Gas-Assisted Superparamagnetic Extraction for Selective Separation

Oct 23, 2013 - Graduate University of the Chinese Academy of Sciences, Beijing 100049, China. Ind. Eng. Chem. Res. , 2013, 52 (46), pp 16314–16320...
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Gas-Assisted Superparamagnetic Extraction for Selective Separation of Binary Mixed Proteins Wensong Li,†,‡ Liangrong Yang,*,† Huacong Zhou,†,‡ Xiaopei Li,†,‡ Fuchun Wang,†,‡ Xingfu Yang,† and Huizhou Liu*,† †

Key Laboratory of Green Process and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China ‡ Graduate University of the Chinese Academy of Sciences, Beijing 100049, China ABSTRACT: In this study, gas-assisted superparamagnetic extraction (GASE) with potential large-scale application was proposed for selective separation of multicomponent proteins. Magnetic poly(glycidyl methacrylate)−iminodiacetic acid−Zn2+ microspheres (MPMs) for selective separation of bovine serum albumin (BSA) and bovine hemoglobin (BHb) are selected as a model. The feasibility of flotation for concentrating BHb-loaded MPMs from dilute mixed proteins solution was proved. The well-concentrated solution of BHb-loaded MPMs could be achieved by simple adjustment of pH values and ionic strength in low regions, without additive detergent. Impurity BSA had the effects of improving the flotability of BHb-loaded MPMs, acting as foaming agent, but decreasing the enrichment ratio in the GASE process. The flotation conditions and the selective adsorption ones showed strong consistency. Under the optimal conditions, the enrichment ratio of 39 and recovery percentage of 98% was obtained within 1.3 min. Furthermore, the enrichment ratio could be further improved by added flotation steps.

1. INTRODUCTION Magnetic separation technology, which combines the use of functionalized superparamagnetic adsorbent particles for the target product with the recovery of these particles in a gradient magnetic field, is rapid, cost-effective, and highly efficient. It has been extensively applied to the fields of protein purification, cell labeling and separation, nucleic acid separation, and immunoassay, etc.1−7 It becomes possible for this technology to directly recover interesting proteins with high selectivity from crude biological process liquors (e.g., fermentation broths, plasma, milk, and whey). There have so far been many reports about magnetic adsorbent particles with good purification for proteins.8 However, these studies are on laboratory-scale analysis and detection. Much less attention is paid to the large-scale application of this technology and there is not yet report about its commercialization. A bottleneck for this is that existing magnetic separators are not suitable for large-scale recovery of magnetic particles from dilute or very dilute biosuspensions.8,9 High gradient magnetic separator (HGMS), which provides powerful attractions to magnetic particles by filling magnetic medium in a uniform background magnetic field, is generally regarded as an ideal candidate for this large-scale recovery task. But it still suffers from some limitations in large-scale application, such as capacity limits at large scale and dramatically increasing costs with the larger scale. Flotation, as an effective technology for solid−liquid separation, has found wide application for separation and enrichment of mineral ores, plastics, coal, fibers, and so on.10−12 Its principles are on the basis of different surface activity of solids. Solids with surface activity (inherent or due to surfactant adsorption) are selectively captured by bubbles rising through the liquid, and are dragged out of the liquid surface, attaining separation and enrichment results. Flotation technology for © 2013 American Chemical Society

solid−liquid separation has some distinct advantages including high separation efficiency, low operation cost, low energy consumption, and easy continuous operation.10 Proteins have inherent surface activity which is dependent on their physicochemical characteristics and environmental conditions such as ionic strength, pH, and the presence of detergents. So the proteins-loaded superparamagnetic particles are expected to have adjustable surface activity with environmental conditions, and it is possible that dilute solution of these particles is enriched by flotation without additional detergents. Thus a possible solution to large-scale recovery problem of magnetic particles is flotation for fast concentration of large volume dilute solution to proper volume, which is practicable in HGMS. This novel integrated technology named as gas-assisted superparamagnetic extraction (GASE) has been proposed in our previous work.13 It has been demonstrated that GASE for separation of single protein has the advantages of potential large-scale application, cost savings, and fast operation.13 However, for GASE to be a viable large-scale protein recovery technique, it is important to demonstrate not only that potential large-scale separation can be achieved for single proteins, but also that potential large-scale selective separation can be achieved for multicomponent mixtures. Most process streams purified are indeed complex multicomponent mixtures, for example, fermentation broths. In this study, GASE technology was proposed with potential large-scale application to selective separation of multicomponent proteins. GASE consists of superparamagnetic adsorption for selectively trapping proteins, and flotation and HGMS for Received: Revised: Accepted: Published: 16314

March 30, 2013 October 16, 2013 October 23, 2013 October 23, 2013 dx.doi.org/10.1021/ie401012c | Ind. Eng. Chem. Res. 2013, 52, 16314−16320

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Figure 1. GASE process for the selective separation of proteins.

obtained from Aldrich were used as monomer and cross-linker, respectively. 2,2′-Azobisisobutyronitrile (AIBN) and polyvinylpyrrolidone (PVP K-30, Mw = 40 000) were used as initiator and stabilizer, respectively. All the reagents were analytical grade, including iminodiacetic acid (IDA), polyethylene glycol (PEG) 6000, ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), and aqueous ammonia (25%). 2.2. Preparation of the MPMs. The MPMs were prepared by the proposed method,16 as follows. First, magnetic PGMA microspheres were synthesized: 11.8 g of FeCl3·6H2O and 4.30 g of FeCl2·4H2O were dissolved in 200 mL of deionized water under nitrogen gas protection with vigorous stirring at 80 °C. Then, 25 mL of NH3·H2O (25%) was quickly added into the solution. After about 2 min, 50 mL of PEG 6000 solution (15 g of PEG dissolved in 50 mL of deionized water) was added to obtain the magnetic Fe3O4 nanoparticles coated with PEG. The product obtained after 2 h was washed two times with deionized water to remove free PEG. The magnetic microspheres were then prepared by dispersion polymerization of GMA in the presence of PEG-stabilized magnetic nanoparticles using AIBN as initiator and PVP-K30 as stabilizer in an ethanol/water medium. The polymerization was carried out in a 250-mL three-necked flask with a mechanical stirrer. PVP (2.5 g) was dissolved in ethanol/water medium (ethanol 80 mL, H2O 9 mL) and 2 g of PEG-coated Fe3O4 was added and sonicated for 20 min to be well mixed. The monomer phase including GMA (12 mL), EGDMA (100 μL), and AIBN (250 mg) was mixed with the above dispersion medium. The mixture was purged with nitrogen for 30 min and shaft sealed. The polymerization was carried out at 70 °C for 24 h under continuous stirring (250 rpm). The resultant microspheres were separated by centrifugation at 5000 rpm and thoroughly

capturing proteins-loaded particles from dilute mixed proteins solution. This GASE process is shown in Figure 1. After equilibrium was achieved for selective adsorption, the target proteins-loaded magnetic particles and partial impurity proteins were concentrated by bubbles onto the surface of the liquid. Then HGMS was used to recover these particles from the small amount of liquid with impurity proteins. Magnetic nanospheres with immobilized Zn2+ as affinity ligands have been successfully applied to selective separation of binary mixed proteins of bovine serum albumin (BSA) and bovine hemoglobin (BHb),14,15 so magnetic poly(glycidyl methacrylate) (PGMA)−iminodiacetic acid (IDA)−Zn2+ microspheres (MPMs) with the mean diameter of 2.5 μm for selective separation of BSA and BHb are selected as a model. The focus of our research is the feasibility of flotation for concentrating proteins-adsorbed magnetic particles from dilute mixed proteins solution. To simplify operation in our smallscale experiments, magnetic separation is carried out by a permanent hand-held magnet with 0.8 T as a substitute for HGMS. Under the conditions of no detergent, the flotability and the consistency among selective extraction conditions and flotation conditions are discussed. Further discussions include effects of impurity BSA in flotation process, effects of flotation on the adsorption selectivity of BHb on the MPMs, and the feasibility of added flotation steps after one flotation. Then, the optimal flotation conditions are obtained based on enrichment ratio and rate with high recovery yield.

2. EXPERIMENTS 2.1. Materials. Unless stated otherwise, all the chemicals were purchased from Beijing Chemical Factory (Beijing, China). Bovine serum albumin (BSA) and bovine hemoglobin (BHb) were obtained from Merck. Glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) 16315

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base of the column via the distributor. The BHb-loaded magnetic microspheres were concentrated into the foam phase. The concentrated solution of MPMs was gained by defoaming of the collected foam phase. Flotation was stopped when the solution became translucent, or no obvious enrichment was observed, which could be easily distinguished by color differences of foam. The volume of the concentrated solution was measured by a 10-mL graduated cylinder. The magnetic microspheres in the retentate were recovered by a hand-held magnet with 0.8 T, then rinsed, dried, and weighed. The calculations of particle concentration were based on mass balance. 2.7. Performance Criteria. The enrichment ratio (Er), recovery (R), and completion time of flotation are used to quantify the separation and concentration efficiency. The expressions of R and Er are given as follows:

washed with hot ethanol and water by repeated magnetic separation. IDA modification and Zn2+ chelating on PGMA microspheres were then done. IDA (11.2 g) was reacted with 7 g of NaOH in 175 mL of H2O to form disodium salt of IDA solution. Then all magnetic microspheres above-obtained were mixed with this IDA solution and pH was adjusted to 10−11 by 2 M Na2CO3 solution. The mixture was stirred at 70 °C for 12 h. The resultant MPMs were centrifuged and washed with water. To chelate Zn2+ on these microspheres, the microspheres were mixed with 200 mL of ZnSO4 solution (60 mg/ mL) under shaking at room temperature for 2 h. The MPMs were then washed with water to remove the excess unbound Zn2+ by magnetic decantation. Finally, the product was dried in a vacuum freeze drier. 2.3. Characterization of Magnetic Microspheres. The size and morphology of magnetic PGMA microspheres were observed by scanning electron microscopy (SEM, JEOL JSM6700F, Japan), and magnetic properties of the MPMs were measured by vibrating sample magnetometry (VSM; Model 4 HF VSM, ADE Technologies, USA). 2.4. Selective Adsorption of BHb on the MPMs. The effects of initial protein concentration, ionic strength, and solution pH on selective adsorption of BHb on the MPMs were studied. The selective adsorption experiments were carried out batchwise in the proteins mixture solutions with equal amount of BSA and BHb. The pH of the solution was varied from 4.0 to 8.0 by different buffer systems (0.02 M CH3COONa CH3COOH for pH 4.0−5.6, 0.02 M Na2HPO4NaH2PO4 for pH 6.8−8.0). The ionic strength was adjusted by the addition of NaCl. In a typical adsorption experiment, 10 mg of magnetic sorbent was mixed with 5 mL of buffer solution with proper concentration of binary mixed proteins. The mixtures were shaken gently at room temperature for 2 h to reach adsorption equilibrium. After a magnetic separation, the supernatant was analyzed for protein concentration. The adsorption capacity of BHb or BSA was determined by the subtraction of the initial and final concentration of the corresponding protein. 2.5. Proteins Concentration Determination. The protein concentrations were measured by the UV−vis spectrophotometer at 280 and 406 nm, respectively.17 BSA absorbance was maximum at 280 nm and negligible at 406 nm while BHb solutions exhibited two maxima: 280 and 406 nm. Thus, the BHb concentration was determined from the absorbance at 406 nm. The BSA concentration was then determined by subtracting the contribution at 280 nm from BHb, which was evaluated directly from the BHb concentration. 2.6. Flotation for the Enrichment of the BHb-Loaded MPMs from Dilute Mixed Proteins Solution. First, a known amount of MPMs was mixed with 100 mL of dilute proteins buffers of BHb (0.1 mg/mL) and BSA (0.1 mg/mL), then NaCl was added to adjust the ionic strength. The buffers were 0.02 M CH3COONaCH3COOH for pH 4.0−5.6 and 0.02 M Na2HPO4NaH2PO4 for pH 6.0−8.0. The mixture was shaken for 2 h at room temperature to reach adsorption equilibrium. The above solution was then transferred to a flotation column. Flotation experiments were carried out in a small glass flotation column (the same device as in our previous work), with the inner diameter of 4 cm, length of 50 cm, and a sintered glass sparger (pore size: 3−4 μm).13 N2 was passed through the

R = (Mi − Mr )/Mi × 100%

Er = Vi /Vf × R

where f, i, and r denote foam phase, initial feed solution, and retentate solution, respectively. Mi represents the mass of magnetic particles in the initial feed solution. Mr represents the mass of magnetic particles in retentate solution. Vi and Vf represent the volume of the initial feed solution and foam phase, respectively.

3. RESULTS AND DISCUSSION 3.1. Characterization of Magnetic Microspheres. The SEM micrograph of synthesized magnetic PGMA microspheres in Figure 2 shows that the magnetic microspheres are spherical, with the average size of about 2.5 μm.

Figure 2. Scanning electron micrograph of magnetic PGMA microspheres.

The magnetization curves of the MPMs at room temperature are shown in Figure 3. The saturation magnetization value of 10.2 emu/g was found for these microspheres, and no hysteresis, remanence, and coercivity also indicated that these microspheres were superparamagnetic. 3.2. Selective Adsorption of BHb on the MPMs. 3.2.1. Effect of Initial Concentration of Binary Proteins Mixture. Figure 4 shows the effects of initial concentration of 16316

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Figure 3. Magnetization curve of MPMs.

Figure 5. Effect of pH on the selective separation and adsorption capacity. The concentration of binary proteins mixture: 0.4 mg/mL BSA and 0.4 mg/mL BHb; 10 mg of sorbent.

point of 4.8. Furthermore, less adsorption of BSA occurred at the values of pH farther from the isoelectric point. The maximal adsorption of BSA at 4.8 could be attributed to least repulsive proteins−particles interactions and proteins−proteins interactions, and minimum conformation change at this pH.19,20 Thus, the high nonspecific adsorption of BSA occurred at pH 4.0−5.6 and little at pH 6.8−8.0. Therefore, good adsorption selectivity of BHb on the MPMs was obtained at pH 6.8−8.0 and poor selectivity was obtained at pH 4.0−5.6. 3.2.3. Effect of Ionic Strength. Figure 6 shows the effect of ionic strength on the selective separation at pH 7.4. It was

Figure 4. Effect of initial concentration of binary proteins mixture on the selective separation and adsorption capacity at pH 7.4.

BSA and BHb on the selective separation. It could be seen that BHb was effectively adsorbed under different initial concentrations at pH 7.4, while BSA was hardly adsorbed. The good adsorption capacity of BHb (more than 260 mg/g) on MPMs was also obtained. The good selectivity of these microspheres is based on the sensitivity of metal affinity ligand to the surface histidine content of BSA and BHb (twenty and four histidine residues on the surface of BHb, while only two histidine residues on BSA).18 3.2.2. Effect of pH. It was observed in Figure 5 that the adsorption capacity of BHb on the MPMs changed very little with varying pH, while the significant nonspecific adsorption of BSA happened at acidic conditions (pH 4.0−5.6) and little at neutral to slightly basic conditions (pH 6.8−8.0), indicating the good adsorption selectivity and capacity of BHb on the MPMs were obtained at pH 6.8−8.0 and poor selectivity at pH 4.0− 5.6. Adsorption of protein on an immobilized metal affinity adsorbent was caused by both the specific affinity interactions between metal ion and protein and the nonspecific interactions of electrostatic interactions. For BHb, the specific affinity interactions were dominant, which was little affected by the value of pH. Thus, the adsorption capacity of BHb on the MPMs changed little with varying pH. However, for BSA, the nonspecific interactions of electrostatic interactions were dominant, which were easily affected by the value of pH and maximal adsorption was generally achieved at its isoelectric

Figure 6. Effect of NaCl concentration on the selective separation and adsorption capacity. The concentration of binary proteins mixture: 0.4 mg/mL BSA and 0.4 mg/mL BHb; 10 mg of sorbent; pH 7.4.

found that the adsorption capacity of BHb on magnetic microspheres decreased with the increase of ionic strength, while the nonspecific adsorption of BSA increased slightly with increasing ionic strength, suggesting that the adsorption selectivity and capacity of BHb on the MPMs became poor at high ionic strengths, but remained good at low ionic strengths (lower than 0.3 M of NaCl concentration). For BHb, the reason for the decrease of the adsorption capacity might be that the increase of ionic strength made the affinity interactions between proteins and metal ions weak. For BSA, there were less 16317

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repulsive electrostatic forces between BSA and magnetic microspheres due to their less negative charges with increasing ionic strength, leading to slightly increasing nonspecific adsorption of BSA on the microspheres. Thus, good selectivity of BHb on the MPMs occurred at low ionic strength. 3.3. Flotation for the Enrichment of BHb-Loaded MPMs from Dilute Mixed Proteins Solution. 3.3.1. Effects of BSA as an Impurity Protein in Flotation Process. Under the conditions of no detergent, the flotation efficiency of MPMs in two protein solutions at different pH was compared to study the effects of impurity BSA in flotation process. The results are shown in Figure 7. At pH 4.0−4.8, the BHb-loaded MPMs in

Figure 8. Effects of pH and NaCl concentration on flotation efficiency. Flotation conditions for dilute mixing solution of 0.1 mg/mL BSA and 0.1 mg/mL BHb: mass of MPMs 70 mg; gas flow rate 20 mL/min; loading volume 100 mL.

suggested that the flotation of BHb-loaded MPMs could be well carried out by adjusting pH values and ionic strength in low regions without any detergent added. The influence of ionic strength on the flotability of BHb-loaded MPMs could be explained in terms of surface activity. Salts could increase hydrophobicity of proteins due to its effect on the water structure surrounding the molecules, whereas the increase of the hydrophobicity of proteins meant the increase of their surface activity, leading to the increase of surface activity of proteins-loaded particles. Thus, the increasing flotability of BHb-loaded MPMs was achieved with increasing ionic strength. It could be also found in Figure 8 that the poor flotation efficiency without NaCl was gained at pH 6.8−8.0, while better flotation efficiency could be achieved in whole pH regions by adding increasing NaCl and reached the maximum value at 0.3 M, then decreased with the increase of NaCl concentration. The results indicated that good flotation efficiency could be achieved at low ionic strength (less than 0.3 M) and pH 6.8− 8.0, which agreed well with adsorption conditions under which good adsorption capacity and selectivity of BHb on the MPMs occurred (as shown in Figures 5 and 6). This consistency further demonstrated that selective adsorption and flotation could be well combined in GASE process. 3.3.3. Feasibility of Added Flotation Steps after One Flotation. If concentrated particles solution could be still enriched by flotation, added flotation steps after one flotation would be feasible. Figure 9 shows flotation efficiency of particles solutions with different concentrations from dilute to concentrated. It was found that the enrichment ratio with good recovery increased with decreasing particles concentration and enrichment ratio of four could be also achieved for concentrated solution as high as 6 mg/mL. The results suggested that added flotation steps are possible and flotation was suitable for concentration of dilute particles solution. Especially for extremely dilute solution, the multistep flotation would greatly improve the enrichment ratio due to total enrichment ratio as the product of enrichment ratio in each step. The reason for high enrichment ratio of dilute particles concentration was that the decreased number of bubbles was needed for transferring particles with the decrease of particles concentration, leading to less liquid entrained by bubbles into the foam phase.

Figure 7. Comparison of flotation efficiency of MPMs in different protein solutions at varying pH. Flotation conditions: mass of MPMs 70 mg in both 0.1 mg/mL BHb solution and mixing solution of 0.1 mg/mL BSA and 0.1 mg/mL BHb (reached adsorption equilibrium before flotation); loading volume 100 mL; gas flow rate 20 mL/min.

two protein solutions had good flotability, and a better enrichment ratio in BHb solution was obtained than that in BSA and BHb solution. However, at pH 6.8−8.0, the BHbloaded MPMs had no flotability in BHb solution, but had the improved flotability in BSA and BHb solution. These comparions suggested that impurity BSA had the effects of improving the flotability of BHb-loaded MPMs but decreasing the enrichment ratio in the GASE process. Judging by the observation of much foam produced in BSA and BHb solution while little foam was produced in BHb solution during flotation experiments, BSA had also the effect of foaming agent. The improvement of the flotability was possibly because BSA with surface activity acted as the role of flotation agent. The decrease of the enrichment ratio was because BSA increased the foam stability and the amount of foam, and made more liquid transfer into the foam phase. 3.3.2. Flotability and the Consistency among Selective Adsorption and Flotation Conditions. The flotability of the BHb-loaded MPMs without additive detergent as the flotation agent and foaming agent was investigated by adjusting pH values and ionic strength in the solution. The results are shown in Figure 8. Under the condition of no NaCl added, the BHbloaded MPMs had good flotability at pH 4.0−4.8, but poor flotability at pH 5.6−8.0 (only less than 60% of recovery obtained in more than 20 min). However, under the condition of NaCl added, high enrichment ratio and good recovery above 95% were achieved in the whole pH region studied, showing the good flotability of BHb-loaded MPMs. These results 16318

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Figure 11. Effect of loading volume on flotation efficiency. Flotation conditions: concentration of MPMs 0.7 mg/mL; gas flow rate 20 mL/ min; concentration of NaCl 0.3 M; pH 7.4.

Figure 9. Effect of concentration of MPMs on flotation efficiency. Flotation conditions: gas flow rate 20 mL/min; loading volume 100 mL; concentration of NaCl 0.3 M; pH 7.4.

3.3.4. Effect of Gas Flow Rate on Flotation Efficiency. Figure 10 shows the effect of gas flow rate on flotation

to higher enrichment ratio. The possible reason for the worse flotation efficiency at the volume exceeding 225 mL was that the rising bubbles collided with the column wall at high liquid column, leading to the detachment of magnetic particles from bubbles. 3.3.6. Flotation Experiment in the Optimal Conditions. We obtained the following optimal conditions for dilute mixed proteins solution with 0.1 mg/mL BSA and 0.1 mg/mL BHb and MPMs concentration of 0.5 mg/mL, based on the discussions above: pH of 7.4, gas flow rate of 20 mL min−1, loading volume of 225 mL, and NaCl concentration of 0.3 M. The flotation results under these optimal conditions were enrichment ratio of 39 and recovery of 98% within 1.3 min. 3.3.7. Effects of Flotation on the Adsorption Selectivity of BHb on the MPMs. To determine whether adsorption selectivity of BHb on MPMs was affected by flotation, the amount of proteins desorbed from MPMs was compared before and after flotation. The results are given in Figure 12. It was noted that the changes of the number of proteins desorbed before and after flotation were inconspicuous, suggesting that adsorption selectivity and capacity of BHb on the MPMs was not influenced by the flotation process.

Figure 10. Effect of gas flow rate on flotation efficiency. Flotation conditions: mass of MPMs 70 mg; loading volume 100 mL; concentration of NaCl 0.3 M; pH 7.4.

efficiency. Good recovery (above 95%) was obtained in all these experiments . Both flotation completion time and enrichment ratio decreased sharply with increasing gas flow rate, indicating that flotation rate could be sped up by increasing gas flow rate. The increase of gas flow rate denoted the increase of the number of bubbles for capturing particles per unit time, thus faster flotation rate was achieved. But this also caused bigger bubble size and less residence time of foam in the column, leading to greater amounts of liquid to be taken into the foam phase. Thus a lower enrichment ratio was achieved with the increase of gas flow rate. 3.3.5. Effect of Loading Volume on Flotation Efficiency. The results about the influence of loading volume on flotation efficiency in Figure 11 showed that the enrichment ratio increased with the increase of loading volume and started to decrease at the volume exceeding 225 mL. In flotation process, the higher the liquid column of the initial solution, the longer the residence time of bubbles in the liquid. These increased times led to the increased capture efficiency of bubbles on particles due to the increase of their collision probability, thus

Figure 12. Comparison of the amount of proteins desorbed from particles before and after flotation. Desorption was carried out at pH 7.4 for 30 min in 0.4 M imidazole solution. 16319

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3.4. Feasibility of Flotation and HGMS for Large-Scale Recovery of Proteins-Loaded Magnetic Particles from Dilute Mixed Proteins Solution. In our previous work, the feasibility of flotation and HGMS for large-scale recovery of proteins-loaded magnetic particles from dilute single protein solution at an acceptable cost has been demonstrated.13 In this work, similarly, flotation could be used to well concentrate proteins-loaded magnetic particles from dilute mixed proteins solution without detergent. Further study indicated that flotation was especially suitable for concentration of dilute particles solution and it was feasible to further improve the enrichment ratio through added flotation steps after one flotation. Therefore, we believe, large-scale recovery of magnetic particles at an acceptable cost from dilute mixed proteins solution can be also obtained through the combination of flotation with HGMS. 3.5. Desorption of Protein. After the BHb-loaded MPMs were recovered from dilute mixed suspension by the combination of flotation and HGMS, desorption of BHb from the MPMs was easy. Imidazole, which can compete with the functional groups of protein for the immobilized metal ions, is usually used as an eluent in metal affinity separation. The recovery of more than 90% for the bound BHb was achieved by two successive elutings with 0.4 M imidazole in binding buffer.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21106162), the National Key Natural Science Foundation of China (21136009), the National High Technology Research and Development Program of China (2009CB219904), and State Key Laboratory of Chemical Engineering (SKL-ChE-11A04).

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REFERENCES

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4. CONCLUSIONS In this work, the GASE process with potential large-scale application was proposed for selective separation of binary mixed proteins. The BHb-loaded MPMs had good flotability by simple adjustment of pH values and ionic strength in low regions, without extra flotation agent and foaming agent. BSA as the impurity protein had the effects of improving the flotability of BHb-loaded MPMs, acting as foaming agent, but decreasing the enrichment ratio in the GASE process. Furthermore, the flotation conditions and selective adsorption ones showed strong consistency, and selective adsorption and capacity of BHb on the MPMs was not influenced by flotation process. Under the optimal conditions, the enrichment ratio of 39 and recovery percentage of 98% were obtained within 1.3 min. These results demonstrated that the BHb-loaded MPMs could be soon enriched directly from dilute large-volume mixed proteins solution by a flotation process. Furthermore, for extremely dilute solution, the enrichment ratio could be greatly improved by multistep flotation. The above results indicate the potential of flotation and HGMS for large-scale recovery of magnetic particles from dilute mixed proteins solution. Therefore, GASE technology for selective separation of multicomponent proteins has the potential for large-scale application.

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*Tel.: +86 10 62642032. Fax: +86 10 62554624. E-mail: [email protected]. *Tel.: +86 10 62642032. Fax: +86 10 62554624. E-mail: hzliu@ home.ipe.ac.cn. Notes

The authors declare no competing financial interest. 16320

dx.doi.org/10.1021/ie401012c | Ind. Eng. Chem. Res. 2013, 52, 16314−16320