Investigation of the Extraction of Hemoglobin by Adsorption onto

Article pubs.acs.org/IECR

Investigation of the Extraction of Hemoglobin by Adsorption onto Nanocellulose-Based Superabsorbent Composite Having Carboxylate Functional Groups from Aqueous Solutions: Kinetic, Equilibrium, and Thermodynamic Profiles Thayyath Sreenivasan Anirudhan,* Sylaja Raveendran Rejeena, and Abdul Rauf Tharun Department of Chemistry, University of Kerala, Karyavattom, Trivandrum 695 581, India ABSTRACT: We have developed a superabsorbent composite based on cellulose, namely poly(acrylic acid)-modified poly(glycidylmethacrylate)-grafted nanocellulose (PAPGNC), via graft copolymerization technique, and it is well characterized. The adsorption of a globular protein, hemoglobin (Hb), onto PAPGNC was studied by batch-adsorption technique. The optimum pH for Hb adsorption was found to be 6.7, and the adsorption attained equilibrium within 3.0 h. The kinetic data were found to follow pseudo-second-order model, and the equilibrium data were found to agree with the Langmuir isotherm model. The maximum adsorption capacity of PAPGNC was found to be 119.92 mg/g at 30 °C. The adsorption capacity of PAPGNC toward Hb was found to increase with an increase in temperature. Spent adsorbent was effectively degenerated using 0.1 M KSCN solution without a loss in adsorption capacity, even after four cycles. The present investigation shows that PAPGNC can be developed as an efficient adsorbent for the extraction and recovery of Hb from aqueous solutions. hydrogel based on polyacrylamide and chitosan,24 has been well-investigated by previous researchers. However, most of these materials lack biodegradability, hydrophilicity, costeffectiveness, and good adsorption capacity. The adsorption using low-cost adsorbent with high adsorption capacities is still under development to reduce the adsorbent dose and minimize the disposal problems. The majority of commercial polymers and ion exchange resins is not always safe or environmental friendly. Today, there is a growing interest in various biosorbent materials, such as fungal or bacterial biomass and biopolymers that can be obtained in large quantities and that are harmless to nature, and special attention has been given to natural polysaccharides.25 Among the available natural polysaccharides, cellulose is marked first, owing to their low density, biodegradability, and availability from renewable sources. However, poor mechanical strength, low adsorption capacity,26−28 and microbial contamination are the main disadvantages faced by the use of bare cellulose. However, these problems can be resolved by the use of cellulose nanofibers because of their exceptional mechanical properties (high specific strength and modulus), large specific surface area, and low coefficient of thermal expansion, high aspect ratio, environmental benefits, and low cost compared to that of cellulose.29 Many workers reported that cellulose-based adsorbents are useful candidates for the separation of proteins from aqueous solutions.30 Niide et al.31 have developed novel adsorbents based on bacterial cellulose (BC) and plant cellulose (PC), namely quaternary ammonium bacterial cellulose (QABC) and quaternary ammonium plant cellulose (QAPC),

1. INTRODUCTION Hemoglobin (Hb) is the physiological oxygen transport metalloprotein that binds oxygen, diffuses into the bloodstream from the lungs, transports it to the outlying tissues, and releases it for aerobic respiration. This globular protein is found mainly in red blood cells in mammals and other animals. It is used to synthesize artificial blood, thereby protecting against blood loss. However, these applications require pure protein samples, for which we have to depend on various separation and purification techniques. The separation and purification of proteins are important because proteins have various sizes, shapes, charges, and hydrophobicities.1−6 The separation of proteins is valuable in biomedical applications such as in vitro analyses, antibody generation, binding assays, and structural studies.1,2,7−10 Ultrafiltration,11 electrophoretic separation,12 adsorption, liquid chromatography, the use of magnetic nanoparticles,13 and membrane chromatography14,15 are the widely accepted methods developed for the separation of proteins; among these, adsorption, which relies on the specific interactions between protein and adsorbent to extract targeted protein from its mixture directly through a simple adsorption process, is used primarily.14 However, in addition to the amount of adsorbed proteins on the surfaces, we should consider the structure or orientation of the adsorbed proteins because it directly expresses the biocompatibility.16,17 Hb is an attractive model for the study of adsorption onto solid surfaces because it is structurally and functionally well characterized and undergoes structural changes. Adsorption of Hb onto a number of valuable adsorbents, such as bentonite clay,18 molecularly imprinted acrylamide functionalized cross-linked chitosan beads,19 molecularly imprinted acrylamide functionalized maleic anhydride modified chitosan beads,20 synthetic Mg/Al hydrotalcite,21 chitosan−anodic aluminum oxide composite membrane,22 SBA-15,23 and a semi-interpenetrating polymer network © 2013 American Chemical Society

Received: Revised: Accepted: Published: 11016

December 30, 2012 July 11, 2013 July 26, 2013 July 26, 2013 dx.doi.org/10.1021/ie303365x | Ind. Eng. Chem. Res. 2013, 52, 11016−11028

Industrial & Engineering Chemistry Research

Article

Scheme 1. Proposed Reaction Mechanism for the Synthesis of PAPGNC from Cellulose

respectively, and QABC showed a higher adsorption capacity for Hb as compared with that of QAPC. However, to our best of knowledge, there has no reported literature so far regarding the adsorption of Hb onto nanocellulose (NC)-based adsorbents. Because NC has more surface area and hydroxyl groups exposed to the surface than cellulose, NC is found to be more hydrophilic and is expected to be biocompatible in nature. However, it exists as a dispersible colloid in solution and prevents its use as a potential adsorbent. Chemical modification such as graft copolymerization is a widely accepted method for achieving adequate structural durability, introducing functionality and thereby increasing the adsorption capacity and thermal stability.32 Because NC contains a larger number of hydroxyl groups than cellulose, the grafting efficiency may be higher in NC. Grafting with monomers like glycidylmethacrylate (GMA)

is considered to be advantageous because it consists of incorporating carbon−carbon π-bonds coming from the GMA onto the structure of the macromolecule and enables it to undergo a gelling process through a radical cross-linking polymerization reaction. 33 GMA is an important vinyl monomer, having a reactive epoxy group, and on addition, the reaction generates new functional groups that find uses in ion exchange, in chelate formation, and as pseudoaffinity ligands.34 Because GMA provides spacer groups that retain the flexibility of the polymer chains, it can be used for the effective adsorption of protein molecules from aqueous solutions. The capacity for holding water and solute molecules within a polymeric gel not only increases the adsorption capacity and biocompatibility but also helps in the removal of toxic materials from our body. A cross-linking reaction helps in the formation of bonds between different chains of linear polymers, leading to 11017

dx.doi.org/10.1021/ie303365x | Ind. Eng. Chem. Res. 2013, 52, 11016−11028


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EGDMA was purged with N2 for 30 min in a 1.0 L reactor equipped with a thermometer and a reflux condenser. Polymerization was initiated by the addition of 2.0 g of BPO at 80 °C for 4 h. The resulting PGNC particles were immersed in 150 mL of 1.0% poly(acrylic acid) aqueous solution whose pH was adjusted to 3.5 with 0.2 mL of 2 M HCl solution. The reaction mixture was stirred vigorously at 50 °C for 3 h. The product was separated by a centrifugation process at room temperature and dried at 50 °C to obtain constant weight. The obtained product, namely PAPGNC, was sieved using 80−230 mesh to get an average particle size of 0.096 mm. PAPGNC was used throughout the adsorption studies. 2.3. Swelling Capacity of PAPGNC. The swelling capacity of a hydrogel is an important parameter concerning its application in the biomedical field. The swelling ability of PAPGNC and CAc was determined by putting 0.1 g of each into a weighd tea bag, immersing the bag in 100 mL of a solution of pH 2.0−9.0 (adjusted using either acetate (50 mM, pH 2.0−6.0) or phosphate buffer (50 mM, pH 6.5−9.0)), and allowing it to soak at 30 °C for 2 h. After soaking, the equilibrated adsorbent particles were allowed to drain by removing the tea bag from water and hung until no drops drained. Swelling (%) can be determined by weight difference method and is expressed by the following equation:38

3D network structures, and thereby improves the water-holding or swelling efficiency. Ethyleneglycol dimethacrylate (EGDMA) is a typical cross-linking agent that adds dimensional stability and stiffness to obtain polymeric material.35 The swelling behavior can be made pH tunable via conjugation with strongly ionizable groups, and this behavior can be utilized for biomedical applications. Functionalization with poly(acrylic acid) (PAA) will generate a large number of strongly ionizable carboxylate (COO−) groups, and the hydrogels exhibit a pH− dependent swelling behavior. PAA-based hydrogels have been widely investigated for biomedical applications due to their biodegradable nature. Also, if the surface is modified with acid groups, the adsorption occurs through an electrostatic interaction, and the anionic character of hydrogels will enhance the amount of protein adsorption.12 In the present study, a cellulose-based superabsorbent composite, poly(acrylic acid)-modified poly(glycidylmethacrylate)-grafted nanocellulose (PAPGNC), was developed, well-characterized, and used for the recovery of Hb from aqueous solutions. PAPGNC was synthesized by the graft copolymerization reaction of glycidylmethacrylate onto nanocellulose (NC) in the presence of EGDMA as cross-linker followed by immobilization of poly(acrylic acid). The PAPGNC was designed so as to adsorb Hb effectively using the ionized carboxyl groups anchored on the surface of the adsorbent through electrostatic attraction before their isoelectric point. Batch adsorption experiments were conducted to observe optimum pH, contact time, initial concentration, and temperature. The regenerating capability of PAPGNC was evaluated by using 0.1 M potassium thiocyanate (KSCN) as the eluent.

swelling (%) =

(Ws − Wd) × 100 Wd

(1)

where Ws (mg) represents the weight of the swollen gel and Wd the (mg) weight of the dry gel. 2.4. Estimation of Epoxy and Carboxyl Group Contents. The pyridine−HCl method was used to determine the amount of epoxy group content in poly(glycidylmethacrylate)-grafted cellulose (PGC) and PGNC.39 About 1.0 g of PGC or PGNC was refluxed with 50 mL of pyridine−HCl mixture (2 mL of concentrated HCl and 123 mL of pyridine) for 20 min. After the solution was cooled, the amount of available epoxy groups was determined by titration of the filtrate with 0.1 M NaOH. The following equation was used to estimate the amount of epoxy groups:

2. MATERIALS AND METHODS 2.1. Materials. All the chemicals used in the present study were of reagent grade and were used without further purification. All stock solutions were prepared in doubledistilled water. As the precursor of cellulose, sawdust of Mangiferra indica (collected from local saw mill, Trivandrum) was taken after thorough washing and drying at 80 °C. Hb (pI = 6.8) with a MW of 68 kDa was obtained from Sino-America Biotechnology. GMA was obtained from Sigma-Aldrich. EGDMA and PAA were received from Aldrich. Cellulose acetate (CAc), benzoyl peroxide (BPO), KSCN, and other chemicals were supplied by E. Merck. 2.2. Preparation of PAPGNC. PAPGNC was prepared by following three steps. The proposed reaction mechanism for the preparation of PAPGNC is represented in Scheme 1. Step 1. Extraction of Cellulose from Sawdust. Sawdust was pretreated with a 10% H2SO4 solution (120 °C, 10 min) and centrifuged to remove a rich pentosanes solution. Delignification was achieved by subsequent treatment with 1% NaOH (100 °C, 1 h). The brown mass obtained was bleached with 5% H2O2 (80 °C, 1 h) and yielded white cellulose as the product.36 Step 2. Preparation of Nanocellulose (NC) from Cellulose. About 5 g of cellulose was dispersed in 250 mL of distilled water under magnetic stirring (20 min). Then 140 mL of 98% sulfuric acid was dropped to the homogenized mixture without heating. After the addition was complete, the mixture was heated at 50 °C for 2 h. The hot mixture was diluted ten times with ice cooled distilled water. The obtained white colloid was centrifuged, washed many times with water, and freeze-dried.37 Step 3. Preparation of PAPGNC. A 100 mL suspension consisting of ∼10.0 g of NC, 71.0 mL of GMA, and 4.0 mL of

epoxy groups (mequiv/g) =

(C NaOHVNaOHV rpy − HCl) V t py − HClW

(2)

where CNaOH is the concentration of NaOH (M), W is the weight of the adsorbent (g), VNaOH is the volume of the NaOH solution used (mL), Vrpy−HCl is the volume of the pyridine− HCl mixture taken for refluxing, and Vtpy−HCl is the volume of the pyridine−HCl mixture taken for titration. The carboxyl group content in the adsorbent was determined by stirring 0.1 g of sample with 20 mL of 0.1 M NaCl aqueous solution for 60 min at room temperature under ultrasonic vibration. After centrifugal separation, the supernatant was titrated against 0.1 M NaOH. The amount of carboxylic acid groups in the adsorbent was estimated using the equation COOH (mequiv/g) =

(C NaOHVNaOH − C HClVHCl) W

(3)

where C is the concentration of HCl or NaOH (M), W is the weight of the adsorbent (g), and V is the volume of the HCl or NaOH solution used (mL). 2.5. Characterization Studies. The X-ray diffraction (XRD) patterns of the samples were recorded using an 11018



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supernatant was analyzed for Hb concentration spectrophotometrically. The reusability of the adsorbent was confirmed by repeating the adsorption−desorption cycles up to four times using 0.1 M KSCN as the desorbing agent. The desorption (%) was calculated using the equation

X’Pert Pro X-ray diffractometer using Cu Kα radiations at a scanning speed of 2°/min and at a wavelength of 1.5406 Å. The Fourier transform infrared (FTIR) spectra were recorded with a Shimadzu FTIR spectrometer in the wavelength range 400− 4000 cm−1 using a KBr window at a resolution of 4 cm−1. The samples were mixed with KBr, and the FTIR spectra was recorded in diffused transmission mode. Dynamic light scattering measurements for the NC were carried out in a laser scattering particle size distribution analyzer from Malvern Instruments Ltd. Water was used as the dispersant, with a refractive index of 1.33 and viscosity of 0.8872 cP. The thermogravimetric (TG) analysis was carried out using a Mettler Toledo STARe system with an air flow over the range from ambient to 900 °C at a scan rate of 5 °C/min. The wet density of cellulose and PAPGNC was determined by the liquid displacement method in a 10 mL gravity bottle using nitrobenzene as the displacing liquid. The surface area was measured by the BET method using a Quantasorb model Q7/S surface area analyzer (Quantachrome). A potentiometric method40 was used to determine the pH at the point of zero charge pHpzc. The concentration of the protein solution was determined spectrophotometrically on a JASCO UV−visible (model V-530) spectrophotometer. All pH measurements were carried out on a Systronic model μ pH system 361 pH meter. A temperature-controlled water bath shaker (Labline) with a temperature variation of ±1 °C was used for the equilibrium studies. Borosil glasswares were used throughout the experiment. 2.6. Adsorption/Desorption Experiments. Batch adsorption experiments were conducted by shaking 0.1 g of adsorbent with 50 mL of Hb solution at the desired concentrations (10−500 mg/L) and at different temperatures (10−40 °C) and pH values (4.0−9.0) in 100 mL glassstoppered Erlenmeyer flasks using a temperature-controlled water bath shaker at a shaking speed of 200 rpm for 3 h. The effect of solution pH on the equilibrium adsorption of Hb was investigated under similar experimental conditions between pH 4.0 and 9.0. The pH was adjusted by preparing Hb solutions in either acetate (50 mM, pH 4.0−6.0) or in phosphate buffer (50 mM, pH 6.5−9.0). The kinetic experiments were carried out at initial concentrations ranging from 25 to 100 mg/L at pH 6.7 and a temperature of 30 °C. Isotherm experiments were conducted with Hb solutions at various concentrations (10− 500 mg/L) at 10, 20, 30, and 40 °C. After the adsorption process, the samples were centrifuged, and the supernatant solutions were analyzed for the residual Hb concentration by using a UV−visible spectrophotometer at a λmax of 406 nm.18 The amount of adsorption qe (mg/g) was calculated by the following equation: qe = (C0 − Ce) ×

V m

(4)

ddsorption (%) =

(C0 − Ce) × 100 C0

(5)

Hb desorbed (%) =

Md × 100 Ma

(6)

where Md (mg/g) is the amount of Hb desorbed by reagents and Ma (mg/g) is the amount of Hb adsorbed onto PAPGNC. The measurements were made for each sample, and the results were averaged. The difference in results for the triplicates was less than 3.9%.

3. RESULTS AND DISCUSSION 3.1. Characterization of the PAPGNC. 3.1.1. X-Ray Diffraction. The XRD patterns of cellulose, NC, PAPGNC, and Hb-PAPGNC are shown in Figure 1. In the XRD pattern of

Figure 1. XRD patterns of cellulose, NC, PAPGNC, and HbPAPGNC.

cellulose, the diffraction maxima at 2θ = 14.6, 15.8, 22.1, and 34.2° can be attributed to the partial crystalline nature of cellulose, like all natural polymers.41 Even though the characteristic peaks of NC are same as that of cellulose, its broadness increased to a small extent, which may be due to the small particle size and strain induced during the conversion to nanoregime. The sharper diffraction peaks are an indication of a high degree of crystallinity in the structure. This is due to the partial removal of the amorphous regions during the acid hydrolysis treatment of cellulose.42 The percent crystallinity of cellulose and NC were compared using the equation:43

where C0 and Ce are the initial and equilibrium Hb concentrations (mg/L), respectively, m is the mass of the adsorbent (g), and V is the volume of the Hb solution (mL). Desorption studies were conducted by shaking 0.1 g of Hbloaded PAPGNC (Hb-PAPGNC) with 50 mL of 0.1 M solutions of KOH, HCl, HNO3, NaOAc, HCOOH, KSCN, and sodium lauryl sulfate for a period of 3.0 h at 30 °C. The agitation rate was fixed at 200 rpm. After centrifugation, the

crystallinity (%) =

(Ic − Ia) × 100 Ic

(7)

where Ic and Ia are the intensity of the crystalline and amorphous peaks, respectively. The percent crystallinity of cellulose and NC using this equation were 67.05% and 81.74%, 11019



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Hb-PAPGNC confirms the presence of hydrogen bonding between the protein and PAPGNC. 3.1.3. Dynamic Light Scattering (DLS) Studies. Dynamic light scattering (DLS) studies (Figure 3) were carried out to

respectively. The high percent crystallinity (14.69%) of NC as compared to that of cellulose may be due to the endoglucanasemediated hydrolysis during their preparation. The average crystallite size of NC calculated from a peak corresponding to the (002) plane, using the Debye−Scherrer formula,44 was found to be 25 nm. This small grain size will increase the surface area and grafting efficiency, which in turn attracts a huge amount of proteins toward it. The broader peaks also support their smaller grain size. The absence of characteristic peaks of cellulose in PAPGNC suggests that exfoliation had occurred during modification. The increased amorphous nature in the diffraction pattern of Hb-PAPGNC indicates that Hb had adsorbed on the surface of PAPGNC. 3.1.2. FTIR Spectra. Figure 2 shows the FTIR spectra of cellulose, PGNC, PAPGNC, Hb, and Hb-PAPGNC. The

Figure 3. Particle size distribution of NC.

measure the particle size of NC, which shows two peaks with average particle sizes of 80.3 and 358.2 nm. This information clearly indicates that the cellulose nanocrystals that were dispersed by water have the same average diameter (D) of 80.3 and length (L) of 358.2 nm. The dimensions of the cellulose nanocrystals closely match the previously reported work of several authors.46,47 The polydispersity index (PDI) of NC was determined to be 0.522. PDI measurement indicates the width of the particle size distribution and shows the value zero for a highly monodispersed sample and one or more for a highly polydispersed sample. The relatively low PDI value obtained for the NC dispersions indicates a high degree of monodispersity of these samples. 3.1.4. TG Analyses. TG scans recorded for cellulose, NC, PAPGNC, and CAc are shown in Figure 4. It is clear from the

Figure 2. FTIR spectra of cellulose, NC, PGNC, PAPGNC, Hb, and Hb-PAPGNC.

absorption bands present in all samples at 3430, 2925, and 1640 cm−1 are due to hydrogen bond O−H stretching vibration, CH stretching from the −CH2 group, and −OH bending vibration, respectively. The bands at 1426, 1382, and 1315 cm−1 are due to the bending vibrations of CH stretching from the −CH2 group. The bands at 1160 and 1033 cm−1 are due to antisymmetric stretching vibrations of the C OC bridge and skeletal vibrations involving CO stretching, respectively; these are assigned to the characteristics of the saccharide structure. Appearance of an absorption band at 897 cm−1 is attributed to the glucosidic ring in the cellulose structure. The new band at 1722 cm−1 in PGNC may be due to the presence of a carbonyl group in the epoxide ring. The presence of the −COOH group in PAPGNC was confirmed by the characteristic CO stretching and COH in-plane bending frequencies of the carboxyl groups at 1705 and 1360 cm−1, respectively. Hb shows bands at 1656 and 1547 cm−1, indicating the vibrations corresponding to the amide I and amide II bands of protein molecules.45 The shifting of the peaks in Hb at 1656 and 1547 cm−1 toward 1642 and 1537 cm−1 in

Figure 4. TG curves of cellulose, NC, PAPGNC, and CAc.

figure that the thermal decomposition of cellulose occurred in three steps: (i) 30−90 °C, (ii) 234−375 °C, and (iii) 375−495 °C. In the first stage of thermal decomposition, a 6.5% weight loss is observed due to the dehydration and decomposition of cellulose. In the second stage, almost 62.5% of mass loss occurs due to splitting of the cellulose structure, chain scission evolving CO and CO2 and forming carbonaceous residues.48 Above 375 °C, only 24.7% of the weight loss is observed due to the oxidation of char. The TG curve of NC shows three 11020



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degradation stages: (i) 30−100 °C, (ii) 140−250 °C, and (iii) 530−695 °C. In the first stage, NC lost 15.2% of its initial weight due to dehydration, whereas about 64.3% of the material is lost during the second stage, which is ascribed to the decomposition and splitting of the cellulose structure. Above 250 °C, only 36.7% of the weight loss is observed due to the oxidation of char. The TG curve of PAPGNC undergoes three degradation stages. The initial weight loss of 7.6% in the 30− 130 °C is due to moisture loss from the polymer network. The second degradation resulting in 85.1% of weight loss at 290− 450 °C implies the degradation of polymer networks. Above 450 °C, 15.0% of weight loss was observed, which is due to the decomposition of carboxyl groups in PAPGNC. For comparing the thermal stability of PAPGNC with that of the commercially available cation exchanger cellulose acetate (CAc), the TG curve of CAc has been analyzed. Three degradation stages were observed for the CAc: (i) 30−275 °C, (ii) 275−390 °C, and (iii) 390−580 °C. A weight loss of 13.6% is observed at the first stage and is due to the generation of acetic acid followed by dehydration. The acid formed catalyzes the dehydration reaction. Upto 86% of the weight was lost during the second step, which may be due to the decomposition and splitting of cellulose structure. It implies that the removal of ester groups and formation of the CC conjugated system had started. Above 390 °C, only 14% of the weight loss was observed, which can be ascribed to the complete removal of ester groups and formation of CC conjugated system as carbonaceous residue.49 However, the TG curves clearly indicate an enhanced thermal stability of the PAPGNC compared to that of cellulose, NC, and CAc. Thus, PAPGNC has been proved a more efficient adsorbent for the adsorption studies than the commercial adsorbents. 3.1.5. Physical Characteristics of PAPGNC. The epoxy content in poly(glycidylmethacrylate)-grafted cellulose (PGC) and PGNC was found to be 1.39 and 2.21 mequiv/g, respectively. The increased grafting efficiency of PGNC may help in the increased adsorption capacity of proteins. The amount of carboxyl groups in PAPGNC was found to be 1.43 mequiv/g. The densities of cellulose and PAPGNC were calculated to be 0.82 and 1.54 g/mL, respectively. The specific surface areas of cellulose and PAPGNC measured by N2 adsorption were 22.7 and 96.2 m2/g, respectively. The zero point charges of cellulose and PAPGNC were observed at pH 5.5 and 4.2, respectively. 3.2. Swelling Capability of PAPGNC in Water. The swelling percentage of PAPGNC and CAc was found to increase with an increase of the pH value from 2.0 to 9.0 (Figure 5). The maximum swelling for both PAPGNC and CAc was achieved at pH 6, and above this pH, the swelling leveled off. It is because, as the pH value increases, the ionization of −COOH functional groups in PAPGNC to −COO− enhanced gradually, and swelling occurs because of large electrostatic repulsion between neighboring ionized −COO− groups. The increase in anion density may also result in raising the osmotic pressure of the network structure, which in turn increases the swelling behavior.50 The maximum swelling capacities for PAPGNC and CAc were found to be 434 and 282%, respectively. The increased swelling capacity for PAPGNC may be due to the increased number of −COOH groups anchored on the surface because of grafting and efficient crosslinking. Also, the ion exchange capacity of PAPGNC (1.43 mequiv/g) was found to be higher than CAc (3.4 × 10−3

Figure 5. Swelling percentage of PAPGNC and CAc with respect to pH.

mequiv/g). The hydrophilicity of NC may also be helped in the increased swelling capacity. 3.3. Effect of Adsorbent Dose on Adsorption. The adsorption experiments were conducted by varying the amount of both poly(acrylic acid)-modified poly(glycidylmethacrylate)grafted cellulose (PAPGC), PAPGNC, and CAc, and the results obtained are shown in Figure 6. It is clear from the figure that

Figure 6. Effect of adsorbent dose on the adsorption of Hb onto PAPGC, PAPGNC, and CAc.

4.0 g/L PAPGNC and 10.0 g/L PAPGC were needed for the complete recovery of 25 mg/L Hb from aqueous solutions. However, only 84.6% of the Hb was recovered even with the use of 10.0 g/L of CAc. The higher adsorption efficiency of PAPGNC (2.5 times greater than PAPGC) may be attributed to the high surface-to-volume ratio of nanocellulose, which in turn increased the grafting efficiency and the number of functional moieties. However, the commercial cation exchanger CAc only shows poor performance when compared to the newly developed adsorbents based on cellulose or NC. Because PAPGNC was more efficient than PAPGC and CAc for the 11021



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recovery of Hb, subsequent adsorption experiments were carried out only with PAPGNC. 3.4. Adsorption of Hb at Different pHs. The pH dependence of the adsorption of Hb onto PAPGNC was studied by varying the pH of the media from 4.0 to 9.0. As the pH value increased, the adsorption percentage also increased, reached a maximum at 6.7, and then decreased slowly (Figure 7). The amount adsorbed was found to be 4.84 (96.8%) and

Figure 8. Time-course profiles of adsorption of Hb onto PAPGNC at different initial concentrations: pseudofirst- and pseudo-second-order kinetic models. Triplicates for each sample were analyzed and each datum point represents the mean value ± standard deviation (n = 3).

However, the equilibrium time was found to be independent of initial concentration. The kinetics models, like pseudo-first-order and pseudosecond-order models, have been applied to describe the mechanism and kinetics of adsorption.

Figure 7. Effect of the solution pH on the adsorption of Hb onto PAPGNC.

pseudo‐first‐order:

11.76 (94.1%) mg/g for initial concentrations of 10.0 and 25.0 mg/L, respectively, at a pH value of 6.7. We have an adsorbent with a zero point charge of 4.2, which means that the adsorbent shows positive behavior below 4.2, and above it shows negative behavior. Also, the carboxyl groups on PAPGNC ionize in the pH range 3.5−4.5 and convert to carboxylate (COO−) groups, possessing a negative charge. The pI value of the Hb we have purchased is 6.8, and it possesses a net positive charge when the medium pH is less than 6.8. Therefore, the adsorption must take place between the pKa of carboxylate 4.5 and pI of Hb 6.8 if the electrostatic interaction has played an important role in the adsorption process of Hb onto PAPGNC. As mentioned in literature, the protein gets strongly adsorbed onto negatively charged species below or near the isoelectric point.51 The maximum adsorption from aqueous solutions is usually obtained at their isoelectric point, as proteins have no net charge at the isoelectric point. Similar results were observed previously when the adsorption of Hb was carried out on bentonite clay18 and SBA-15 surfaces,23 and they have provided satisfactory explanations for the adsorption characteristics of Hb at each pH value. The adsorption capacity gets decreased when deviating from the isoelectric point, and this may be caused by the increase in the conformational size of the protein molecules and the lateral electrostatic repulsions between adjacent adsorbed protein molecules. 3.5. Effect of Contact Time on Hb Adsorption. In batch experiments, kinetic study is very important to find out the effective contact time of the adsorbent with the adsorbate and to evaluate the reaction coefficients. As seen in Figure 8, Hb adsorption rate was rapid at the beginning of adsorption; thereafter adsorption was gradual, and equilibrium was reached within 3.0 h. This adsorption trend may be due to the abundant availability of reaction sites and gradual utilization of these sites.

pseudo‐second‐order:

qt = qe(1 − e−k1t )

qt =

(8)

k 2qe 2t 1 + tk 2qe

(9)

where qe and qt (mg/g) are the amounts of Hb adsorbed on PAPGNC at equilibrium and time t (min), respectively. k1 (min−1) is the rate constant for the pseudo-first-order adsorption process. k2 (g/(mg min)) is the rate constant for the pseudo-second-order kinetics. The comparison of pseudo-first-order and pseudo-secondorder parameters are given in Table 1, from which it can be seen that the values of k2 decreased from 2.30 × 10−2 to 0.3 × 10−2 g/(mg min) as the initial concentration of Hb increased from 25 to 100 mg/L. The decrease in values of k2 with an increase of initial concentration may be due to lowering the probability of collision between the Hb molecules, which speeds up the movement of Hb toward the adsorbent surface, at lower concentrations. The closeness of theoretical values (qe,calc) with experimental data (qe,exp) proves the applicability of the pseudo-second-order kinetic model rather than the pseudo-first-order model. Also, the independent behaviors of k1 with the initial concentration itself indicates the unsuitability of the pseudo-first-order kinetic model in the adsorption process of Hb onto PAPGNC. The R2 values closer to unity and very low χ2 values confirmed the applicability of the pseudo-secondorder kinetic model in the present study. When the rate of reaction of an adsorption process is controlled by chemical exchange, the pseudo-second-order model can be better adjusted to the experimental kinetic data.52 Thus, the kinetic study reveals that the adsorption of Hb onto PAPGNC follows the mechanism of ion exchange followed by complexation.53 Many authors have been reported that the adsorption of proteins commonly occurs via an ion exchange mechanism.54,55 11022



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Table 1. Kinetic Parameters for the Adsorption of Hb onto PAPGNCa pseudo-first-order

a

C0 (mg/L)

qe,exp (mg/g)

25 50 75 100

11.775 23.302 34.398 45.169

k1 (min−1) 0.162 0.107 0.136 0.103

± ± ± ±

0.023 0.010 0.019 0.013

pseudo-second-order

qe,calc (mg/g)

R2

χ2

± ± ± ±

0.958 0.979 0.950 0.960

0.524 1.125 5.027 7.35

11.175 22.397 32.557 43.108

0.250 0.388 0.749 0.937

k2 (g/mg min) 0.023 0.007 0.006 0.003

± ± ± ±

0.0022 0.0002 0.0006 0.0002

qe,calc (mg/g)

R2

χ2

± ± ± ±

0.993 0.999 0.992 0.996

0.091 0.032 0.771 0.807

11.818 24.033 34.584 46.145

0.134 0.087 0.377 0.407

± standard deviation; n = 3.

Figure 9. Comparison of different isotherm models for the adsorption of Hb onto PAPGNC. Triplicates for each sample were analyzed and each datum point represents the mean value ± standard deviation (n = 3).

3.6. Effect of Concentration of Hb Solution. The effect of the concentration of Hb on the adsorption process (adsorption isotherm) was investigated at 30 °C under the optimized conditions of pH 6.7 with 3.0 h shaking time, and the results are plotted in Figure 9. Hb concentration was varied between 10 and 500 mg/L. It was observed from Figure 9 that the amount adsorbed increased with an increase in initial concentration. As the concentration of Hb increased more, the amount adsorbed was found to increase only slightly, which may be attributed to the saturation of active sites of the adsorbent. The adsorption behaviors of PAPGNC were investigated with following adsorption isotherm models:56−58 Langmuir:

Freundlich:

Q 0bCe 1 + bCe

(10)

qe = KFCe1/ n

(11)

qe =

Dubinin−Radushkevich (D−R):

qe = χm (ε 2)−β (12)

where qe (mg/L) is the amount adsorbed at equilibrium, Ce (mg/L) is the equilibrium concentration of the Hb on adsorption, Q0 (mg/g) is the maximum adsorption capacity at a complete monolayer, and b (L/mg) is the Langmuir constant related to the affinity of binding sites and is a measure of the energy of adsorption.59 KF ((mg1−1/n L1/n)/g) and 1/n are the Freundlich constants related to adsorption capacity and intensity of adsorption, respectively. χm (mg/g) is the maximum adsorption capacity, β is the activity coefficient related to mean sorption energy, and ε is the Polanyi potential, which is equal to ε = RT ln(1 + 1/Ce)

(13)

where R is the gas constant (kJ/(mol K)) and T is temperature (K). The adsorption potential is independent of temperature 11023



0.939 0.923 0.925 0.942 1.796 1.826 1.863 1.953 0.004 0.005 0.000 0.016 ± ± ± ± 0.155 0.150 0.144 0.131 1.012 2.329 2.738 4.716 ± ± ± ± 0.951 0.933 0.932 0.939 0.037 0.040 0.033 0.009 ± ± ± ± 0.359 0.340 0.321 0.244 2.198 3.115 3.754 0.000 ± ± ± ± 12.060 15.974 20.683 35.829 6.80 11.59 12.49 23.81 0.993 0.991 0.993 0.989 ± ± ± ± a

± standard deviation; n = 3.

2.486 2.883 2.911 3.512 96.659 107.615 119.921 129.543 10 20 30 40

T (°C)

0.027 0.039 0.049 0.092 ± ± ± ±

0.002 0.004 0.004 0.010

1/n b (L/mg) Q (mg/g)

R

2

χ

2

KF ((mg

1−1/n

1/n

L )/g)

Freundlich

R

2

χ 11024

Langmuir

The positive values of Ea in the adsorption process were found to be increased with increase in temperature, which shows that the higher solution temperature will favor the adsorption process. The sorption capacity for a D−R adsorption isotherm on the basis of the Polanyi potential theory of solutions was found to be nearer to the Freundlich isotherm than to the Langmuir isotherm. Thus, the good fit of equilibrium data with the Langmuir isotherm model as compared to Freundlich and D−R isotherm models confirms the monolayer coverage process of Hb onto PAPGNC. Previous researchers also confirmed Langmuir as the typical isotherm for explaining the adsorption behavior of Hb onto polymeric surfaces.18,20

0

Table 2. Isotherm Parameters for the Adsorption of Hb onto PAPGNC at Different Temperaturesa

(15)

48.36 90.42 119.11 124.47

(14)

where C0 is the initial Hb concentration (mg/L) and b is the Langmuir adsorption equilibrium constant (L/mg). RL > 1, RL = 1, 0 < RL < 1, and RL = 0 indicate that the adsorption system studied is unfavorable, linear, favorable, and irreversible, respectively. In the present case, RL values were calculated to be 0.671, 0.449, 0.289, 0.214, 0.169, 0.119, 0.093, 0.075, 0.063, 0.049, and 0.039 for initial concentrations of 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 mg/L, respectively. This indicates that a favorable adsorption of Hb onto PAPGNC had occurred, and therefore the PAPGNC can be referred as a favorable adsorbent for Hb adsorption. The Freundlich constant 1/n is usually dependent on the nature and strength of the adsorption process as well as on the distribution of active sites. The Freundlich isotherm parameter 1/n < 1 represents a favorable adsorption.61 A smaller 1/n value indicates a more heterogeneous surface, whereas a value closer to or equal to 1 indicates the adsorbent has relatively more homogeneous binding sites.59 The 1/n value (0.24−0.35) obtained for the present study represents a favorable adsorption and corresponds to a heterogeneous surface with an exponential distribution of energy of the adsorbent sites. The mean free energy of adsorption (Ea) can be computed from the Dubinin−Radushkevich equation using the relationship Ea = (2β)−1/2

15.436 19.453 24.213 32.802

R2 Ea (kJ/mol) β (mol2/kJ2)

Dubinin−Radushkevich

χm (mg/g)

1 1 + bC0

2

RL =

χ2

and varies according to the nature of the adsorbent and adsorbate. The experimental data and the nonlinear forms of the Langmuir, Freundlich, and Dubinin−Radushkevich (D−R) isotherm models are plotted in Figure 9, and the values of nonlinear parameters are listed in Table 2. As revealed in Table 2, the Langmuir equation fits well for Hb adsorption onto the PAPGNC for the concentration range studied. The value of Q0 was found to be increased with increasing temperature, indicating that the adsorption density was higher at higher temperatures. The b values indicate the extent of the affinity between the adsorbent and adsorbate, and large values of b indicate strong binding. The increase in b values with an increase in temperature implies that adsorption is more feasible at higher temperatures. An important application of a Langmuir isotherm is that it can be used for predicting if an adsorption system is favorable or unfavorable because it can be expressed in terms of a dimensionless constant separation factor or equilibrium parameter, RL,60 which is defined by

54.87 93.19 118.59 131.77

Article



Article

According to the Langmuir model, the maximum Hb adsorption capacity obtained was reported to be 32.5, 35.7, 88, 17.5, 36.53, 34.19, and 43.86 mg/g for adsorption onto molecularly imprinted acrylamide functionalized cross-linked chitosan beads,19 molecularly imprinted acrylamide functionalized maleic anhydride modified chitosan beads,20 synthetic Mg/Al hydrotalcite,21 chitosan−anodic aluminum oxide composite membrane,22 semi-interpenetrating polymer network hydrogel based on polyacrylamide and chitosan,24 plant cellulose, and bacterial cellulose,31 respectively. Compared to these materials, PAPGNC shows higher adsorption capacity (119.92 mg/g), which represents it as a good candidate for the recovery of Hb from aqueous solutions. 3.7. Effect of Temperature on the Adsorption of Hb onto PAPGNC. In order to explain the effect of temperature on the adsorption process, the equilibrium data obtained from isotherm experiments held at different temperatures (Figure 9 and Table 2) have been analyzed. It was observed that the amount of Hb adsorbed increased from 96.66 to 129.54 mg/g as the temperature increased from 10 to 40 °C, and higher temperatures favored the adsorption of Hb onto PAPGNC. This can be explained in different ways: (a) the Hb molecules at higher temperatures may unfold and cause agglomeration due to the entanglement of their chains, thus resulting in an increased adsorption at higher temperature; (b) because protein adsorption is normally a diffusion-controlled process, the mobility of the Hb molecules increases with an increase in temperature and results in greater adsorption; (c) at higher temperatures, Hb molecules may unfold so that their hydrophobic parts interact with the hydrophobic part of the adsorbent, and thus, the involvement of hydrophobic forces in addition to the electrostatic attraction enhance the protein adsorption.62 An increase in adsorption capacity was also observed with an increase in temperature for the adsorption of Hb onto bentonite clay surfaces.18 To describe the feasibility and nature of adsorption process, the thermodynamic parameters such as the changes in free energy (ΔG0), enthalpy (ΔH0), and entropy (ΔS0) were estimated with the help of a van’t Hoff plot using the following formulas: ΔG 0 = ΔH 0 − T ΔS 0

(16)

ΔS 0 ΔH 0 − R RT

(17)

ln KL =

Figure 10. Plot of ln KL versus 1/T for the adsorption of Hb onto PAPGNC.

the increase in the negative ΔG0 value with temperature suggests that the adsorption is more favorable at higher temperatures. The isosteric heat of adsorption, ΔHX (kJ/mol), is the heat of adsorption determined at constant coverage and can be calculated using the Clausius−Clapeyron equation:64

ΔHx +K (18) RT where Ce is the equilibrium protein concentration in solution (mg/L), R is the gas constant (J/(mol K)), and T is the absolute temperature (K). The isosteric heat of adsorption is calculated from the slope of the plot ln Ce versus 1/T (Figure 11) and was found to be 6.79 kJ/mol. The positive value of ΔHX confirms the endothermic nature of the adsorption process. 3.8. Reuse of the PAPGNC via Desorption. A desorption study will help to regenerate the spent adsorbent and can repeatedly be used to adsorb the proteins. Desorption efficiency of the spent adsorbent was checked with 0.1 M solutions of ln Ce =

where ΔG is the free energy change of specific adsorption (J/ mol), R is the gas constant (J/(mol K)), T is the absolute temperature (K), KL (L/g) is an equilibrium constant obtained by multiplying the Langmuir constants Q0 and b, and ΔS0 (J/ (mol K)) and ΔH0 (J/mol) are the standard entropy and enthalpy changes of adsorption, respectively. ΔH0 and ΔS0 values were calculated from the slope and intercept, respectively, of the plots of ln KL versus 1/T (Figure 10). The value of ΔH0 (35.93 kJ/mol) was found to be positive, which indicated the endothermic nature of adsorption process. The value of ΔS0 (134.85 J/(mol K)) was found to be positive due to the exchange of protein molecules with more mobile ions present on the PAPGNC, which would cause an increase in entropy, during the adsorption process.63 The values of ΔG0 were found to be −2.23, −3.58, −4.93, and −6.27 kJ/mol for the adsorption process at 10, 20, 30, and 40 °C, respectively. The negative values of ΔG0 confirm the feasibility and spontaneity of the adsorption of Hb onto PAPGNC. Also, 0

Figure 11. Plot of ln Ce for the adsorption of Hb as a function of 1/T. 11025



Article

increased randomness at the solid−liquid interface, respectively. The adsorbent can be regenerated by treating with 0.1 M KSCN without a significant loss in adsorption efficiency. In the near future, PAPGNC can be developed as a valuable cation exchanger for the recovery of Hb from aqueous solutions.

KOH, HCl, HNO3, NaOAc, HCOOH, KSCN, and sodium lauryl sulfate, and the desorption percentage was found to be 88.3, 82.1, 84.6, 71.7, 78.5, 93.4, and 85.2, respectively. The results demonstrated that the adsorbed proteins could be effectively desorbed from the spent adsorbent using 0.1 M KSCN. KSCN is a widely accepted desorbing agent for the recovery of valuable proteins,54,65 because it retains the structural activity of proteins.65 For obtaining the versatility of the PAPGNC, the adsorption−desorption experiments were repeated for four cycles using 0.1 M KSCN. During the four cycles, the adsorption capacity of PAPGNC was found to be increased from 94.1 to 88.6%, whereas the desorption capacity decreased from 93.4 to 86.3% (Figure 12). This result showed

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +91 471 241 6472. Fax: +0 471 230 7158. E-mail: tsani@ rediffmail.com. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank the University Grants Commission, Government of India, New Delhi, for financially supporting this research under Major Research Project MRP F. No. 37425/2009 (S.R.R.).

■

REFERENCES

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Figure 12. Adsorption−desorption cycles of Hb using 0.1 KSCN as the desorbing agent.

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4. CONCLUSIONS Cellulose-based superabsorbent composites are biocompatible and biodegradable materials that show promise for a number of industrial uses, especially in biomedical applications. The present study concentrated on the synthesis and characterization of a novel superabsorbent composite of cellulose, poly(acrylic acid)-modified poly(glycidylmethacrylate)-grafted nanocellulose (PAPGNC), for the recovery of Hb from aqueous solutions. The adsorption capacity of PAPGNC for the adsorption of Hb was tested by a batch adsorption technique. The pH 6.7 was found to be optimum for the adsorption of Hb on PAPGNC. The kinetics of adsorption of Hb onto PAPGNC can be better described with the help of a pseudo-second-order kinetic model, which follows an ion exchange mechanism. The good agreement of isotherm data with the Langmuir isotherm model confirmed a monolayer coverage process. The adsorption capacity for Hb follows the endothermic nature of adsorption. The Gibbs free energy possesses negative values for all interactions, indicating the spontaneous nature of adsorption. The positive values of ΔH0 and ΔS0 indicate the endothermic nature of adsorption and 11026



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Investigation of the Extraction of Hemoglobin by Adsorption onto

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