dextrin (B), buffer containing 50 mM HP--CD and 15% dextran (C). 1, R-Sabutamol; 2, S-Sabutamol. Other conditions: fused silica capillary 50 m (48.5 cm × 40 cm), UV detected at 200 nm, 16 kV of operation voltage and 25°C of cassette temperature.
Figure 3. Fluorescence enhancement of salbutamol by HP--CD without dextran (A), with 5% dextran (B), with 10% dextran (C) with 15% dextran (D), and fluorescence quenching of salbutamol by dextran (E). From 1 to 7, the concentration of HP--CD were 0, 4, 8, 12, 16, 20, 24 mM, and the concentration of dextran were 0, 1, 2, 3, 4, 5, 6%, respectively. Operation conditions: excitation and emission slit width, 3 nm; excitation wavelength, 280 nm; phosphate buffer solution, 60 mM, pH 2.5.
3.3.1 Fluorescence spectroscopy studies The interaction between salbutamol and HP--CD in the presence of dextran with different concentrations was initially investigated using fluorescence spectroscopy. It was found that within the concentration range of dextran studied, the fluorescence intensity increased along with the increase of HP--CD concentration. The corresponding spectra were shown in Fig. 3 and the results revealed that salbutamol was transferred from the polar water solution into the apolar cavity of HP--CD, which can protect the guest molecular from quenching or other processes occurring in the bulk solvent. To quantitatively compare the interacting intensity of inclusion reaction at different concentrations of dextran, we C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
fitted the spectrum data to the Benesi–Hildebrand expression. The calculated association constants were summarized in Table 1, with a good linearity. Thus the 1:1 binding ratio between HP--CD and salbutamol can be confirmed. By comparing the association constants listed in Table 1, it can be seen that the intensity of combination was enhanced dramatically along with the increase of dextran. Accordingly, the result of fluorescence studies was in line with the expectation that molecular crowding can facilitate the association reaction between HP--CD and salbutamol. On the other hand, as shown in Fig. 3E, the fluorescence intensities of salbutamol decreased with the increase of dextran concentration and the quenching effect indicated the analyte can interact with dextran. The quenching www.electrophoresis-journal.com
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Table 1. The association constants between salbutamol enantiomers and HP--CD calculated by the Benesi–Hildebrand equation
Buffer components
Linear equation
R2
Kb (×10−3 L/g)
HP--CD HP--CD and 5% dextran HP--CD and 10% dextran HP--CD and 15% dextran Dextran
y = 1.18x + 4.71 × 10−3 y = 6.29 × 10−1 x + 5.40 × 10−3 y = 7.39 × 10−1 x + 9.58 × 10−3 y = 7.51×10−1 x + 1.25 × 10−2 y = 1.50x + 7.21 × 10−4
0.9960 0.9990 0.9963 0.9985 0.9948
3.99 8.58 12.96 16.64 0.48
Table 2. The association constants between salbutamol enantiomers and HP--CD determined by Scatchard analysis
Kb (×10−3 L/g)
Buffer containing HP--CD
Buffer containing 15% dextran and HP--CD
Buffer containing 6% dextrin and HP--CD
K1 (correlation coefficient) K2 (correlation coefficient) K2 /K1
4.52 (0.9923) 4.98 (0.9947) 1.102
8.46 (0.9866) 9.71 (0.9889) 1.148
7.83 (0.9890) 8.89 (0.9871) 1.135
mechanism was confirmed as static quenching based on the results of fluorescence analysis at different temperatures (data not shown). Hence, the related association constant can be calculated based on the Lineweaver–Burk equation. The result listed in Table 1 showed that the interaction intensity of salbutamol with dextran was weak as compared with HP-CD. Therefore, the side interaction introduced by dextran may have no significant impact on the chiral separation. Besides, to confirm the reliability of fluorescence studies, we further carried out the ACE analysis.
Table 3. The experimentally determined parameters for the calculation based on Eq. (2)
Types of additives
HP--CD Dextran
Kb (×10−3 L/g)
(×10−9 m2 V−1 s−1 )
K1
K2
ep,AC /ep,AD
A ep
4.52 0.36
4.98 0.36
8.20 1.58
18.32 18.32
3.3.2 ACE analysis The ACE technique and Scatchard analysis were utilized to evaluate the differences of two salbutamol enantiomers that interacted with HP--CD. By preparing a series of running buffers containing different concentrations of HP--CD in the presence or absence of dextran and dextrin, we determined the change in apparent mobility of each salbutamol enantiomers and calculated respective association constants using Scatchard equation. As shown in Table 2, both the association constants and their ratio were increased significantly after adding dextran or dextrin to the buffer containing HP-CD. The magnitude of association constants determined by ACE was in good accordance with the results of fluorescence studies. Moreover, the higher ratio of association constants also corresponded with the anticipation that molecular crowding can enhance the interacting differences of two enantiomers with cyclodextrins. Hence, the ACE analysis not only confirmed the reliability of fluorescence studies, but also offered more evidences that molecular crowding was an effective strategy to enhance the enantioselectivity of cyclodextrin in chiral CE. Not only interaction of salbutamol with HP--CD, the ACE technique also can be used to investigate the potential side interaction introduced by the crowding-inducing agents [27, 34, 35]. Since salbutamol interacted not only with cyclodextrins but also with crowding-inducing agents, the side interaction would shift the association constants of salbu C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 4. Theoretical and experimental dependence of mobility ratios on the concentration of HP--CD in the system containing different concentrations of HP--CD with or without 15% dextran.
tamol with HP--CD measured by ACE and may bring about better resolution [34,35]. Therefore, it is necessary to evaluate the side interaction based on Eq. (2) listed in Section 2.4, and the related parameters were determined by ACE and listed in Table 3. The small association constants indicated the weak interaction of salbutamol with dextran, which was consistent with the result of fluorescence analysis. Beyond that, based on these parameters and Eq. (2), the theoretical mobility ratios of the enantiomers were calculated in the presence of different concentrations of HP--CD with or without 15% dextran. As shown in Fig. 4, the side interaction introduced by dextran www.electrophoresis-journal.com
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can lead to a slight decrease of the calculated mobility ratios, which was unfavorable to the enhancement of enantioseparation. Conversely, the experimentally determined ratios were significantly higher than the calculated values. Thus, the observed enhanced enantioselectivity can be attributed to the effect of molecular crowding rather than the weak interaction of salbutamol with dextran compared with HP--CD. Molecular crowding not only can explain why the enantioselectivity was enhanced significantly, but also can illustrate the different effects of dextran and dextrin on chiral separation as the change of concentration. Molecular crowding, which retards diffusional motion, also can decrease the inclusion reaction rate on the condition that the overall rate of the inclusion reaction is controlled by the diffusion mobility of reactant molecules [14]. Thus, considering the two opposite effects and the different constitution of dextran and dextrin, it was reasonable that they had different impact on the chiral separation of salbutamol with the change of concentration. For the dextran, the positive effect of molecular crowding occupied a dominant position in the whole concentration range studied. Consequently, the stronger stability of formed complexes enlarged the interacting differences of two enantiomers with HP--CD and enhanced the enantioselectivity of HP--CD. As for dextrin, the aforementioned two opposite effects achieved a balance at the moderate concentration, i.e. 6% w/v, and the separation effectiveness reached optimum simultaneously.
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tioseparation of salbutamol, the model drug, was enhanced by using HP--CD as chiral selector and dextran or dextrin as the crowding-inducing agents. Compared with the results obtained without crowding-inducing agents, the presence of dextrin and dextran, especially the dextran, can significantly improve the enantioselectivity of HP--CD. The mechanism of enhanced enantioselectivity was also investigated intensively through fluorescence spectroscopy and ACE technique. It can be attributed to the effect of molecular crowding which can promote the interaction of enantiomers with cyclodextrins and intensify the interacting differences of two enantiomers with cyclodextrins. It should be noted that the analysis time was long in the presence of dextran. Hence, further work should be considered in the future, for example, searching for more available crowding-inducing agents in chiral CE. In conclusion, molecular crowding was an effective strategy to enhance the enantioselectivity of cyclodextrin in chiral CE and has potential for more extensive use such as applications in greater number of analytes. This work was supported by the National Natural Science Foundation of China (21175159, 21275169 and 81202886). The authors have declared no conflict of interest.
5 References 3.3.3 The viscous effects in chiral CE Apart from molecular crowding, the change of buffer viscosity caused by the addition of crowding-inducing agents also deserved attention. There were two opposing effects of increased viscosity on the resolution of enantioseparation. Higher viscosity will increase the analysis time and decrease the diffusion coefficient of solutes, and it seemingly contributed to the improvement of resolution. But on the other hand, the mobility differences of the enantiomers decreased with the growing viscosity, which would be expected to lower the resolution. To have an insight into the viscous effects on separation in CE, Schure et al. [36] synthesized the two aspects and developed a theory to describe the viscous effect of buffer additives on the resolution of small molecules. They found that the resolution was independent of the viscosity of the running buffer. Moreover, Shimizu et al. [37] investigated the relation between mobility, , diffusion coefficient, D, of a small molecule and the viscosity of the buffer containing PEG. The results also showed that the ratio /D related to the resolution was nearly independent of viscosity. Therefore, we can further confirm the predominant role of molecular crowding in the enhanced chiral separation of salbutamol.
4 Concluding remarks In the present study, we revealed the application of molecular crowding for enantioseparation in chiral CE. The enan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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