Lead and Chromium Adsorption from Water using L-Cysteine Functionalized Magnetite (Fe3O4) Nanoparticles

L-Cysteine functionalized magnetite nanoparticles (L-Cyst-Fe3O4 NPs) were synthesized by chemical co-precipitation using Fe2+ and Fe3+ as iron precursors, sodium hydroxide as a base and L-Cysteine as functionalized agent. The structural and morphological studies were carried out using X-ray powder diffraction, transmission electron microscopy, dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and UV-Vis spectrophotometric techniques. The zeta potential of bare Fe3O4 and functionalized L-Cyst-Fe3O4 NPs were +28 mV and −30.2 mV (pH 7.0), respectively. The positive surface charge changes to negative imply the presence of L-Cyst monolayer at particle interface. Band gap energy of 2.12 eV [bare Fe3O4NPs] and 1.4 eV [L-Cyst-Fe3O4 NPs] were obtained. Lead and chromium removal were investigated at different initial pHs, contact time, temperatures and adsorbate-adsorbent concentrations. Maximum Cr6+ and Pb2+ removal occurred at pH 2.0 and 6.0, respectively. Sorption dynamics data were best described by pseudo-second order rate equation. Pb2+ and Cr6+ sorption equilibrium data were best fitted to Langmuir equation. Langmuir adsorption capacities of 18.8 mg/g (Pb2+) and 34.5 mg/g (Cr6+) at 45 °C were obtained. Regeneration of exhausted L-Cyst-Fe3O4 NPs and recovery of Pb2+/Cr6+ were demonstrated using 0.01 M HNO3 and NaOH. L-Cyst-Fe3O4 NPs stability and reusability were also demonstrated.


L-Cyst-Fe
Biocompatibility Studies. A549 human lung epithelial cancerous cells were incubated in RPMI 1640 medium with 10% FBS and 1% antibiotic-antimycotic solution 100X in 5% CO 2 at 37 °C. Once cells achieved 90% confluency in T-20 culture flask then moved forward for MTT assay in 96-wells tissue culture treated plate. Initially, 5 × 10 3 cells/well with 10% (FBS) RPMI-1640 were plated in 96-well tissue culture plate and incubated for next 24 h in 5% CO 2 incubator at 37 °C. After achieving of 90% confluency, the media was replaced with 1% RPMI-1640 containing Fe 3 O 4 and L-Cyst-Fe 3 O 4 at different concentration ranges from 10 to 100 µg/mL and again incubated for next 24 h. Then 30 µL of MTT (5 mg/mL in DPBS) were added to each well. The tissue culture plate was kept in dark condition for 4 h in 5% CO 2 incubator at 37 °C to allow the reduction of MTT dye to formazan crystal by living cells. After 4 h, whole media was removed and 200 µL of DMSO added to solubilize crystal. Absorbance was measured at 570 nm with the help of ELISA plate reader. The percentage (%) change in proliferation was calculated on control cells that were not exposed to NPs (i.e., only cells). All experiments were done in triplicate sets.
Adsorption experiments. Adsorption studies for Pb 2+ and Cr 6+ removal by L-Cyst-Fe 3 O 4 NPs were conducted in batch mode. Effect of pH on Pb 2+ and Cr 6+ was investigated at various initial pHs (pH 2.0 to 7.0 for Pb 2+ ) and (pH 2.0 to 8.0 for Cr 6+ ). Solutions pHs were adjusted using 1 M NaOH and 1 M HCl. The effect of contact time and adsorbent dose on Pb 2+ and Cr 6+ sorption were performed at different time intervals [5,10,15,20,25  were filtered using a Whatman no. 42 filter paper. Equilibrium Pb 2+ and Cr 6+ concentrations were measured on AAS. The amount of Pb 2+ and Cr 6+ adsorbed on L-Cyst-Fe 3 O 4 NPs per unit mass was calculated using eq. 1.
Where, q e is the amount of adsorbate adsorbed on per gram of adsorbent (mg/g), C i & C e are the initial and equilibrium concentrations (mg/L) of Pb 2+ and Cr 6+ in solution, V is the volume (L), and W is the weight (g) of the adsorbent.  31 . The sample for analysis was prepared by dispersing pinch of NPs in 15 mL of distilled water. The hydrodynamic radius was obtained from Stoke-Einstein relation (eq. 2).

L-Cyst-Fe
Where, η is the solvent viscosity is, k B is the Boltzmann constant, and T is the absolute temperature. The point zero charge (pHpzc) of L-Cyst-Fe 3 O 4 NPs were obtained from zeta-potential measurements performed on an electrophoresis instrument (model ZC-2000, Microtec, Japan). The samples were prepared by dispersing trace amount of NPs in 20 mL of distilled water followed by agitation for 48 h 32 . The pHpzc value was determined from a plot of initial pH versus pH of the supernatant.

Results and Discussion
Characterization of L-Cyst-Fe 3 O 4 NPs. X-ray powder diffraction (XRD). XRD patterns of synthesized  Where, 0.9 is the value assigned for constant 'K' , which is related to the Miller index of crystallographic planes, λ is the wavelength of X-ray, θ is half of the diffraction angle and β is the angular width (in radians) at half-maximum intensity.
Transmission electron microscopy (TEM). Figure 1  NPs 34 . The broad peak at 300-365 nm is due to Fe 3 O 4 NPs local oxygen vacancies present in the lattice, indicating iron NPs formation 34,35 . Wide peak was observed in the range 470-500 nm, which is attributed due to the formation of pair excitation of magnetically coupled Fe 3+ ions 36,37 . There was a decrease in the peak intensities at 228 and 300-365 nm after L-Cyst functionalization of magnetite. The peaks became wide. This indicated a decrease in electronic excitation state due to the formation of Fe-S bond. The optical band gap energy was determined using the Tauc's relation (eq. 4).
Where, α is the absorption coefficient of NPs at a certain wavelength λ, h is Planck's constant, C is a proportionality constant, Ѵ is the incident light frequency, E g is the band gap energy, and the exponent, n = 1/2 and 2 for direct and indirect band gaps of NPs, respectively. The band gaps of Fe 3 O 4 and L-Cyst-Fe 3 O 4 NPs were obtained by plotting curves between (αhѴ) 2 and hѴ [ Fig. S6(b); Supplementary Information]. The energy band gap (E g ) of Fe 3 O 4 (2.12 eV) was higher than those of bulk iron (2.0 eV) 35 . The band gap further decreased (1.4 eV) after L-Cyst impregnation. This could be possibly due to the hydrophilic nature of L-Cyst that resulted in peak broadening. Band gap widening is because of the striking quantum confinement effect observed in many other semiconducting mate rials that possess delocalized electronic states close to the Fermi level 38 . This confirms that L-Cyst is successfully funtionalized onto magnetite surface. . The expected negative surface charge is due to the presence of carboxyl group above its isoelectric point [5.07] 27 . L-Cyst, like other amino acids, is a zwitterionic molecule 25,27 . Thus, zeta potential of L-Cyst varies with pH 25,30 . L-Cyst is negatively charged due to the presence of carboxyl group [pH < pH pzc (5.07)] and positively charged due to the presence of ammonium group [pH > pH pzc (5.07)] 27 . Thus, at pH 6.0, L-Cyst-Fe 3 O 4 NPs exhibit a negative zeta potential 39 . This again confirmed the successful loading of L-Cyst on magnetite surface.     Fig. 4(a,b). Fig. 3(b). Pb 2+ and Cr 6+ sorption experiments were conducted using 50 mL Pb 2+ or Cr 6+ solution (50 mg/L) and L-Cyst-Fe 3 O 4 NPs (dosage: 1.0, 1.5, 2.0, 2.5 and 3.0 g/L) at optimal conditions [temperature: 25 °C, agitation speed: 200 rpm, equilibrium time: 60 min]. Pb 2+ and Cr 6+ removal increased considerably on increasing the dose (from 1.0 to 3.0 g/L) due to increase in the number of sorption sites 40,46,47 . Pb 2+ removal increased from 45% to 99% on increasing dose from 1.0 to 2.0 g/L. No further Pb 2+ uptake was observed on increasing adsorbent dose to 2.5 and 3.0 g/L. Similarly, Cr 6+ uptake increased from 62% to 96% on increasing dose from 1.0 to 2.0 g/L. Therefore, 2.0 g/L dose was used as optimum dose for further Pb 2+ and Cr 6+ adsorption.  Here, k 1 and k 2 (min −1 ) are the first-order and second-order adsorption rate constants, q e is the amount adsorbed at equilibrium, and q t is the amount of Pb 2+ and Cr 6+ adsorbed at time 't' . Figure 3(d,e) shows the pseudo-first and second-order kinetic plots obtained for Pb 2+ and Cr 6+ adsorption on L-Cyst-Fe 3 O 4 NPs. The slopes and intercepts as calculated from the plots were used to determine the regression coefficient (R 2 ) and kinetic rate constants (k 1 and k 2 ) [eqs 5 and 6]. The kinetic parameters obtained are summarized in Table 1 40,46 .

Effect of L-Cyst-Fe 3 O 4 NPs Dose. The effect of L-Cyst-Fe 3 O 4 NPs dose on Pb 2+ and Cr 6+ uptake is shown in
Cr 6+ and Pb 2+ Sorption Equilibrium Studies. Sorption isotherm models provide information about the adsorption mechanisms and adsorbate-adsorbent interactions. Cr 6+ and Pb 2+ sorption equilibrium data were fitted  Where, b (L/mg) is the Langmuir adsorption constant and q max (mg/g) is the monolayer adsorption capacity of the adsorbent. K F and 1/n are Freundlich constants which correspond to adsorption capacity and adsorption intensity, respectively. All the Freundlich and Langmuir constants are given in Table 2. The Langmuir separation factor (R L ) (eq. 9) can also be used to predict the affinity between the adsorbate and adsorbent.  Table 3. Thermodynamic parameters for Pb 2+ and Cr 6+ adsorption onto L-Cyst-Fe 3 O 4 NPs. Where b is the Langmuir constant and C i is the initial concentration. The value of R L tells the type of isotherm to be irreversible (R L = 0), linear (R L = 1), unfavorable (R L > 1), or favorable (0 < R L < 1) 48 . R L values between 0 and 1 indicate favorable Pb 2+ and Cr 6+ adsorption on L-Cyst Fe 3 O 4 NPs [ Fig. S8(a,b)] . The linear plots of both Langmuir and Freundlich isotherm model of Pb 2+ and Cr 6+ adsorption are shown in Fig. 5(a-d). The Langmuir adsorption equilibrium constants of Pb 2+ and Cr 6+ were obtained from linear plots between C e /q e and C e [eq. 7] [ Fig. 5(a,b)]. The Freundlich equilibrium constants were determined from the plot of ln q e versus ln C e [eq. 8] [Fig. 5(c,d)]. The Pb 2+ and Cr 6+ uptake increased with temperature rise from 25 °C to 45 °C indicating the endothermic nature of adsorption. The adsorption constants and correlation coefficients (R 2 ) of Langmuir and Freundlich models are given in Table 2. The Langmuir equation best fitted the data obtained for Pb 2+ and Cr 6+ adsorption onto L-Cyst-Fe 3 O 4 NPs [ Table 2], thereby indicating a monolayer adsorption of Pb 2+ and Cr 6+ . A maximum adsorption capacity (q e ) of L-Cyst-Fe 3 O 4 NPs was 18.78 mg/g (for Pb 2+ ) and 34.48 mg/g (for Cr 6+ ).
Considering that ΔH does not change much with change in temperature over the temperature range of study, the integration of (eq. 10) results into: Where, K is the thermodynamic equilibrium constant, ΔH° is the standard molar sorption enthalpy at temperature T, R is the universal gas constant (8.314 J/mol K), C is integration constant and T is the absolute temperature (K). The values of K were determined by plotting ln (q e /C e ) against q e [ Fig. 6(a,b)], where q e is the amount of Pb 2+ and Cr 6+ adsorbed and C e is the Pb 2+ and Cr 6+ equilibrium concentrations. The plot of ln K against 1/T [ Fig. 6(c,d)] theoretically yields a straight line allowing calculation of ∆H° and ∆S° from the respective slope (equal to −ΔH°/R) and intercept (equal to ΔS°/R) of eq. 12.
The value of standard Gibbs free energy (ΔG°) was calculated using eq. 13.
C Where, R is the universal constant (8.314 J/mol K) and T is the absolute temperature (K). The values of the thermodynamic parameters are given in Table 3.   However, after Pb 2+ and Cr 6+ uptake on L-Cyst-Fe 3 O 4 NPs, some shifting in the peaks between 1624 and 1401 cm −1 (corresponding to asymmetric and symmetric stretching of COO − and -NH 2 , respectively) were observed. A sharp peak at 1509 disappeared [curve (d, e)]. This could be probably due to binding of Pb 2+ and Cr 6+ with COO − and -NH 2 groups. The peaks between 3410 to 3416 cm −1 weakens after Pb 2+ and Cr 6+ uptake. It may be due to surface precipitation of lead hydroxide and chromium hydroxide that decreased the intensity of O-H stretching frequencies 46 . Figure 7(B,C) shows the SEM-EDX spectra of Pb 2+ and Cr 6+ loaded NPs. Inset Tables in Fig. 7(B,C) summarizes intense EDX peaks of lead and chromium, which clearly confirmed the adsorption of Pb 2+ and Cr 6+ on L-Cyst-Fe 3 O 4 NPs surfaces.

SEM-EDX and mapping.
SEM mapping micrographs of L-Cyst-Fe 3 O 4 NPs before and after uptake of Pb 2+ and Cr 6+ are shown in Fig. 8(a-d). The elemental distribution specifies iron in red, oxygen in fluorescent green, carbon in blue, nitrogen in cyan and sulfur in dark greens [ Fig. 8(a,c)]. The elemental mapping also confirmed the Pb 2+ uptake (in yellow) [ Fig. 8(b)] and Cr 6+ (in cyan) [ Fig. 8(d)]. It is clear, Pb 2+ and Cr 6+ ions are uniformly distributed on L-Cyst-Fe 3 O 4 NPs surfaces [ Fig. 8(b,d)].
Recovery of exhausted L-Cyst-Fe 3 O 4 NPs from aqueous system using a simple magnet is demonstrated in Fig. 8(e). The NPs were readily attracted to the magnet. Therefore, the NPs can easily be recovered using a magnet rather traditional filtration. Regeneration and Stability of L-Cyst-Fe 3 O 4 NPs. The efficiency of NPs was investigated for multiple adsorptions-desorption cycles. Briefly, 3.0 g/L NPs were agitated with 50 mg/L Pb 2+ or Cr 6+ solution (40 mL) for 15 minutes. Equilibrium concentration was measured and the spent NPs were desorbed using 0.01 M NaOH or HNO 3 . The desorbed NPs were dried and reused for another adsorption cycle (Pb 2+ and Cr 6+ separately). This adsorption-desorption procedure was repeated five times [ Fig. 9(a,b)]. The adsorption yield decreased in every cycle. Lead adsorption yield was 83% in first cycle that reduced to 37% in fifth cycle [ Fig. 9(a)]. However, chromium removal yield was slight better. It was 22% in the first cycle that reduced to 15% in the fifth cycle [ Fig. 9(b)]. Almost 40% of the total desorption was achieved in first 20 mL HNO 3 or NaOH for lead [ Fig. 9(c)]. Lead desorption was favorable using an acidic eluent because at low pH, there is a competition between H + ions and metal ions as demonstrated in Fig. 4(a) where the surface FeO − react with H + or Pb 2+ . A 46% and 71% of total chromium desorption was achieved using first 20 mL HNO 3 and NaOH, respectively [ Fig. 9(d)]. Thus, magnetite nanoparticles can be regenerated and reused for Cr 6+ and Pb 2+ removal and recovery.
Furthermore, TEM studies were also performed to check the stability and morphology of spent nanosorbents after five successive adsorption-desorption cycles. Figure S9(b,c) (Supplementary Information) shows the TEM images of regenerated L-Cyst-Fe 3 O 4 NPs retained almost the original morphology after adsorption-desorption cycles. These results suggest that L-Cyst-Fe 3 O 4 NPs has good reusability and stability properties.

Conclusions
Highly stable L-Cyst functionalized magnetite NPs were successfully synthesized by co-precipitation. L-Cyst effectively prevents magnetite NPs from oxidation, aggregation and also increased its biocompatibility. SEM, energy band gap, zeta potential, and hydrodynamic size studies confirmed the successful impregnation of L-Cyst onto magnetite NPs surface. The absence of NH 3 + vibrational peak and shifting of IR bands implied the successful L-Cyst functionalization onto magnetite surface as well as Pb 2+ and Cr 6+ loading. L-Cyst interacts with the NPs via its thiol group and heavy metals via its amino and carboxyl groups. SEM-EDX and SEM mapping images confirmed successful adsorption of Pb 2+ and Cr 6+ onto L-Cyst-Fe 3 O 4 NPs. Sorption equilibrium was reached within 25 min at 25 °C. Langmuir equation best described the sorption equilibrium data. Langmuir adsorption capacities of 34.5 and 18.8 mg g −1 were obtained for Cr 6+ and Pb 2+ , respectively. Sorption dynamics data were best described using pseudo-second-order rate equation. Maximum Pb 2+ and Cr 6+ removal was achieved at pH 6.0 and 2.0, respectively. Thermodynamic studies illustrated that Cr 6+ and Pb 2+ adsorption is endothermic in nature. The manipulation of exhausted L-Cyst-Fe 3 O 4 NPs from aqueous media was also demonstrated. L-Cyst-Fe 3 O 4 NPs stability and reusability were also investigated. L-Cyst-Fe 3 O 4 NPs can be considered as fast, efficient, and biocompatible nano-adsorbent for Cr 6+ and Pb 2+ removal from contaminated waters.