Adsorptive Separation of Myoglobin from Aqueous Solutions Using

Article pubs.acs.org/jced

Adsorptive Separation of Myoglobin from Aqueous Solutions Using Iron Oxide Magnetic Nanoparticles Modified with Functionalized Nanocrystalline Cellulose T. S. Anirudhan,* S. R. Rejeena, and J. Binusree Department of Chemistry, University of Kerala, Kariavattom, Trivandrum 695 581, India ABSTRACT: Protein adsorption is an effective method for the separation of proteins and hence widely accepted in biotechnological applications. The adsorption characteristics of a heme protein, myoglobin (Mb) on a newly developed adsorbent, poly(2-methylpropenoic acid−copolymerized-ethylenesulfonic acid)−grafted−magnetic nanocrystalline cellulose composite [P(MAAco-VSA)-g-MNCC] was studied. Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy studies have helped to characterize the adsorbent. The influence of pH and temperature on the swelling behavior of P(MAA-co-VSA)-g-MNCC was investigated. The specific surface area, the point of zero charge, and the amount of acidic functionalities present on the adsorbent surface were estimated. The effects of some external parameters on the adsorption of Mb were also investigated. Different kinetic and isotherm models were used in this study. The regeneration experiments were conducted with 0.3 mmol·kg−1 NH4OH for five cycles with minimal adsorbent loss. The results from the present investigation proved that Mb can be effectively separated from aqueous solutions by means of P(MAA-coVSA)-g-MNCC.

1. INTRODUCTION Myoglobin (Mb) is a well characterized, water-soluble, heme protein with a molecular mass of 16 951 g·mol−1 and pI 7.2.1−3 Mb is present in skeletal, cardiac muscles of human body, and helps in the transport, short-term storage of oxygen and increases the diffusion of oxygen into cells.4−7 This important physiological function helps with the healing of wounds in skin, muscles, and tissues. Mb is widely taken as a model protein for many studies of heme proteins, biosensors, and electrocatalysis because of its small size, well-known structure, and commercial availability.6,8−12 However, these applications require pure protein samples and so have to separate from their bulk mixtures. Among various methods, adsorption is single-step, efficient, and cost-effective isolation and purification.13 Protein adsorption has greater importance in medical, biological, and technological areas.14−16 Adsorption of protein onto the surface depends on the nature of both the protein and adsorbent surface. Magnetic iron oxide nanoparticles (MNPs) have a wide range of application in medicine, biology, and material science due to their unique magnetic properties, nontoxicity, biocompatibility, and low cost of production.17 Among various MNPs, magnetite (Fe3O4) displays interesting superparamagnetic behavior particularly on the nanometer scale. Encapsulation of Fe3O4 imparts magnetic susceptibility, enhancing potential applications in magnetic bioseparation, which was developed in 1993.18 The hydrophobic surface of MNPs results in their agglomeration in biological medium and in a magnetic field limits their application.19 The hydrophobic nature of MNPs can be reduced by a coating of suitable materials. © XXXX American Chemical Society

Different polymers including natural polysaccharides are used as coating materials on Fe3O4 in order to avoid the clustering of MNPs. Many polysaccharides such as chitosan, carboxymethyl chitosan, carboxymethyl cellulose sodium, agar, and soluble starch were used individually as stabilizers during the synthesis of Fe3O4 nanoparticles in order to improve the chemical and thermal stability, biocompatibility and biodegradability of MNPs.20 Also, the surface of polysaccharide is highly suitable for the separation of biomolecules. There are reports regarding the adsorption of proteins with the use of chitosan21 and dextran22 coated MNPs. Recently researchers have given much attention to the cellulose-based green magnetic nanocomposites because of their positive impact on environment along with ubiquity, renewability, and other unique properties.23 But the uncontrolled water uptake, low sorption capacity, high susceptibility for microbial attack, and poor mechanical stability reduces the application of cellulose as a substrate for the preparation of highly specific adsorbent. These drawbacks can be overcome by complete removal of the amorphous region. Nanocrystalline cellulose (NC) produced by concentrated sulfuric acid has a high crystalline nature, high stability in solution by preventing agglomeration, and high mechanical properties.24−28 The dispersibility of NC in different solvents can further be enhanced by graft copolymerization using vinyl monomers Received: January 28, 2013 Accepted: March 16, 2013

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dx.doi.org/10.1021/je400088g | J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

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and the introduction of ionizable and hydrophobic functional groups onto composite which makes them pH sensitive. The present article describes the preparation and application of a new water-insoluble core−shell magnetic adsorbent, poly(2-methylpropenoic acid−copolymerized-ethylenesulfonic acid)−grafted−magnetic nanocrystalline cellulose composite [P(MAA-co-VSA)-g-MNCC] for the separation of Mb from aqueous solutions. Here Fe3O4 acts as a core and P(MAA-coVSA)-grafted on NC acts as shell. The presence of strongly ionizable −COOH and −SO3H functionalities enhances the pH sensitivity of newly developed adsorbent. Here we aim at the use of both acidic functionalities from the adsorbent for the separation of Mb at optimized adsorption conditions, and separate the adsorbent via magnetic fishing after use.

Scheme 1. Proposed Reaction Mechanism for the Synthesis of P(MAA-co-VSA)-g-MNCC

2. MATERIALS AND METHODS 2.1. Materials. Saw dust of Mangiferra indica (collected from the local saw mill, Trivandrum) was used after thorough washing and drying at 353 K. Myoglobin (Mb), 2methylpropenoic acid (common name, methacrylic acid; abbreviation, MAA), ethylene sulfonic acid (common name, vinylsulfonic acid; abbreviation, VSA) and 2-(2-methylacryloyloxy)ethyl 2-methyl-acrylate (EGDMA) were obtained from Sigma Aldrich, USA, and used as received. Dipotassium (sulfonatooxy)sulfonate (KPS), sulfuric acid, azane (ammonia solution), diaminomethanal, hydrochloric acid, NaCl, KCl, thiourea, K2SO4, Na2SO4, and NaOH were procured from E. Merck, India. Iron(III) trichloride hexahydrate (FeCl3·6H2O) (0.98 mass fraction pure), iron(II) sulfate heptahydrate (FeSO4·7H2O) (0.99 mass fraction pure) were obtained from Fisher, USA. Analytical grade chemicals were used throughout the investigation without further purification. All aqueous solutions were prepared in double distilled water. When not in use the pure Mb powder was stored under desiccating conditions at 273 K. Mb solution was prepared in pure water. When not in use it was stored in the refrigerator and used only within the same day of preparation. 2.2. Preparation of P(MAA-co-VSA)-g-MNCC. Scheme 1 illustrates the general procedure for the preparation of P(MAAco-VSA)-g-MNCC. This procedure consists of three steps. Step 1. Preparation of NC from Saw Dust. Cellulose extracted from saw dust by acid-alkali treatment of and subsequent bleaching using 1.47 mol·kg−1 hydrogen peroxide29 is converted into NC by acid hydrolysis. About 5 g of cellulose was dispersed in 250 mL of distilled water in a flask by magnetic stirring for 20 min. To the homogenized mixture kept in an ice bath was added 140 mL of 9.99 mol·kg−1 sulfuric acid drop by drop. After complete addition of the acid, the mixture was heated at 323 K under vigorous stirring for 4 h. The mixture was diluted times 10 with distilled water. The obtained colloid was washed and centrifuged. Step 2. Preparation of MNC. Magnetic nanocrystalline cellulose (MNC) was prepared by the coprecipitation of Fe2+ and Fe3+ ions in an aqueous solution containing NC. About 1.5 g of NC was added to 200 mL of distilled water and stirred well for 10 min. An amount of 1.49 g of FeCl3·6H2O and 0.745 g of FeSO4·7H2O were added to the solution which acts as a source of iron present in MNC. The solution was magnetically stirred for 10 min at 333 K. Chemical precipitation was achieved by adding NH3·H2O under vigorous stirring. An orange color suspension changed to black at the end of the reaction. During the reaction, the pH of the solution was maintained at about 10. After 4 h, the mixture was cooled to room temperature with

stirring, and the resulting MNC were separated and washed with distilled water and finally with ethanol. The obtained product was dried. Step 3. Preparation of P(MAA-co-VSA)-g-MNCC. The MNC was further modified by free radical graft copolymerization using VSA and MAA. About 0.5 g of MNCC is stirred well in 100 mL of distilled water in a 250 mL stoppered bottle and heated at 333 K (∼20 min). KPS (0.92 g; initiator) was added and kept for 10 min. After cooling the suspension to 313 K, a mixture of MAA (8.5 mL), VSA (7.8 mL), and EGDMA (2.83 mL; cross-linker) were added. The pH was adjusted by NaOH to 10. The temperature was raised to 343 K and maintained for 2 h to complete the reaction. The obtained product was filtered under suction, washed with distilled water and ethanol, and dried in vacuum at 323 K. The dried sample was then ground and sieved to obtain −80 + 230 mesh size particles (average diameter of 0.096 mm). 2.3. Characterization. The structural information of the adsorbent can be obtained by measuring various physical and surface properties. The Fourier transform infrared (FTIR) spectra were recorded at (400 to 4000) cm−1 wavelength range using a KBr pellet technique with a Shimadzu FTIR spectrometer at a resolution of 4 cm−1 in diffused transmission mode. The X-ray diffraction (XRD) patterns were obtained with an X’Pert Pro X-ray diffractometer using Cu Kα radiations at a scanning speed of 2°·min−1 and at a wavelength of 1.5406 Å. Adsorbent was examined by scanning electron microscopy (SEM) analysis done using Philips XL 30 CP scanning electron microscope. The surface area was calculated by the Brunauer− Emmet−Teller (BET) protocol from the N2 adsorption isotherm (taking 16.2 Å as cross sectional area for a nitrogen molecule) using a Quantasorb (USA) surface area analyzer (model Q7/S). A potentiometric method30 was used to determine the pH of point of zero charge (pHpzc). The B



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surface charge density σ0 (C·cm−2) was calculated from the acid−base titration curve.31 The point of intersection of σ0 against pH curves for different concentrations of NaCl give the pHpzc. Swelling measurements were carried out by using 0.1 g of P(MAA-co-VSA)-g-MNCC at different temperatures (283, 293, 303, 313 K; optimum pH) as well as at different pH (2.0−9.0; at room temperature). The swelling capacity was calculated using the following equation.

Q eq =

W0 − WT WT

mL of aqueous Mb solution having a concentration 11.7·10−3 mmol·kg−1. 2.5. Desorption and Regeneration Studies. A 1.0 g sample of P(MAA-co-VSA)-g-MNCC was shaken with 500 mL of Mb solution having a concentration 5.9·10−3 mmol·kg−1 (pH, 7.0; temperature, 303 K) for 30 min. The solution was filtered using Whatmann No. 40 filter paper and the Mb-loaded P(MAA-co-VSA)-g-MNCC was dried at room temperature. To 0.1 g of Mb-loaded P(MAA-co-VSA)-g-MNCC, 50 mL of desorbing agent was added. After being shaken for 30 min at 303 K, the suspensions were filtered and analyzed for Mb. To regenerate the spent adsorbent, the same procedure was followed for the five cycles. After each desorption cycle, spent adsorbent was washed with distilled water, to remove any Mb which may be weakly sorbed on the surface of the adsorbent, and dried. Desorption percentage was calculated by using the following equation.

(1)

where Qeq is the equilibrium water adsorption (grams of water per grams of sample); W0 and WT are the weight of swollen and dry sample, respectively.32 The total amount of acidic group and carboxylic acid group in the adsorbent can be estimated by Boehm titration method using NaOH and NaHCO 3 , respectively.33 Hence we can find out the total sulfonyl group present in the adsorbent. A temperature controlled water bath shaker (Labline, India) with temperature variation of ± 1 °C was used for the equilibrium studies. Concentration of Mb in solution was measured on a JASCO UV−vis (model V-530, India) spectrophotometer at 409 nm.34 Borosil glasswares were used throughout the experiment. All pH measurements were carried out on a Systronic model μ pH meter (model 361). 2.4. Adsorption Studies. Adsorption experiments were carried out by agitating 0.1 g of P(MAA-co-VSA)-g-MNCC with 50 mL of Mb solution of desired concentration and pH at 303 K in a temperature controlled water bath shaker at a speed of 200 rpm. The effect of pH on the adsorption of Mb on the adsorbent was studied over the range 2.0−9.0 using solutions with an initial concentration of 5.9·10−3 mmol·kg−1 and 8.8·10−3 mmol·kg−1. The concentration of Mb in the supernatant liquid was determined by using a UV−visible spectrophotometer at 409 nm. The adsorption results were evaluated by using the following equations. adsorption percentage(%) =

(C0 − Ce) ·100 C0

adsorption capacity (Q) (mmol·kg −1) = (C0 − Ce)

desorption (%) =

amount of Mb desorbed by the reagent (mg ·g −1) amount of Mb adsorbed onto the adsorbent (mg·g −1) ·100

(4)

3. RESULTS AND DISCUSSION 3.1. Preparation and Properties of the Adsorbent. Adsorbent was prepared through the core−shell mini emulsion polymerization method. The MNC was prepared by a chemical coprecipitation method and then encapsulated into NC. Graft copolymerization of functional monomers, MAA and VSA, onto the MNC was carried out by using K2S2O8 as a radical initiator and EGDMA as a cross-linking monomer. Initially persulfate initiator decomposes under heating to generate the free radical (KSO4•). This radical abstracts hydrogen from the hydroxyl group of MNC to form an alkoxy radical. Vinyl monomers adjacent to the reaction site first become a radical acceptor and initiate the chain growth. Thereafter they act as radical donor to other monomers. During the chain propagation the end vinyl groups of the EGDMA react with the polymeric chain and results in a three-dimensional network system being formed with both −SO3H and −COOH functionalities. MAA, a vinyl monomer with a high hydrophilic nature compared to VSA, reacts faster with the hydrophilic hydroxyl group of MNC. The amount of −SO3H and −COOH functional group in P(MAA-co-VSA)-g-MNCC was estimated to be 1.92 meq·g−1 and 1.36 meq·g−1, respectively, was an evidence for proper grafting of PMAA and PVSA on to MNC. The specific surface area for Cellulose and P(MAA-co-VSA)-g-MNCC was found to be 22.7 m2·g−1 and 112.36 m2·g−1, respectively. These values indicate that surface area of adsorbent is 5 times greater than cellulose. The pHpzc of cellulose and P(MAA-co-VSA)-gMNCC were observed at pH 5.5 and 3.6, respectively, which indicates that the surface of the adsorbent become more negative after the modification and grafting. The low pHpzc exhibited by P(MAA-co-VSA)-g-MNCC indicates that it has more acidic functionalities compared to cellulose, and this helps for the removal of Mb before its pI. 3.2. Characterization of P(MAA-co-VSA)-g-MNCC. The FTIR spectra of cellulose, MNC, P(MAA-co-VSA)-g-MNCC, and Mb-P(MAA-co-VSA)-g-MNCC are presented in Figure 1. The IR spectra of cellulose shows the adsorption peak at 3441

(2)

W w (3)

where Q is the amount of Mb adsorbed onto P(MAA-co-VSA)g-MNCC, C 0 and C e are the concentrations of Mb (mmol·kg−1) at initial and equilibrium time, respectively, W is the mass (kg) of solvent, and w (g) is the adsorbent mass.35 The kinetic experiments were carried out with different concentrations ((2.9·10−3, 5.9·10−3, 8.8·10−3, and 11.7·10−3) mmol·kg−1) of Mb solution. At time zero and at predetermined time intervals up to 4 h, known volumes of the sample (∼2 mL) were collected, and the concentration of Mb in the supernatant liquid was determined using a spectrometer. The equilibrium isotherms were determined by varying the concentration of Mb solution ((1.5·10−3 to 47.2·10−3) mmol.kg−1). The adsorption was studied at (283, 293, 303, and 313) K. To study the effect of adsorbent dose, the adsorption experiment was carried out by using varying amount of P(MAA-co-VSA)-g-MNCC ranging from 0.25 g·kg−1 to 6.0 g·kg−1 with 50 mL Mb solution having initial concentration of 5.9·10−3 mmol·kg−1. The effect of ions on the adsorption of Mb was studied by mixing 25 mL of 0.1 mmol·kg−1 KCl, NaCl, Na2SO4, K2SO4, diaminomethanal or thiourea solution with 25 C



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peaks of both amorphous (2θ = 15.8°) and crystalline nature (2θ = 22.1° and 34.2°). The NC also gave sharper peaks at the same 2θ values, indicating the partial removal of the amorphous region in cellulose by acid hydrolysis. The distinct peaks occurring at 2θ of 18.1°, 30.1°, 35.5°, 42.8°, 57.2°, and 62.6° reveal that the prepared MNC has pure Fe3O4 with spinel structure.42 The sharper diffraction peaks of MNC indicate a high degree of crystallinity. The grafting of amorphous VSA and MAA reduces the crystalline nature of MNC. This grafting may improve the physical and mechanical properties of MNC to some extent. The adsorption Mb onto P(MAA-co-VSA)-gMNCC completely removes the crystallinity of adsorbent without causing any degradation to the adsorbent. The crystalline size of MNC was calculated using Debye− Scherrer’s formula.43 D=

0.89λ β cos θ

(5)

where λ is the wavelength of X-rays (1.5406 Å), β is the full width at half-maximum of the XRD peaks, and θ is the diffraction angle. The crystallite size of MNC thus calculated to be 23.8 nm, at an angle of 35.5°. SEM images of cellulose, MNC, P(MAA-co-VSA)-g-MNCC, and Mb-P(MAA-co-VSA)-g-MNCC (Figure 3) shows that the morphology of each of them is quite different. Cellulose displayed a spindle-like structure because of strong intramolecular H-bonding. MNC are highly porous in nature with microscale interstitial spaces. The SEM image of P(MAA-coVSA)-g-MNCC has an undulant and coarse structure. This surface is convenient for the adsorption of Mb onto the polymeric network. The porous nature of adsorbent is completely removed by the adsorption of Mb. The presence of Mb onto the surface of P(MAA-co-VSA)-g-MNCC did not significantly change the morphology of adsorbent indicating its high stability of the adsorbent. 3.3. Swelling of P(MAA-co-VSA)-g-MNCC. The swelling ratio of P(MAA-co-VSA)-g-MNCC was found to be increased with an increase in temperature and pH as shown in Figure 4. Enhancement of swelling at higher temperature may be due to the increase in osmotic pressure between the polymeric network and repulsion between similarly charged functionalities. The maximum swelling ratio for the P(MAA-co-VSA)-gMNCC was observed at pH 9.0. Under acidic pH, the H+ ion strength will be very high and suppress the ionization of most of the acidic groups. The H-bonded interaction between −SO3H and −COOH group strengthened the polymeric network and the repulsion between the functional groups was restricted. So the network shrinks and results in a decrease in swelling capacity. With an increase in pH, the ionization of both −COOH and −SO3H enhanced gradually. Thus the electrostatic repulsion between groups increases. Increasing in the anion density in the P(MAA-co-VSA)-g-MNCC, results in high swelling capacity at higher pH. 3.4. Effect of pH on Mb Adsorption. Adsorption of Mb onto P(MAA-co-VSA)-g-MNCC was studied by varying the pH of the medium from 2.0 to 9.0 with two different concentrations (5.9·10−3 mmol·kg−1 and 8.8·10−3 mmol·kg−1) and the results obtained were shown in Figure 5. It is clear that pH has a significant effect on the adsorption of Mb onto P(MAA-co-VSA)-g-MNCC. The maximum adsorption of Mb occurred at pH 7.0, which is close to the isoelectric point of Mb (pI = 7.2). At pH 7.0, the Mb exists as a positive molecule and the surface of the adsorbent has negative charge which

Figure 1. FTIR spectra of cellulose (A), MNC (B), P(MAA-co-VSA)g-MNCC (C), and Mb-P(MAA-co-VSA)-g-MNCC (D): υ, wavenumber; T, transmittance.

cm−1 due to the H- bonded O−H stretching, and peaks at 2925 and 1030 cm−1 could be attributed to the C−H stretching and C−H bending of the CH2 group. The peaks at 1636 cm−1 and 668 cm−1 are due to the CO stretching of hemicelluloses and β-glycosidic linkage, respectively. The band at 897 cm−1 is characteristic of glucosidic ring in the cellulose structure. The FTIR spectra of MNC having a peak at 563 cm−1 is due to the stretching vibration mode and the torsional vibration mode of Fe−O bonds in the tetrahedral sites and in the octahedral sites of the Fe3O4 nanoparticles. A weak band at 855 cm−1 is due to an iron nanoparticles skelton.36 The small shift in the O−H stretching peaks from 3441 cm−1 to 3256 cm−1 indicate an interaction between the cellulose and magnetic core. The characteristic absorption bands of cellulose are present in MNC, where a small decrease in intensity indicates the degradation of the H-bond between the cellulosic chains during the acid hydrolysis treatment.37 A strong peak at 1029 cm−1 is due to O−H bending vibration band. The characteristic absorption peaks of the −SO3H group ((1247, 1034, and 751) cm−1), −COOH group (1705 cm−1, 1463 cm−1), CH3 group ((2941, 1543, 1394) cm−1) and C−C vibrations (1169 cm−1) in P(MAA-co-VSA)-g-MNCC indicate the proper grafting of MAA and VSA on MNC. The shifts in the adsorption peaks of Fe−O and O−H clearly confirm the existence of grafting.38−40 The spectrum of Mb-P(MAA-coVSA)-g-MNCC showed the presence of amide bands at 1642 cm−1 (amide I) and 1535 cm−1 (amide II), which can be related to the protein matrix formation.41 The amide I band represents the stretching vibrations of CO/C−N bonds in the backbone of the protein. Further, the amide II band arises from the combination of C−H stretching and N−H bending vibrations of the protein backbone. The Mb-P(MAA-co-VSA)-g-MNCC spectrum has characteristic bands of adsorbent indicating the high stability of adsorbent even after the loading of Mb. XRD patterns of cellulose, NC, MNC, P(MAA-co-VSA)-gMNCC, and Mb-P(MAA-co-VSA)-g-MNCC are given in Figure 2. The XRD pattern of cellulose show characteristic D



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Figure 2. XRD patterns of cellulose (A), NC (B), MNC (C), P(MAA-co-VSA)-g-MNCC (D), and Mb-P(MAA-co-VSA)-g-MNCC (E): I = intensity.

enhances the electrostatic attraction and thereby adsorption. As already reported, the maximum adsorption of protein onto negatively charged adsorbents was observed below or nearer to the isoelectric point.41,44 At very low pH, the lower adsorption should be associated with the repulsion between positively charged Mb and P(MAA-co-VSA)-g-MNCC. As the pH increases, the ionization of both adsorbent and Mb also increases. The surface of P(MAA-co-VSA)-g-MNCC become negative only after pH 3.6. A small increase in the adsorption of Mb on the pH range 4.0 to 7.0 is due to the slight decrease in repulsion between Mb and the adsorbent surface. After pH 7.0, both the adsorbent and Mb have negative charge. Hence electrostatic repulsion begins to operate between Mb and P(MAA-co-VSA)-g-MNCC. At pH 7.0, the amount of Mb adsorbed was found to be 49.79 mg·g−1 (99.58 %) and 72.92 mg·g−1 (97.23 %) for an initial Mb concentration of (5.9·10−3 and 8.8·10−3) mmol·kg−1, respectively. The final pH of each solution was recorded after the adsorption process and was found that the final pH of the solution was lowered compared to that of initial value. This result points out that the adsorption of Mb onto P(MAA-co-VSA)-g-MNCC took place through the

exchange mechanism. The Mb molecule was adsorbed on the P(MAA-co-VSA)-g-MNCC surface by the release of H+. In view of electrostatic interaction between a negatively charged adsorbent and a positively charged adsorbate system, it was decided to maintain the pH at 7.0 for further experiments. 3.5. Effect of Adsorbent Concentration on Mb Adsorption. The effect of adsorbent dose on the adsorption of 5.9·10−3 mmol·kg−1 Mb onto P(MAA-co-VSA)-g-MNCC with various amounts of adsorbent ranging from 0.25 to 6.0 g·kg−1 were studied (Figure 6). The percentage recovery of Mb was found to be (94.0, 95.57, 98.23, 99.94 and 100) % for an adsorbent dose of (0.25, 0.5, 1.0, 1.5, and 2.0) g·kg−1, respectively, and reaches a saturation level at high dose (2.0 g·kg−1). The increase in adsorption is due to the increase in availability of adsorption sites at a high dosage. For the complete removal of 5.9·10−3 mmol of Mb from 1 kg of solvent, a minimum dose of 2.0 g was only required. So the adsorbent dose on adsorption of Mb onto P(MAA-co-VSA)-gMNCC was minimized to 2.0 g. 3.6. Kinetic Study for the Mb Adsorption. The effect of contact time on adsorption of Mb by P(MAA-co-VSA)-gE



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Figure 3. SEM images of cellulose (A), MNC (B), P(MAA-co-VSA)-g-MNCC (C), and Mb-P(MAA-co-VSA)-g-MNCC (D).

Figure 5. Effect of pH on the adsorption (x) of Mb onto P(MAA-coVSA)-g-MNCC (equilibrium time, 30 min; temperature, 303 K; adsorbent dose, 2 g·kg −1 . Initial concentrations: ■ , 5.9·10 −3 mmol·kg−1; ●, 8.8·10−3 mmol·kg−1).

Figure 4. Swelling (S) of P(MAA-co-VSA)-g-MNCC with respect to time (T) (▲, 313 K; ●, 303 K; ■, 293 K; ▼, 283 K) and pH.

MNCC obtained at pH 7.0 and 303 K is presented in Figure 7. The system reaches equilibrium within 30 min. The adsorption is higher in the initial stage due to the greater availability of reaction sites for the adsorption of Mb. After 30 min, rise in adsorption capacity was negligible and hence, shaking time was fixed at 30 min for the remainder of the batch experiments to

make sure that equilibrium is reached. With an increase of initial concentration of Mb from (2.9·10−3 to 11.7·10−3) mmol·kg−1, the adsorption capacity increases from (25.5 to 94.10) mg·g−1, indicating that the contact time for the Mb adsorption depends on the initial concentration of Mb. As the F



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To examine the mechanism of sorption, adsorption of Mb onto P(MAA-co-VSA)-g-MNCC was studied using pseudofirst-order45 and pseudo-second-order46 rate equations, which can be expressed as follows: Pseudo-first-order:

qt = qe[1 − e−k1t ]

(6)

Pseudo-second-order: qt =

k 2qe 2t 1 + k 2qet

(7)

where k1 is the Lagergren rate constant of adsorption (min−1), k2 is the pseudo-second-order rate constant of adsorption (g·mg−1·min−1), qe and qt are the amount of Mb adsorbed (mg·g−1) at equilibrium and at time t (min), respectively. The kinetic parameters for different concentrations and temperatures were determined and presented in Table 1. From the graph it is clear that for the pseudo-second-order model there exists a good agreement between calculated (qe,cal) and experimental value (qe,exp). The value of regression coefficients closer to unity also showed that pseudo-second-order kinetic model47 can be applied for the entire adsorption process. The applicability of the pseudo-second-order kinetic model substantiates the ion exchange and complexation reaction mechanism predicted for the adsorption of Mb onto P(MAAco-VSA)-g-MNCC. The rate limiting step for the pseudosecond-order reaction may be chemisorption, involving sharing or exchange of the electrons between adsorbate and adsorbent.48 −COOH and −SO3H groups on the P(MAAco-VSA)-g-MNCC are the multiactive sites involved in the complexation. The adsorption through the ion exchange mechanism is also confirmed by the results from pH study; the final pH was lower than the initial pH. The lowering of pH supports the exchange of H+ ions from the P(MAA-co-VSA)-gMNCC by the positively charged Mb molecule. From the table it is clear that the value of k2 decreases and the value of qe increases with an increase in the initial concentration of Mb. The initial adsorption rate h 0 (mg·g−1.min−1) from pseudosecond-order kinetic model can be written as:

Figure 6. Effect of adsorbent dose (m) onto the adsorption (x) of Mb onto P(MAA-co-VSA)-g-MNCC (initial pH, 7.0; initial concentration, 5.9·10−3 mmol·kg−1; temperature, 303 K; equilibrium time, 30 min).

Figure 7. Adsorption kinetics of Mb onto P(MAA-co-VSA)-g-MNCC at different initial concentrations: initial pH, 7.0; temperature, 303 K; adsorbent dose, 2 g·kg−1. Initial concentrations: ■, 11.7·10−3 mmol·kg−1; ★, 8.8·10−3 mmol·kg−1; ●, 5.9·10−3 mmol·kg−1; ⧫, 2.9·10−3 mmol·kg−1; ---, pseudo-first-order, , pseudo-second-order.

h0 = k 2qe 2

(8)

The pseudo-second-order kinetic analysis reveals that the values of the initial adsorption rate, h0, increases with increase in the initial concentration of Mb. At lower sorbate concentration the probability of collision with sorbent is very low and hence very few Mb could be bonded to the active sites on the surface of

concentration increased, the chance of effective collision between the adsorbent and the adsorbate also increased, which in turn will improve the adsorption.

Table 1. Kinetic Parameters for the Adsorption of Mb onto P(MAA-co-VSA)-g-MNCC at 303 Ka pseudo-first-order Co

qe,exp

mmol·kg

−1

−3

2.9·10 5.9·10−3 8.8·10−3 11.7·10−3

mg·g

−1

25.00 49.79 72.92 94.10

k1 min 0.296 0.244 0.178 0.151

± ± ± ±

pseudo-second-order

qe,cal

−1

0.033 0.019 0.015 0.010

mg·g 24.69 48.99 71.29 91.98

± ± ± ±

−1

0.291 0.467 0.887 1.005

k2 R

χ

0.981 0.988 0.981 0.986

0.92 2.28 7.67 9.43

2

2

−1

qe,cal −1

g·mg .min 0.004 0.001 0.005 0.003

± ± ± ±

0.0031 0.0005 0.0002 0.0001

mg·g 25.30 50.68 74.34 96.66

± ± ± ±

−1

0.176 0.181 0.321 0.460

h0 −1

mg·g ·min−1

R2

χ2

17.92 25.67 27.63 28.03

0.995 0.998 0.998 0.998

0.22 0.22 0.64 1.25

a

C0, initial concentration of Mb solution; qe,exp, experimental adsorption capacity; k1, pseudo-first-order rate constant; qe,exp, calculated adsorption capacity; k2, pseudo-second-order rate constant; h0, initial adsorption rate. All the adsorption experiments were done in triplicates and the mean cumulative values (± standard deviation) are reported. The difference in results for triplicate measurements was less than 4.0 %. G



Article

Figure 8. Comparison of adsorption isotherm data for the adsorption of Mb onto P(MAA-co-VSA)-g-MNCC (adsorbent dose, 2 g·kg−1; initial pH, 7.0; equilibrium time, 30 min. Temperature: ⧫, 313 K; ●, 303 K; ★, 293 K; ■, 283 K; , Langmuir; ···, Sips; ---, Freundlich.

−4.926, −5.094, and −5.263) kJ.mol−1 at (283, 293, 303, and 313) K, respectively. The observed increase in negative values of ΔG0 with rise in temperature shows that the adsorption becomes more favorable at higher temperature. The negative ΔG0 values indicate the feasibility and spontaneity of the adsorption process.38 3.8. Adsorption Isotherm. The adsorption isotherms of Mb by P(MAA-co-VSA)-g-MNCC at pH 7.0 and 303 K was given in Figure 8. The equilibrium isotherm model is very important in describing the interactive nature of adsorbent and adsorbate. Analysis of isotherm data is important for predicting the adsorption capacity. Langmuir,50 Freundlich,51 and Sips52 isotherm models were used for this purpose. The nonlinear equations are

the adsorbent. The equilibrium adsorption capacity, qe, however increased with an increase in initial concentration of Mb due to large number of available adsorption sites. 3.7. Adsorption Thermodynamics. The adsorption capacity of Mb onto P(MAA-co-VSA)-g-MNCC increased from 25.0 mg·g−1 to 94.1 mg·g−1 with an increase in temperature from 283 to 313 K (Figure 8). This indicates that adsorption is endothermic in nature. Thermodynamic parameters were calculated from van’t Hoff equation: ΔG 0 = ΔH 0 − T ΔS 0

ln K ads =

−ΔH 0 ΔS 0 + RT R

(9)

(10)

where ΔH0 and ΔS0 are the enthalpy and entropy changes, respectively, R is the universal gas constant (8.314 J·mol−1·K−1) and T the absolute temperature (K). The positive value of ΔH0 (3.724 kJ·mol−1) indicates an endothermic adsorption process. The positive value of ΔS0 (16.827 J·mol−1·K−1) refers to an increase in entropy during the adsorption of Mb onto P(MAAco-VSA)-g-MNCC. The exchange of the Mb molecule in the solution with more mobile H+ ions from adsorbent results in increased entropy.38 TΔS0 > ΔH0 at all temperatures indicates that the adsorption process is entropic driven rather than enthalpic.49 The values of ΔG0 were found to be (−4.758,

Langmuir isotherm:

qe =

Q 0bCe 1 + bCe

(11)

Freundlich isotherm:

qe = KFCe1/ nf H

(12)



a

Parameters: T, temperature; Q0, Langmuir adsorption capacity; b, Langmuir constants; KF, Freundlich adsorption capacity; 1/nF, Freundlich constants; Qs, Sips maximum adsorption capacity; Ks, Sips equilibrium constant; 1/ns, Sips model exponent. All the adsorption experiments were done in triplicate and the mean cumulative values (± standard deviation) are reported. The difference in results for triplicate measurements was less than 4.0 %.

0.994 0.992 0.993 0.997 0.022 0.028 0.036 0.036 ± ± ± ± 0.219 0.273 0.366 0.513 0.017 2.204 2.531 2.842 ± ± ± ± 252.77 268.92 297.33 305.41 951.09 790.36 679.75 670.73 0.896 0.92 0.937 0.942 0.0318 0.030 0.025 0.023 3.561 3.687 3.052 2.943 ± ± ± ± 76.36 80.04 91.1 103.95 95.37 136.39 299.01 327.55 0.989 0.986 0.972 0.971 ± ± ± ± 241.82 252.22 259.70 262.66

3.260 2.519 2.105 2.909

0.177 0.228 0.306 0.600 ± ± ± ± 283 293 303 313

0.197 0.028 0.059 0.120

KF b

L·mg−1

Q

mg·g−1

T

K

R2

χ2

mg1−1/n·L1/n·g−1

0.21 0.228 0.216 0.205

± ± ± ±

Ks Qs

mg·g−1 χ2 R2 1/nf

Freundlich

Table 2. Isotherm Parameters for the Adsorption of Mb onto P(MAA-co-VSA)-g-MNCC at Different Temperaturesa I

L·mg−1

Sips

1/ns

where qe (mg·g−1) and Ce (mmol·kg−1) are the equilibrium concentrations, Q0 (mg·g−1) and b (L·mg−1) are Langmuir constants, KF (mg1−1/n·L1/n·g−1) and 1/nF are the Freundlich constants, and Qs (mg·g−1) is the Sips maximum adsorption capacity, Ks (L·mg−1) is the Sips equilibrium constant and 1/ns is the Sips model exponent. The better isotherm model was evaluated based on the study of χ2 and R2. The values of adsorption isotherm constants calculated using nonlinear regression analysis along with χ2 and R2are reported in Table 2. The low value of χ2 and high values of R2 for the Sips model indicate that it is better fit isothermal model. The isotherm graph of Sips adsorption isotherm showed that the experimental data fitted well with theoretical data. The Sips equation used as a combination of Langmuir and Freundlich isotherm models and used to represent the adsorption on heterogeneous surface. The 1/ns value is a measure of surface heterogeneity. The deviation of 1/ns from unity refers a highly heterogeneous surface.48 From Table 2, it is clear that 1/ns decreased from 0.814 to 0.585 as the temperature increased from 283 to 313 K. Thus increase in surface heterogeneity of P(MAA-co-VSA)-g-MNCC with temperature is another factor that favors the adsorption of Mb. At low solute concentration, Sips model effectively reduces to a Freundlich isotherm and at high adsorbate concentrations, it predicts a monolayer sorption capacity characteristic of the Langmuir isotherm. Sips isotherm indicates a multilayer adsorption at lower concentration and monolayer at higher concentration of Mb onto P(MAA-co-VSA)-g-MNCC. The The ns values above unity show that adsorption data obtained in this study follow Langmuir form rather than that of Freundlich.53 The maximum adsorption capacity obtained from the Sips model is slightly greater than that obtained from the Langmuir model at all temperatures. The fitness of isotherm model follows the order: Sips > Langmuir > Freundlich. 3.9. Effect of Ionic Strength. The adsorption experiments were conducted by mixing 11.7·10−3 mmol·kg−1 Mb solution, whose percentage adsorption alone is 94.1 %, with 0.1 mmol·kg−1 NaCl, KCl, Na2SO4, K2SO4, diaminomethanal, and thiourea. The percentage adsorption was found to be decreased in the presence of coexisting ions. The percentage of adsorption in the presence of NaCl, KCl, Na2SO4, K2SO4, diaminomethanal, and thiourea (each of 0.1 mmol·kg−1 concentration) was found to be (77.9, 79.3, 80.5, 81.2, 76.9, and 78.9) %, respectively. The effect of ions can be explained on the basis of electrostatic interaction that exists between the adsorbent and Mb.54 The protein will be dehydrated in the presence of salt molecules,44 this in turn decreases the hydrophilic interaction that exists between Mb and P(MAAco-VSA)-g-MNCC. There were earlier reports regarding the increased amount of adsorption of proteins onto surfaces in the presence of Na2SO4, as compared to other anionic components, which enhance the protein affinity for the surface through enhanced hydrophobic interaction. Among the inorganic salts, the Na+ ions have a pronounced effect in decreasing the adsorption of Mb compared to K+. This is because of the high positive charge density that exists on Na+ rather than on K+. The presence of organic salts like diaminomethanal and thiourea also decreases the adsorption of Mb onto P(MAA-

0.047 0.077 0.060 0.037

R2

(13)

Langmuir

1 + K sCe1/ ns

± ± ± ±

Q sK sCe1/ ns

0

qe =

0.814 0.768 0.613 0.585

χ2

Sips isotherm:

58.45 88.59 89.05 39.69

Article



Article

Commision (New Delhi) granted Major Research Project MRP F. No. 37-425/2009 (SR).

co-VSA)-g-MNCC. Mb is a soft protein and the use of organic salts denatures the protein, hence lowering the adsorption capacity.55 But the exact nature of interaction between these denaturing salts and protein is not known completely. 3.10. Desorption and Regeneration Studies. Desorption efficiency of the spent adsorbent was checked with different reagents such as KOH, NH4OH, CH3COOH, HNO3, NH4Cl and K2(OX) having a concentration of 0.3 mmol·kg−1 and found to be (89.5, 97.3, 63.9, 65.9, 80.0 and 81.2) %, respectively. Among these 0.3 mmol·kg−1 NH4OH was proved to be the most suitable desorbing reagent. Therefore, we conducted adsorption−desorption experiments only with 0.3 mmol·kg−1 NH4OH for five cycles. During five cycles, the adsorption capacity of P(MAA-co-VSA)-g-MNCC declined from 99.58 to 95.44 % and the recovery of Mb molecules in 0.3 mmol·kg−1 NH4OH decreased from 97.28 to 94.12 %. The loss of adsorbent during five adsorption−desorption cycles was minimal, and this stabilized behavior of the P(MAA-co-VSA)-gMNCC is highly beneficial for the design of a batch reactor.

Notes

The authors declare no competing financial interest.

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4. CONCLUSIONS The novel adsorbent, P(MAA-co-VSA)-g-MNCC, prepared by the modification of MNPs was found to be effective in the recovery of Mb from aqueous solutions. NC layer and P(MAAco-VSA) act as coating layers on MNPs and stabilize the Fe3O4 core. The adsorption was affected by time, temperature, concentration, and pH. The best recovery of Mb was found to be at pH 7.0, near the isoelectric point of Mb, indicating the existence of an electrostatic interaction between adsorbent and protein. Complete recovery of Mb from aqueous solutions having 5.9·10−3 mmol·kg−1 Mb concentration is possible with 2 g·kg−1 of the adsorbent, at 303 K within 30 min. A small amount of adsorbent was only required for the complete separation within a small reaction time. The formation of pure Fe3O4 core, P(MAA-co-VSA)-g-NC shell and adsorption of Mb onto adsorbent was confirmed by FTIR, SEM, and XRD analyses. A surface area analyzer and potentiometric titration method gave evidence for proper grafting of polymer onto the surface of MNC. The kinetics of adsorption of Mb onto P(MAA-co-VSA)-g-MNCC can be best suited with a pseudosecond-order kinetic model, indicating that a chemical interaction exists between sorbent and sorbate. The adsorption of Mb onto newly developed adsorbent has an endothermic nature, with negative ΔG0, positive ΔH0, and ΔS0. The experimental data suited the Sips model, confirming multilayer and monolayer coverage at lower and higher Mb concentration, respectively. The spent adsorbent can be recycled for multiple uses by treatment with 0.3 mmol·kg−1 NH4OH. Thus the surface properties of P(MAA-co-VSA)-g-MNCC govern the adsorption. The adsorption−desorption process was found to be a fast process which implies a better application in industrial fields.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel.: +91 471 2416472. Fax: +0471 2307158. E-mail: tsani@ rediffmail.com. Funding

The authors thank Department of Chemistry, University of Kerala for providing laboratory facilities. S. R. Rejeena gratefully acknowledges the financial support from the University Grants J



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K


Adsorptive Separation of Myoglobin from Aqueous Solutions Using

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