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Characterization of the Ligand Exchange Reactions on CdSe/ZnS QDs by Capillary Electrophoresis Nannan Wei, Ling Li, Huige Zhang, Weifeng Wang, Congjie Pan, Shengda Qi, Hongyi Zhang, Hongli Chen, and Xingguo Chen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03856 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Characterization of the Ligand Exchange Reactions on CdSe/ZnS QDs by Capillary Electrophoresis

Nannan Wei†, Ling Li†, Huige Zhang*,†, Weifeng Wang‡, Congjie Pan†, Shengda Qi†, Hongyi Zhang§, Hongli Chen*,†, Xingguo Chen†

†

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and

Chemical Engineering, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, Lanzhou University, Lanzhou 730000, China ‡

Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou

730000, P. R. China § Key

Laboratory of Analytical Science and Technology of Hebei Province, College

of Chemistry and Environmental Science, Hebei University, Baoding 071002, China *Corresponding author *E-mail: [email protected] Tel.: +86-931-8912763 Fax: 86-931-8912582

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Abstract The continuous development of semiconductor quantum dots (QDs) in biochemical research has attracted special attention and surface functionalizing becomes more important to optimize their performance. Ligand exchange reactions are commonly used to modify the surface of QDs for their biomedical applications. However, the kinetics of ligand exchange for semiconductor QDs remain fully unexplored. Here, we describe a simple and rapid method to characterize the ligand exchange reactions on CdSe/ZnS QDs by capillary electrophoresis (CE). The results of ultraviolet-visible (UV–vis) absorption spectra, fluorescence spectra and fourier-transform infrared spectroscopy (FT-IR) indicated that the successful implementation of ligand exchange process. The dynamics of ligand exchange of OA-coated CdSe/ZnS QDs with 4-mercaptobenzoic acid (4-MBA) was monitored by CE and the observed ligand exchange trends were fitted with logistic functions. When the ligand exchange reactions reached equilibrium, the ligand density of QDs can be quantified by CE. It is anticipated that CE will be a new powerful technique for quantitative analysis of the ligand exchange reactions on the surface of QDs. Keywords: capillary electrophoresis, quantum dots, ligand exchange reactions, ligand density

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Introduction Semiconductor quantum dots (QDs) are fluorescent nanoscale inorganic particles composed of groups II-VI or III-V elements with typical diameters ranging from 1 to 10 nm, which have good photo stability, broad absorption and narrow emission wavelengths. QDs have shown a broad application in the fields of bioimaging, diagnostics,1-3 optoelectronics such as light-emitting diodes4,

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and solar cell6 , etc.

However, QDs were often synthesized in the organic solvents with hydrophobic surface of organic ligands, these hydrophobic ligands are not ideal for many QDs applications. Therefore, it’s necessary to modify the surface of QDs with hydrophilic ligands for further applications. To date, the most popular methods used to modify the surface of hydrophobic QDs can be mainly categorized by three different approaches: (i) ligand exchange, (ii) encapsulation, and (iii) silica coating.7-11 Up to now, ligand exchange reactions are commonly used method to replace the original hydrophobic ligands on the surface of QDs by hydrophilic ligands. For typical ligand exchange reactions, ligands can be classified as L-, X-, or Z-type.12 L-type ligands have two lone pairs, such as RNH2 and PR3, etc. Z-type ligands are long-chain carboxylates or phosphonates that accepting lone pairs. X-type ligands (O2CR, Cl, SR, etc) have a lone electron. Thiolated ligands, a typical class of X-type ligands, have been the most frequently utilized functional groups as anchors on QDs surfaces due to their strong affinity to the metal elements such as Cd.13-16 Despite most studies usually focus on the improvement of the properties of QDs, there is also an increasing interest in understanding the surface chemistry of QDs. Currently, various analytical techniques have been conducted monitoring the surface chemistry of QDs, such as fourier transform infrared spectrometer (FT-IR), X-ray 3


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photoelectron spectroscopy (XPS), energy-dispersive X-ray spectroscopy (EDS), inductively coupled plasma-mass spectrometry (ICP-MS) and thermogravimetric analysis (TGA).17-20 In spite of these techniques have obtained useful information on characterizing the surface chemistry of QDs, they were subject to some restrictions. For instance, FT-IR plays an important role in analyzing the functional groups bound to QDs, however, most of the FT-IR analyses were performed on thin films and the equilibrium of solution-phase had been broken. Radio-analytical techniques were also used to characterize ligand exchange on magnetic nanoparticles (MNPs). Powell’s group utilized a radiotracer technique to track the exchange of a radiolabeled 14C-oleic acid ligand with hydrophilic ligands on iron oxide nanoparticles.21,22 Although isotopic labeling could provide an interesting alternative to characterize the ligand exchange reactions, it should be noted that the radiation effect of radioactive isotopes is also inevitable. Additionally, the above-mentioned methods often require complicated purification steps, and the dynamic changes of the analytes cannot be directly observed during the ligand exchange process. To better understand the surface chemistry of QDs, the investigation of the dynamics of ligand exchange is also essential because of its crucial implications in the design of an important class of novel functionalized nanoparticles. That is to say, the dynamic equilibrium between the surface-bound ligands and free ligands in solution can’t be neglected. Nuclear magnetic resonance (NMR), an effective technique for measuring the structures of molecule, is the most commonly used method to characterize the surface chemistry of QDs.23-29 However, over the past decade, relatively few studies have focused on the dynamic study of ligand exchange on semiconductor QDs by NMR. Owen et al. investigated the kinetics of the ligand exchange reaction on CdSe 4


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nanocrystals by 1H NMR and showed that the extent of ligand exchange is nearly complete (> 90%).30 However, in order to avoid broadening the proton resonances of surface-bound species, NMR requires relatively high concentrations of QD-complexes to achieve suitable signal.31,32 In addition, further purification was also needed when using NMR to study the ligand exchange on QDs.33,34 However, many works have indicated that the purification process would cause loss of ligands on the surface of QDs, and further results in deficiency on the surface of QDs.23,35,36 Capillary electrophoresis (CE) is one of the principal separation and analysis techniques with versatile advantages of high efficiency, short analysis time, low sample and reagent consumption. It has been used as a mean for the characterization and separation of nanomaterials and nanomaterials-bioconjugates, including carbon dots (CDs),37,38 gold and silver nanoparticles (AuNPs and AgNPs),39 CdSe QDs40and CdSe/ZnS QDs-conjugates,41 etc. Typically, Miguel et al. proposed a simple method to separate CdSe QDs by CE, which was able to separate QDs that differed by only 0.5 nm in diameter and 19 nm in fluorescence emission maximum wavelength.40 It can be seen that CE has rapidly emerged as one of the most powerful approach for high efficient separation of nanomaterials. By virtue of its unique advantages, CE should be a perfect tool to characterize the ligand exchange reactions on the surface of QDs. In the present work, we used CE to investigate the ligand exchange of QDs (Scheme 1). Following a typical ligand exchange process,42 the OA-coated CdSe/ZnS QDs was exchanged with 4-mercaptobenzoic acid (4-MBA). Unlike most of other methods mentioned above, no additional purification is required for characterizing the ligand exchange by CE. Moreover, the developed CE method has been successfully applied 5


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to study the ligand exchange kinetics on CdSe/ZnS QDs. The ligand density of QDs was also evaluated. To the best of our knowledge, this is the first work applying CE as a tool for characterizing the ligand exchange on QDs.

Scheme 1. Schematic diagram for the ligand exchange procedures of CdSe/ZnS QDs monitored by CE.

Experimental Section Chemicals and materials CdSe/ZnS core/shell quantum dots (QDs, 8.0 nm) were purchased from Tianjin Nanocomy Co., Ltd. (Tianjin, China). 4-Mercaptobenzoic acid (4-MBA) was purchased from TCI (Shanghai, China). Oleic acid (OA) and tetramethylammonium hydroxide pentahydrate were supplied by Sigma-Aldrich (Shanghai, China). NaOH and HCl were obtained from Chengdu Kelong Chemical Co., Ltd. (Chengdu, China). Methanol and tris(hydroxymethyl)aminomethane (Tris) were commercially available from Tianjin Guangfu Fine Chemical Industry Research Institute (Tianjin, China). 6


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Ether and ethyl acetate were bought from Rionlon Co., Ltd. (Tianjin, China). Sodium dodecyl sulphate (SDS) was purchased from Shanghai Test Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals and reagents were analytical grade and used without further purification (unless otherwise stated). Fused silica capillary (375 μm o.d. × 75 μm i.d.) was purchased from Yongnian Photoconductive Fiber Factory (Hebei, China). The ultrapure water used throughout the experiments was purified through an 18202 VAXL water purification system (Chongqing, China).

Apparatus and characterization The ultraviolet-visible (UV-vis) absorption spectra were recorded on a TU-1901 UV-vis spectrophotometer with a 1 cm quartz cell (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). Fluorescence spectra were recorded by a RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) with both excitation and emission slit set at 5 nm, the excitation wavelength was set at 467 nm. Infrared spectra were collected on a Nicolet Nexus 670 Fourier-transform infrared spectrometer (FT-IR, America) using KBr pellets. Transmission electron microscopy (TEM) micrographs were performed on an FEI Talos system.

Solutions preparation Stock standard solutions of 4-MBA (2 mg/mL) and OA (5 mg/mL) was prepared by dissolving the compounds in methanol and refrigerated at 4 ℃. Working solutions of the standards were prepared by dilution of the respective stock solutions with methanol.

CE conditions All capillary electrophoretic experiments were performed on Beckman PA 800 plus CE analysis system (Beckman, Fullerton, CA, USA) equipped with a diode array 7


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detector (detection wavelength, 214 nm). Data acquisition and instrument control were carried out using the Beckman 32 Karat software. Before the first use, the bare capillary (375 μm o.d. × 75 μm i.d., 40.0 cm effective length and 50.2 cm total length) was conditioned by rinsing sequentially with methanol (10 min), ultrapure water (10 min), 1 M HCl (10 min), ultrapure water (10 min), 1 M NaOH (10 min) and ultrapure water (10 min). Between two runs, the capillary column was rinsed with 1 M NaOH for 3 min, ultrapure water for 3 min and running buffer for 3 min. The working solution was injected under a pressure of 0.5 psi for 3 s, and then a running voltage of +20 kV was applied. The capillary temperature was 25℃. All of the solutions except the test sample were filtered through a 0.45 μm pore size membranes prior to the CE experiments.

Results and Discussion Characterization of the thiol-coated QDs The ligand exchange process of CdSe/ZnS QDs with 4-mercaptobenzoic acid (4-MBA) and the subsequent precipitation/centrifugation steps were according to the Peng’s group42 with little modifications (Supporting Information). The purification steps are essential for FT-IR, UV-vis and fluorescence spectrometer characterizations. Upon purification, Figure S1 indicated the successful transferring of QDs to the aqueous phase. Moreover, the purified QDs had good water solubility as the molar ratio of 4-MBA:QDs was higher than 14000:1. The successful ligand exchange process has also been confirmed by UV-vis absorption, FT-IR and fluorescence spectra. Figure 1A shows the UV-vis absorption spectra of the CdSe/ZnS QDs before and after ligand exchange. It can be seen that new absorption band appeared around 8


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264 nm for the thiol-coated QDs, which originated from 4-MBA. We also found that the first excitonic absorption band exhibited no distinguishable shift (see the inset), indicating the size of CdSe/ZnS QDs before and after ligand exchange remained unaffected, which was further manifested by TEM images in Figure S2A and S2B.43 In addition, the absorbances between 400 to 700 nm for the purified QDs increased. The slight aggregation after ligand exchange should be responsible for that (see Figure S2B). It is reported that the purified QDs are more likely to lose ligand during storage or use, which results in a considerably uncovered surface and induces the aggregation.42,44 From Figure 1B, the fluorescence intensity of QDs after ligand exchange was significantly reduced, this indicated that the thiol group was bound to the QDs.45 However, a higher MBA-to-QD ratio led to stronger fluorescence intensity, this should be due to the excess ligand can repare the surface defects.44 In order to further verify the original OA ligands on the surface of QDs were efficiently replaced by 4-MBA, the FT-IR spectra were also measured for CdSe/ZnS QDs before and after ligand exchange (Figure 2). It was evident that the typical absorption peaks from the C-H stretching (2849 cm−1 and 2918 cm−1), C-O symmetric and asymmetric vibration (1450 cm−1 and 1537 cm−1) of the native OA ligands almost disappeared, and the new peaks of thiol-coated QDs showed similar spectra features with 4-MBA, indicating that the native ligands were mostly replaced by 4-MBA. The disappearance of the S-H band at 2557 cm−1 also manifested that 4-MBA was bound to the QDs. Taken together, 4-MBA was successfully coated on the surface of CdSe/ZnS QDs.

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Figure 1. (A) UV-vis absorption spectra and (B) fluorescence spectra of the CdSe/ZnS QDs with the original hydrophobic ligands, 4-MBA and the purified hydrophilic QDs after ligand exchange at different 4-MBA:QD ratio. The inset in Figure 1A shows the absorption spectra of the first excitonic absorption peak of the corresponding sample. Note that the QDs were purified after ligand exchange.

Figure 2. FT-IR spectra of the CdSe/ZnS QDs with the original ligands, 4-MBA and the purified hydrophilic QDs after ligand exchange at different 4-MBA:QD ratio. Note that the QDs were purified after ligand exchange.

Optimization of CE separation conditions On the basis of the successful ligand exchange process, the QDs solution after ligand exchange without further purification steps were separated by CE, and the separation 10


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conditions were optimized carefully. Possible substances in the mixture solution after ligand exchange were CdSe/ZnS QDs with original OA ligands (OA-coated QDs), free OA, thiol-coated QDs and free 4-MBA. Micellar electrokinetic chromatography (MEKC), an effective method for separating charged and uncharged compounds, was an ideal candidate and was introduced for the separation. In order to achieve high separation efficiency, various separation buffers, such as borate, phosphate and Tris were taken into account (Figure S3A). Finally, Tris buffer was chosen for further studies as it enabled to obtain better peak shapes in a shorter analysis time. Notably, all the separation conditions were optimized at the fixed 4-MBA:QD (14000:1). SDS is the most popular anionic surfactant used in MEKC and the concentration of SDS is a very critical parameter affecting the separation efficiency. Therefore, separation conditions were firstly optimized at various SDS concentrations (20 to 60 mM) (Figure 3A). It can be seen that higher SDS concentrations resulted in longer migration time. The use of high concentration of SDS contributed to increase the probability of interactions between analytes and micelles accompanied by the electroosmotic flow (EOF) decreasing. Thus, compromising between the peak shape and the migration time, 50 mM was selected as the optimum concentration of SDS. In addition, the concentration of buffer can also affect the MEKC separation. From Figure S4, increasing the buffer concentration from 3 mM to 20 mM, it only exhibited little contribution to the separation. However, the peak shapes were better at 5 mM Tris. Finally, the optimum concentration of Tris was selected as 5 mM. The effect of pH on the separation was also investigated in this work. The pH value of running buffer can adjust the velocity of EOF and affect the mobilities of the analytes in MEKC. It has been reported that the degradation of QDs would occur in an acidic 11


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medium.46,47 Considering the stability of CdSe/ZnS QDs, the effect of running buffer pH on the separation was examined in the range of 9.5-11.0 (Figure 3B), 10.0 was found to be a suitable pH value. Moreover, no adjustment of pH was required under this running buffer conditions (5 mM Tris, 50 mM SDS). Therefore, the optimum separation conditions were as following: 5 mM Tris, 50 mM SDS, pH 10.0.

Figure 3. The MEKC electropherograms of the QDs mixture solution under the separation conditions: (A) 10 mM Tris with different concentrations of SDS, pH 9.5; (B) 5 mM Tris, 50 mM SDS with different pH. Injection: 0.5 psi, 3 s; Applied voltage: +20 kV; detection wavelength: 214 nm; capillary: 75 μm i.d., 50.2 cm total length (40 cm effective length); capillary temperature: 25℃. Peak identifications: (1) thiol-coated CdSe/ZnS QDs; (2) free OA; (3) free 4-MBA.

Under the optimum separation conditions, the typical electropherogram of the QDs mixture solution was shown in Figure 4 and its peak identification was shown in Figure S3B. Obviously, the free 4-MBA migrated more slowly than the thiol-coated QDs owing to its higher charge/mass ratio, although both of them were negatively charged. As for the free OA, it was hydrophobic and could effectively interact with SDS micelle, therefore, it migrated between thiol-coated QDs and free 4-MBA. 12


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Figure 4. Typical electropherogram of the QDs mixture solution after ligand exchange. Peak identifications: (1) thiol-coated CdSe/ZnS QDs; (2) free OA; (3) free 4-MBA. Separation conditions: 5 mM Tris, 50 mM SDS (pH 10.0). Other conditions were the same as those in Figure 3.

Kinetic studies of the ligand exchange process by CE For a given QDs and ligand, there are two primary factors influencing the extent of ligand exchange: ligand concentration and reaction time. Under the optimum separation conditions, ligand exchange kinetics was also investigated by MEKC using a constant concentration of the OA-coated CdSe/ZnS QDs (3.8 μM), and 4-MBA was used as model substitute ligand with a varied molar ratio of 4-MBA:QD. The concentration of CdSe/ZnS QDs was calculated from the UV-vis absorption spectrum,48 and the detailed process was provided in Supporting information. The dynamics of ligand exchange of QDs were monitored at various time by varying the molar ratio of 4-MBA:QD. The resulting data were fitted with logistic functions (Figure 5). As shown in Figure 5A, at a molar ratio of 4-MBA:QD ≈ 28000:1 (the corresponding electropherograms was shown in Figure S5), the peak area of thiol-coated QDs increased as a function of time to reach a plateau in 10 h, and that of 13


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free 4-MBA decreased as a function of time and also reached a plateau in 10 h, which demonstrated that the ligand exchange reaction has reached the equilibrium in 10 h. Control experiments were also conducted under the optimum separation conditions with different molar ratio of 4-MBA:QD (14000:1, 42000:1). From Figure 5B, it could be concluded that the ligand exchange reaction reached equilibrium more quickly with the higher concentration of ligand used (i.e. the higher molar ratio of 4-MBA:QD). This implied that the concentration of ligand affected not only the extent of ligand exchange, but also the reaction rate. However, when too few ligands were used (such as the molar ratio of 4-MBA:QD at 5700:1), the precipitation was observed during the ligand-exchange process (Figure S6). Quantifying ligand density is a good point to understand the surface chemistry of QDs. In order to quantify the ligand density of QDs, a series of 4-MBA (0.02-1.5 mg/mL) and OA (0.1-5.0 mg/mL) standard solutions were prepared, and the good calibration relationships were established by plotting peak areas vs concentrations of 4-MBA (Figure S7A) and OA (Figure S7B). In the QDs mixture solutions after the ligand exchange, the amounts of free 4-MBA could be obtained by calculating from the corresponding calibration curve. Furthermore, the number of 4-MBA that are bound to the QDs can be evaluated by subtracting the free 4-MBA from the total 4-MBA (see the equation (S1) in Supporting information). Meanwhile, the free OA stripped from the surface of OA-coated CdSe/ZnS QDs after the ligand exchange was quantified according to its calibration curve. From Figure 5B, it could be seen that the peak areas of thiol-coated CdSe/ZnS QDs were basically unchanged with reaction for 12 h as the molar ratio of 4-MBA:QD were 42000:1 and 28000:1, which indicated that the number of 4-MBA ligands on the 14


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surface of QDs have reached the saturation. However, the ligand exchange hadn’t reached equilibrium in 12 h as the molar ratio of 4-MBA:QD was 14000:1. Comparing the peak area of stripped OA at the molar ratio of 4-MBA:QD of 28000:1 and 14000:1 (Figure S8), they were very close. That is to say, with reaction of only 12 h, nearly all original OA on QDs were stripped off at 14000:1 of 4-MBA:QD, however, 4-MBA could bond to QDs with the larger quantities and reached the saturation at 28000:1 of 4-MBA:QD. The ligand density of 4-MBA on CdSe/ZnS QDs can be calculated as about 125 MBA/nm2 (see the equation (S2) in Supporting information28). Similarly, according to the number of stripped OA from the corresponding calibration curve, the initial ligand density of commercial CdSe/ZnS QDs was calculated as 21 OA/nm2. These values are higher than those measured by Mattoussi’s group and other group (4.6 molecules per nm2 for green QDs, 5.1 molecules per nm2 for yellow QDs).23 The main reasons should be as follows: (1) the extinction coefficient used for calculating the concentration of the QDs can affect the surface ligand density.25 Therefore, the actual extinction coefficient of the selected CdSe/ZnS QDs should be strictly determined in the future study; (2) many works have indicated that the purification process would cause loss of ligands on the surface of QDs, and further results in deficiency on the surface of QDs.23,35,36 In our work, OA-coated CdSe/ZnS QDs were purchased and used directly without any processing. Furthermore, CE was used to detect the ligand exchange on QDs surface without the need of complex purification steps, so the ligand density on the surface of QDs was relatively higher than other reports; (3) the shape of nanomaterials has large effect on the total surface area, and further to the ligand density. From TEM images in Figure S2, it can be observed that our purchased QDs are with diverse shapes, but not all 15


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spherical. However, we assumed that the shape of QDs were spherical for the convenience of calculation; (4) the nanoparticle size has important effect on the ligand density. In Figure S2, the unpurified QDs after ligand exchange (Figure S2C) had more QDs with larger size than the purified QDs (Figure S2B). Furthermore, the hydrodynamic radius of QDs in the reaction solution should be larger than the radius obtained by TEM. However, we all adopted the average diameter of 8.0 nm provided by company to calculate the ligand density; (5) 4-MBA has higher affinity to the core metal elements of QDs, and it may be bonded to the QDs by multiple layers but not by a single layer. Especially in the QDs mixture solution without any purification, multiple-layer coating should be more in line with the actual situation. Besides, the different synthetic methods of QDs should also contribute to the different ligand density.

Figure 5. (A) The change in peak area of thiol-coated CdSe/ZnS QDs and free 4-MBA at the different ligand exchange time (4-MBA:QD ≈ 28000:1). (B) The change in peak area of thiol-coated CdSe/ZnS QDs at the different ligand exchange time. The different colors correspond to different 4-MBA:QD ratio. Error bars are the standard deviation of three parallel experiments.

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Conclusions In this work, ligand exchange reactions on the surface of CdSe/ZnS QDs have been characterized by CE. The approach allows to simultaneously distinguish native ligand, hydrophobic CdSe/ZnS QDs, new ligand and thiol-coated QDs in the mixture solution, and the laborious purified processes are not necessary. We have studied the effect of reaction time on the ligand exchange kinetics and found the peak areas of bound and free ligands are as a function of time. The effect of ligand concentrations on ligand exchange dynamic was also investigated, which indicated that both the extent of reaction and reaction rate can be affected by the ligand concentrations. Furthermore, the ligand density of QDs can be quantified by the developed method. Thus, CE is expected to be a promising technique for characterizating the ligand exchange of QDs and can be used to guide the selective functionalization of QDs for various applications.

Associated Content Supporting Information Figure S1-S8, photos of hydrophobic CdSe/ZnS QDs transferred to aqueous phase with different molar ratio of 4-MBA:QD, under room-light and UV conditions; TEM images; the electropherograms of the QDs mixture solution; the MEKC electropherograms of the QDs mixture solution with different concentrations of Tris; the electropherograms of the QDs mixture solution under different reaction times for the ligand exchange; photographs of ligand exchange reaction after 12 h; the calibration curve between peak areas and concentrations of 4-MBA and OA; the electropherograms of the QDs mixture solution after ligand exchange at different 17


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4-MBA:QD ratio.

Acknowledgments The authors are grateful for financial support from the National Natural Science Foundation of China (No. 21475053, 21874060, 21705156).

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