Diethylaminoethyl-Modified Magnetic Starlike Organic Spherical

Feb 27, 2019 - Diethylaminoethyl-Modified Magnetic Starlike Organic Spherical Adsorbent: Fabrication, Characterization, and Potential for Protein Adso...
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Diethylaminoethyl-Modified Magnetic Starlike Organic Spherical Adsorbent: Fabrication, Characterization, and Potential for Protein Adsorption Liangzhi Qiao, Liangshen Zhao, Hao Ai, Yaling Li, Yi Liu, and Kaifeng Du* Department of Pharmaceutical & Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, P.R. China

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ABSTRACT: This work reports the synthesis of a magnetic weak anionic spherical adsorbent with large size and starlike structure by magnetic induced self-assembly of organic latexes followed by modification with diethylaminoethyl hydrochloride (DEAE-HCl). The prepared adsorbent features unique starlike long chains stacked by anisotropic latexes (1 μm), large particle size (250 μm), and magnetic response. According to N2 adsorption−desorption analysis, the starlike adsorbent possesses both specific surface area of 55.2 m2 g−1 and total mesopore volume of about 2.7 cm3 g−1. After being modified with DEAE, the weak anionic adsorbent is evaluated for its adsorption performance. The analysis proves that the adsorbent approaches not only high static adsorption capacity of 113.05 mg mL−1 for BSA adsorption but also fast adsorption rate for approaching the adsorption equilibrium within less than 40 min. Additionally, the large particle size and magnetic response of the adsorbent facilitate the easy recovery of adsorbent from aqueous solution by simple filtration and/or external magnetic force. Hence, the prepared magnetic starlike spherical adsorbent is expected to have potential application in highperformance and large-scale purification of proteins. biological effluent by a simple filtration in batch adsorption. In addition, if being filled into a chromatographic column, the packed macrospheres still endow the chromatography column with relatively high bed permeability, which contributes to both low backpressure and reduced operational cost.19 In this way, the large spherical adsorbent is especially prominent in industrial applications. To realize high separation efficiency, the macroshperes should have suitable porous channels throughout their networks for providing both high adsorption capacity and fast mass transfer. However, introducing the inner porous structure faces the risk of reducing intraparticle diffusion within the macrospheres, which compromises not only the dynamic adsorption capacity of desired proteins but also the separation resolution because of the serious diffusion resistance occurring in the adsorbent. A potential solution for this problem is using adsorbents with small size. In general, an adsorbent with small size often possesses the advantages of flow homogeneity, short diffusion distance, and relatively large specific surface area, which relates strongly with high separation efficiency.20−22 However, because small particle size endows the adsorbents with a poor capability of processing

1. INTRODUCTION Proteins are important biomolecules that play crucial roles in various physiological processes, and the enrichment and purification of high value-added proteins are currently hot topics. Traditionally, adsorption is regarded as a universal technique and widely applied to separate varied proteins from biological sources in the downstream processes. For adsorption, the adsorbent serves as the key point and determines both the separation efficiency and the production cost.1−5 To date, a bewildering variety of adsorbents are currently available for purification of proteins in product catalogs of many manufacturers.6−11 The adsorbents are expected to possess both high adsorption capacity and fast mass transfer toward high-performance separation. For this purpose, great efforts have been devoted to the design and construction of varied adsorbents with suitable porous structure and external morphology for excellent adsorption performance.12−14 Currently, the adsorbents are rich in various shapes, such as porous beads, irregular particles, membranes, monoliths, and so on. Among them, the spherical adsorbent is considered as the mainstream. In general, spherical adsorbents are classified mainly into micro- and macrospheres depending on their bead size.15−18 The macrospheres often feature large size over dozens of micrometers and porous structure.18 Expectedly, the large bead size allows easy recovery of adsorbent from complex © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

December 3, 2018 January 25, 2019 February 27, 2019 February 27, 2019 DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

added rapidly into the solution. The resulting suspension was vigorously stirred for 5 min, and then 5 mL of oleic acid was added dropwise into the suspension. The solution was kept at 80 °C for another 1 h. Afterward, cooling the solution to room temperature gave the black magnetic precipitate. The magnetic precipitate was distilled in 150 mL of ethanol at 65 °C for 1 h followed by washing with deionized water three times, by which the excess oleic acids were removed. Finally, these magnetic nanoparticles were dried in a vacuum drying chamber and dispersed in cyclohexane. 2.3. Synthesis of Magnetic Starlike Poly(GMA-EDMA) Macrospheres (MPGE). The magnetic starlike poly(GMAEDMA) macrospheres (MPGE) were prepared by a magnetic induced dispersion polymerization.26 In the typical procedure, PVP ethanol solution (3 wt %) served as a continuous water phase. AIBN (0.05 g) was dissolved in the organic mixture of 3 mL of GMA, 10 mL of toluene, 3 mL of dodecanol, and 2 mL of cyclohexanol together with a portion of magnetic colloidal particles, which was used as a dispersion phase. Subsequently, the dispersion phase was mixed ultrasonically with the continuous water phase at 60 °C with nitrogen protection for 30 min. Under both external magnetic field and mechanical stirring, EDMA was added into the reaction system to initiate the cross-linking polymerization by increasing temperature to 75 °C. After the completion of polymerization, the magnetic starlike poly(GMA-EDMA) macrospheres (denoted as MPGE) were obtained and washed with excess acetone and deionized water to remove the unreacted monomers and oilphase reagents, which was followed by drying in a vacuum oven. 2.4. Modification of MPGE with DEAE-HCl. The weak anionic adsorbent was prepared by modifying DEAE onto MPGE by ring-opening reaction.27 Briefly, 5.0 g of dried MPGE was mixed with 2.5 M DEAE-HCl at pH 12 with gentle stirring. The suspension was heated to 65 °C and kept for 4 h for DEAE immobilization. Finally, the obtained DEAE adsorbent (denoted as D-MPGE) was washed thoroughly with ethanol and water and then dried in a vacuum drying oven. After that, the weak anionic adsorbent was dispersed in 0.05 M Tris-HCl buffer (pH 8.6) and was ready to use. 2.5. Protein Adsorption Evaluation. The adsorption isotherm is a fundamental method that describes the protein adsorption on adsorbent.27 For this purpose, three isotherm models were applied to analyze the experimental adsorption data and then gave the adsorption parameters. In the study, the adsorption equilibrium experiment of D-MPGE was performed in finite batch adsorption. That is, 20 mM Tris-HCl buffer (pH 8.6) served as the adsorption buffer for BSA adsorption. Then, 50 mg of D-MPGE was mixed with 10 mL of BSA solution (0.2−3.0 mg mL−1 in adsorption buffer) and then incubated for 150 min. After the adsorption was completed, the supernatant was collected by filtration and analyzed at 280 nm with an ultraviolet−visible (UV−vis) spectrophotometer. The static adsorption capacities were determined based on mass balance (eq 1). Then, fitting these experimental adsorption data into three isotherms models (Langmuir, Sips, and Freundlich) gave the adsorption isotherm curves and the parameters.

large-volume effluent (high backpressure and difficult recovery), they are less applied in the industrial separation field.23 As described above, macrospheres and microspheres are not in conflict in structure but complementary, each with their own competencies for high separation efficiency. Therefore, a good strategy is the combination of the structural characteristics of macro- and microspheres into one adsorbent with suitable geometry. Inspired by this, a novel synthesis strategy of magnetic starlike macrospheres was proposed by a modified magnetic induced dispersion polymerization. According to the strategy, the prepared macrospheres were composed of both starlike polymer chains and magnetic Fe3O4 cores, in which the starlike, long organic polymer chains originated from magnetic induced self-assembly of organic latexes by second polymerization. During the adsorption process, these flexible long organic polymer chains expanded fully and dispersed well into the protein solution, in which the adsorption character is similar to the adsorbent in small size. Therefore, it was expected that the starlike macrospheres possessed both relatively large specific surface area for high capacity and short diffusion distance for fast separation. Meanwhile, the large size together with magnetic cores offers easy recovery of adsorbent from complex biological effluents by simple filtration or external magnetic response. Therefore, the novel starlike macrospheres are expected to be more suitable in purifying proteins in the industrial grade. The as-prepared magnetic starlike macrospheres were studied in terms of structure and physical characteristics and served as the weak anionic adsorbent to evaluate their adsorption properties. Herein, bovine serum albumin (BSA) was chosen as model protein for the adsorption evaluation because of its structural homology with human serum albumin, cost effectiveness, and easy availability. To the best of our knowledge, this is the first report on the preparation of magnetic starlike macrospheres. Regarding protein separation, it is expected that this novel adsorbent has great potential in water treatment and catalysis fields.24

2. MATERIALS AND METHODS 2.1. Materials. Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), ammonium hydroxide (NH3·H2O, 28 wt %), oleic acid, ethanol, dodecanol, toluene, cyclohexanol, poly(vinyl alcohol) (PVA, degree of polymerization 1788), and polyvinylpyrrolidone (PVP, K30) were purchased from Changzheng Chemical (Chengdu, Sichuan, China). Glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EDMA), 2,2′-azobis(isobutyronitrile) (AIBN), and 2-diethylaminochloroethane hydrochloride (DEAE-HCl) were purchased from SigmaAldrich. Bovine serum albumin (BSA, reagent grade) was purchased from Chengdu Biochemical Reagent. GMA and EDMA were purified by reduced pressure distillation and stored in a refrigerator prior to use. AIBN was recrystallized in hot methanol before it was used, and other chemicals were used as received. All the reagents were analytical grade unless otherwise specified. 2.2. Preparation of Magnetic Nanoparticles. The oildispersed magnetic nanoparticles were prepared by the classical coprecipitation method.25 Typically, 4.6 g of FeCl2· 4H2O and 11.8 g of FeCl3·6H2O were dissolved in 350 mL of deionized water, and the solution was heated to 60 °C under the nitrogen atmosphere for 20 min. Then, the solution was stirred at 80 °C for 1 h, and 20 mL of 28 wt % NH3·H2O was

qt = B

(C0 − Ct )V m

(1) DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

Figure 1. Schematic illustration of the fabrication process of MPGE via self-assembly and dispersion polymerization.

Langmuir:

Sips:

qt =

Freundlich:

qt =

eq 5. k2 is the rate constant (g mg−1 min−1) of the pseudosecond-order kinetic model for adsorption. The slope and intercept of the linear plots of t/qt against t yield the values of 1/qe and 1/k2qe2 for eq 6. 2.6. Characterization. Fourier transform infrared (FTIR) spectra were acquired by Fourier transform infrared spectroscopy (FT-IR, Spectrum Two Li10014). The microscopic morphology of samples was examined by scanning electron microscopy (SEM, JSM-7500F). X-ray diffraction (XRD) patterns of the samples were analyzed on an X-ray diffractometer (Rigaku D/max-2550pc, Japan) with a scan range between 10° and 80°. The magnetic content of samples was determined through thermogravimetric analysis (TGA, HTG-2) in the temperature range from room temperature to 600 °C with a heating rate of 10 °C/min. The magnetic properties of samples were performed using a vibrating sample magnetometer (VSM, LakeShore 7307) at room temperature. The N2 analysis was used to evaluate the specific surface area and pore size distribution of adsorbent by the Brunauer− Emmett−Teller method (NOVA 2000 porosimeter, Quantanchrome, United States). The zeta potentials of DEAEmodified adsorbent at different solution pH values were analyzed by a Zeta-Plus4 analyzer (Brookhaven Co., United States). The concentrations of BSA were determined from the absorbance at 280 nm by using a UV−vis spectrophotometer (TU-1901, Beijing Purkinje General Instrument Co., Ltd., China).

qmc t Kd + c t

(2)

qs(K sc t )n 1 + (K sct )n

(3)

qt = K f ct 1/ n −1

(4) −1

where c0 (mg mL ) and ct (mg mL ) are the initial and equilibrium BSA concentrations, respectively; v (mL) is the used volume of BSA solution, and m (mg) is the mass of DMPGE; Kd (mg mL−1) and qm (mg g−1) in eq 2 are the dissociation constant and equilibrium adsorption capacity from Langmuir model, respectively; qs (mg g−1), Ks, and n in eq 3 are the equilibrium adsorption capacity, the binding constant, and the surface heterogeneity factor from the Sips model, respectively; KF and n in eq 4 are the equilibrium adsorption capacity and characteristic constant related to adsorption intensity from the Freundlich model. Dynamic adsorption analysis of BSA on D-MPGE was performed in a 20 mL centrifuge tube at room temperature as described in a previous report.27 In brief, 50 mg of D-MPGE was placed into a flask containing 10 mL of BSA solution to start the adsorption with stirring at 200 rpm. Here, varied BSA concentrations were studied at 0.4, 0.6, and 0.8 mg mL−1. At a defined time interval, the BSA solution was separated from the suspension and analyzed quickly at 280 nm to determine the BSA concentration in real time. Afterward, the adsorbed BSA capacity (qt) on D-MPGE at each time interval was calculated by mass balance. These adsorbed capacities along with varied time were recorded as the adsorption kinetics data. Fitting these experimental adsorption data into two kinetics models, the Lagergren first-order equation (eq 5) and pseudo-secondorder equation (eq 6) gave the corresponding kinetic curves and parameters (k1 and k2).28−30 log(qe − qt ) = log qe − k1t

3. RESULTS AND DISCUSSION 3.1. Preparation Strategy of MPGE. This work aims to develop a novel technique for the production of magnetic starlike polymeric macrospheres (MPGE) for high-performance adsorbent support. The novel magnetic starlike macrospheres feature a controlled anisotropic structure and are suitable for highly efficient protein separation, in which the fabrication route is illustrated in Figure 1. As seen here, the fabrication process starts with the synthesis of magnetic poly(glycidyl methacrylate, GMA) latexes through dispersion polymerization. That is, the magnetic colloidal particles are imported into the monomers reaction system under vigorous stirring. When the polymerization reaction is initiated, the prepolymers of GMA molecules are formed and then captured by magnetic colloidal particles, leading to the formation of Fe3O 4/poly(GMA) latexes (Figure S1). Second, these magnetic latexes are swollen with ethylene glycol dimethacry-

(5)

t 1 1 = + qt qe k 2qe 2

(6) −1

where qe and qt are the amounts of BSA adsorbed (mg g ) at equilibrium and time t (min), respectively; k1 is the rate constant of the Lagergren first-order kinetic model (min−1), which was calculated from the plots of log(qe − qt) versus t for C

DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

Figure 2. SEM images of magnetic starlike macrospheres (MPGE): (a) ×100 and (b) ×3000. (c) Optical microscopy (OM, ×40) image in wet state.

late (EDMA) and further cross-linked each other. During the cross-linking reaction, combining the external magnetic force with mechanical stirring is the key point for constructing the stacked magnetic starlike macrospheres. It can be explained by noting that these magnetic swollen latexes are driven to selfassemble into the orientated latex-stacked material with chainlike structure by the dipolar interaction under the external magnetic field. With continuous stirring, the orientated latex-stacked material is then broken into many macrospheres with anisotropic structure. The final size of macrospheres is controlled by the force balance between the external magnetic field and the mechanical stirring. By this synthesis route, the magnetic starlike macrospheres are formed with excellent magnetic property, stable framework, and large morphology. It is expected that the unique structure would endow the magnetic starlike macrospheres with great potential and be convenient for practical application. 3.2. Characterization of MPGE. Figure 2 shows the scanning electron microscopy (SEM) images of final magnetic starlike macrospheres after drying in air and their optical microscopy (OM) image in the wet state. It is revealed from Figure 2a that the prepared macrospheres exhibit an irregular spherical morphology in particle size of about 250 μm. Different from the conventional ones, this type of macrospheres seemed like a relatively loosely interwoven network with a starlike anisotropic structure. In the highly magnified SEM image (Figure 2b), the anisotropic structure was seen to originate from the accumulation of many latexes in the size range of about 1 μm. When immersed into water, the dried magnetic starlike macrospheres expanded from 250 to 450 μm in size and displayed more obvious anisotropic structure from inside to out (Figure 2c). Although it is difficult to observe the inner structure of MPGE, from the results we can deduce that these macrospheres in the swollen state possessed high porosity and wide interwoven porous channels. Apparently, the anisotropic porous structure characteristic endowed the MPGE with fast mass transfer for target molecules during the separation operation. According to the results reported above, what we proposed in this work was the combination of two aspects for final magnetic starlike macrospheres with a dramatically anisotropic structure. The first aspect originated from the diffusion-driven accumulation into irregular colloidal material together with the shearing force from continuous stirring, which was conducive to generating the random and branched fractal-like structure. The second was the magnetically driven self-assembly of colloidal particles, leading to the formation of latex-stacked long chains in the direction of applied magnetic field. These latex-stacked long chains were cross-linked chemically together by the cross-linker agent, creating the stable anisotropic structure observed in Figure 2. According to the formation

mechanism, we can regulate a controlled starlike structure of macrospheres by tuning magnetic strength, stirring speed, and concentration of cross-linking agent. The specific surface area and pore size are regarded as two important parameters that describing the structure characteristics of MPGE. Figure 3 shows the nitrogen adsorption−

Figure 3. Nitrogen adsorption−desorption isotherm of magnetic starlike macrospheres (MPGE). The corresponding pore size distribution (inset) was obtained from the desorption branch by using the Barrett−Joyner−Halenda (BJH) method. P0 = 101.325 kPa; Ds represents the differential specific area obtained by the BJH method.

desorption isotherm and pore size distribution of MPGE. It reveals that MPGE exhibits a typical type IV hysteresis loop occurring at P/P0 = 0.9−1.0, confirming the existence of large mesopores within MPGE. This observation is also proven by the pore size distribution plot in the inset of Figure 3, which displays a broad mesopore size distribution centered at about 8 nm. Furthermore, the total pore volume and specific surface area of MPGE were determined to be about 2.7 cm3 g−1 and 55.2 m2 g−1, respectively. This is as expected because the MPGE would benefit from a relatively specific surface area for large adsorption capacity for the desired proteins. Figure 4 shows the FTIR spectra of varied samples, including bare Fe3O4, oleic acid (OA) coated Fe3O4, poly(GMA) latexes, and magnetic starlike macrospheres (MPGE). In the case of Fe3O4 nanoparticles with and without oleic acid coating (Figure 4), a strong characteristic band and two weak bonds were observed. Among these bonds, the relatively strong peak at 583 cm−1 was assigned to vibration of the Fe−O bond from crystalline lattice of Fe3O4, while the weak absorption peaks appearing at 3436 and 1617 cm−1 were D

DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

X-ray diffraction analysis is a common technique that can elucidate the additional information about the crystal structure of the samples. In this way, XRD was performed on OA-coated Fe3O4 and MPEG, and the results are listed in panel 1 of Figure 5. As seen here, there were six characteristic diffraction peaks (2θ = 30.18°, 35.46°, 43.26°, 53.84°, 57.23°, and 62.77°) observed on both samples, which corresponded to the reflections of (220), (311), (400), (422), (511), and (440) of Fe3O4.33 Coating polymer on Fe3O4 reduced further the XRD peak intensity, which indicated the fact that the crystalline nature of Fe3O4 nanoparticles was partly shielded by the coated polymer. Meanwhile, a relatively broad peak at about 20.1° was found in Figure 5(1)b, demonstrating the presence of an amorphous polymer coating.34 Together with the results, we concluded that the magnetic Fe3O4 nanoparticles were successfully incorporated into the MPGE through the dispersion polymerization. Thermogravimetric analysis was further applied to explore the chemical composition and structure change of MPGE. As shown in panel 2 of Figure 5, there were two stages of weight loss for both samples when the temperature increased from room temperature to 600 °C. For the first stage, a tiny weight loss was found from the initial temperature to about 230 °C, which accounted for the evaporation of small molecules such as water and residual organic solvent (toluene). The second weight loss from 230 to 500 °C was attributed to the pyrolysis of polymer chains occurring in the air atmosphere. Resolving the TGA results gave the conclusion that the polymer chains began to decompose at about 230 °C and that the final degraded temperature was approximately 480 °C. As compared with the sample of pure poly(GMA-EDMA) polymer in Figure 5(2)a, in which the weight reduced to approximately zero, the weight of MPGE reduced to about 28% in mass after the calcination in air. In this way, the coated polymer shell was estimated to be about 72% in total mass of MPGE. The magnetic response analysis of MPGE was carried out by a VSM measurement at room temperature, and the results are shown in Figure 6a. It revealed that the magnetic hysteresis loop with an S-like curve was symmetrical to the origin and that there was no hysteresis; both remanence and coercivity were approximately determined to be zero. These results confirmed that the prepared magnetic starlike macrospheres possessed of an excellent superparamagnetic property. In

Figure 4. FT-IR spectra of (a) bare Fe3O4, (b) OA-coated Fe3O4, (c) poly(GMA) latexes, and (d) magnetic starlike macrospheres (MPGE).

assigned to the hydroxyl groups on the surface of Fe3O4. In contrast, there were three new characteristic peaks at 2927, 2850, and 1710 cm−1 observed on the sample of OA-coated Fe3O4, which were assigned to the asymmetric CH2 stretch, symmetric CH2 stretch, and stretching vibration of C=O in oleic acid, respectively.31 The results indicated that the molecules of OA have been covered successively on Fe3O4. With the formation of MPGE, more characteristic peaks were observed, as shown in Figure 4d. Resolving the spectra, we knew that the peaks of 846, 903, and 1260 cm−1 correspond to the symmetrical stretching and bending vibration of epoxy group, and the peaks of 1146, 749, and 1640 cm−1 correspond to the C−O−C stretching vibration of ester group, vibration of adjacent methylene groups, and stretching vibrations of double bonds, respectively.32 Those intrinsic absorption peaks from MPGE indicated that the organic polymer was successfully coated on Fe3O4 nanoparticles, and the results were consistent with the SEM observation. From the above analysis, the magnetic starlike macrospheres were prepared as the proposed fabrication process, as described in Figure 1.

Figure 5. (1) XRD patterns of the OA-coated Fe3O4 (a) and magnetic starlike macrospheres (MPGE) (b). (2) TG analysis of macrospheres without magnetic particles (a) and MPGE (b). E

DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

Figure 6. Magnetization curve of MPGE by a vibrating sample magnetometer (LakeShore 7307) at room temperature (a) and a photograph of magnetic separation (WMPGE:Wwater = 1:100) (b).

Figure 7. BSA adsorption as a function of incubation time for D-MPGE (a) and Lagergren-first-order (b) and pseudo-second-order (c) kinetic models at different initial BSA concentrations. Conditions: 50 mg of D-MPGE; 10 mL BSA solutions of 0.4, 0.6, and 0.8 mg mL−1.

addition, the saturation magnetization values were determined to be about 9.6 emu g−1, indicating that a very thick polymer shell was coated on the magnetic nanoparticles, as also confirmed by TGA. The excellent superparamagnetic property together with high saturation magnetization was expected for high-efficiency separation because it ensured the easy recovery of adsorbent from the complex system by an external magnetic field. Figure 6b displays the recovery process of MPGE from solution in vial by a magnet. It reveals that a brown homogeneous dispersion of MPGE was found in a vial without the magnet (left-hand image). When subjected to an extra magnetic field, the brown magnetic macrospheres were attracted to the wall of the vial within several seconds, and the dispersion became clear and transparent (right). Meanwhile, the magnetic starlike particles could disperse well again when the magnet was taken away (data not shown). These results further confirmed that the obtained MPGE was highly superparamagnetic and that it was very convenient for the magnetic recovery from adsorption solution. 3.3. Adsorption Evaluation. The zeta potential of DEAEmodified magnetic starlike macrospheres (D-MPGE) was explored before the adsorption analysis (Figure S2). It is found from Figure S2 that the zeta potentials on D-MPGE were positive and reduced gradually along with an increase of pH values from 2 to 10. The positive values indicated the

DEAE groups on MPGE were dissociated and carried the positive charge, in which the electrostatic interaction facilitated the adsorption of BSA molecules on D-MPGE. When exceeding pH 10, the zeta potential approached to zero point and even become negative, as a result of transformation of surface charges on D-MPGE. This can be explained as follows: The dissociation constant (pKa) of DEAE ligands is in the range of 9.5−10.5. In the alkaline condition (pH > 9.5), excess free hydroxyl ions (OH−) inhibit the dissociation of weak anionic terminals (DEAE-) and then lead to the reduction of surface charge on adsorbent. Therefore, the adsorbent of D-MPGE would lose its adsorption ability toward the negatively charged proteins. Given the moderate adsorption interaction between BSA and D-MPGE, pH 8.6 for the protein solution was chosen in the following adsorption process. Adsorption kinetics is of fundamental importance in providing sufficient information for exploring the adsorption mechanism and optimizing the separation process. Figure 7 shows the adsorption kinetics of D-MPGE for BSA adsorption. As seen here, the adsorption rate on D-MPGE increased rapidly during the initial 20 min for varied BSA solutions with different concentrations (0.4−0.8 mg mL−1) after immersing the adsorbents into BSA solutions, which accounted for about 90% of the total equilibrium adsorption capacity. After that, the F

DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research Table 1. Kinetic Parameters of BSA Adsorption onto D-MPGE at Different Initial BSA Concentrations pseudo-first-order kinetic model

pseudo-second-order kinetic model

C0 (mg mL−1)

qe (mg mL−1)

qe1 (mg mL−1)

k1 (min−1)

R2

qe2 (mg mL−1)

k2 (g mg−1 min−1)

R2

0.4 0.6 0.8

80 102 113

78.5 94.8 105.5

0.393 0.321 0.374

0.951 0.985 0.977

84.4 103.8 114.6

0.00758 0.00459 0.00497

0.993 0.991 0.996

BSA adsorption increased slowly and reached the adsorption equilibrium after about 40 min. This can be explained as follows: In the initial adsorption stage, the adsorption sites on adsorbent were sufficient and easily available for the desired proteins. With the accessible adsorption sites were occupied, the adsorption reached saturation eventually. According to this result, the adsorption time was determined to be about 40 min in the following adsorption operation. Obviously, the prepared starlike magnetic adsorbent exhibited faster mass transfer of BSA than other similar ones.26,35 The fast mass transfer can be ascribed to the unique starlike structure of D-MPGE because its loosened and expanded network enhanced strongly protein permeation through the adsorbent. The adsorption kinetics plots of BSA on D-MPGE are shown in Figure 7, and the relevant calculated results are listed in Table 1. As seen here, the values of R2 for the pseudosecond-order kinetic model were larger than those for the Lagergren first-order model, regardless of varied BSA concentrations. The fitted curves of th epseudo-second-order kinetic model (thin solid lines) matched better with the experimental adsorption data (Figure 7c). These results indicated that the pseudo-second-order kinetic model is more feasible for describing the adsorption behavior of BSA on D-MPGE, which implied that the adsorption rate was mainly determined by the ion-exchange interaction between BSA and D-MPGE. The result was consistent with other similar adsorption process.27 To optimize the adsorption process, it is of fundamental importance to establish the most appropriate correlation for the equilibrium adsorption data. For this purpose, the experimental adsorption data were analyzed using three isotherm models: Langmuir, Sips, and Freundlich. The fitting curves and calculated parameters are shown in Figure 8 and Table 2, respectively. As seen from Figure 8, the experimental equilibrium capacities of BSA on D-MPGE increased along with an increase in the initial BSA concentration and reached a plateau around 1 mg mL−1 BSA. A comparison of the experimental adsorption data and fitted isotherm curves proved that the Sips model described the adsorption more successfully than other models (Langmuir and Freundlich) according to the R2 values (>0.99) in Table 2. By the fitted results of the Sips model, the maximum equilibrium adsorption capacity, binding parameter (Ks) and surface heterogeneity factor (n) of BSA adsorption on D-MPGE were determined to be about 113.05 mg mL−1, 3.667 L mg−1, and 2.161, respectively. In general, the Sips model introduces a power law expression of the Freundlich model into the Langmuir model. That is, at low BSA concentration it approaches a Freundlich isotherm, while at high BSA concentration it predicts a monolayer adsorption characteristic of the Langmuir model. In addition, the static adsorption capacities of other similar adsorbents from the reported literature are tabulated in Table 3 for comparison. As expected, the static adsorption capacity for BSA is high enough for the proposed DEAEmodified spherical adsorbent, exhibiting great potential for

Figure 8. Adsorption isotherm curves of BSA on D-MPGE fitted by Langmuir, Sips, and Freundlich models. Adsorption conditions: 10 mL BSA solutions (0.2−3.0 mg mL−1) in 20 mM Tris-HCl buffers; 50 mg of D-MPGE; 150 min of adsorption time.

Table 2. Adsorption Parameters of BSA on D-MPGE with Langmuir, Sips, and Freundlich Models isotherm models Langmuir Sips

Freundlich

adj. R-squared (R2)

parameters −1

qm (mg mL ) Kd (L mL−1) Qs (mg mL−1) Ks (L mg−1) n Qf (mg1−1/n L1/n g−1) n

128.74 0.274 113.05 3.667 2.161 95.39 4.389

0.954 0.996

0.873

Table 3. Comparison of Static Adsorption Capacities toward BSA between Varied Adsorbents27,36−38 adsorbents

static adsorption capacities (mg g−1)

DEAE methacrylate monolith polyethylenimine organic monolith gigaporous polystyrene microspheres cellulose porous beads starlike spherical adsorbent (our work)

21.55 41.5 89.55 67.2 113.05

adsorption application. Obviously, the excellent adsorption ability was ascribed to the unique expanded starlike structure of D-MPGE, which provided an adsorption surface different from that of other traditional adsorbents. Reusability of an adsorbent is of crucial importance for practical protein separation. From a view of low production cost, the adsorbent should be reusable for a maximum number of cycles. For the evaluation of reusability, the prepared adsorbent of D-MPGE was investigated by repeating adsorption−desorption cycles using a batch adsorption system G

DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research Notes

(Figure 9). As seen from Figure 9, the BSA adsorption capacity on DEAE MPGE decreased less than 10% after 23 cycles.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was funded by Natural Science Foundation of China (Grant 21676170). We thank the Analytical & Testing Center of Sichuan University for analyzing the external morphology of the prepared samples, and we are grateful to Shuping Zheng for her help with the SEM images.

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Figure 9. Repeated use of DEAE-modified starlike spherical adsorbent for BSA adsorption.

Furthermore, the repeated 23-cycle adsorbent exhibited intact external morphology, as observed by SEM measurement (Figure S3). All these results proved that the adsorbent possessed excellent structural stability and allowed the repeated use for separation applications.

4. CONCLUSION The starlike magnetic macrospheres were prepared with the entrapment of magnetic particles under a homogeneous initiation system via a controllable dispersion polymerization, in which the assisted magnetic field played an important role in the formation of controllable anisotropic structure. After being modified with DEAE, the magnetic starlike macrospheres were converted into anion exchanger and evaluated with BSA for adsorption efficiency. It showed that the static adsorption capacity was determined to be up to 113.05 mg g−1 by Sips analysis. In addition, the adsorption process was completed in less than 40 min and matched well with the pseudo-secondorder model. The effect of starlike structure on the adsorption efficiency was discussed on the basis of these results, proving that the prepared adsorbent was suitable for high-speed and high-capacity protein separation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.8b05967. SEM image of magnetic poly(GMA) latexes, plot of Zeta potentials of DEAE MPGE versus pH, and SEM image of DEAE-MPGE after 23 adsorption−desorption cycles (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-28-85405221. Fax: +86-28-85405221. E-mail: [email protected]. ORCID

Kaifeng Du: 0000-0002-7402-4334 H

DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

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DOI: 10.1021/acs.iecr.8b05967 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX