Ind. Eng. Chem. Res. 2007, 46, 439-445
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MATERIALS AND INTERFACES Antibacterial and Adsorption Characteristics of Activated Carbon Functionalized with Quaternary Ammonium Moieties Zhilong Shi, K. G. Neoh,* and E. T. Kang
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Department of Chemical and Biomolecular Engineering, National UniVersity of Singapore, 10 Kent Ridge, Singapore 119260
Activated carbon (AC) was functionalized with two types of quaternary ammonium groups to achieve antibacterial properties. The first route utilized covalently coupled 3-(trimethoxysilyl)propyldimethyloctadecylammonium chloride (QAS) on the AC surface, while the second route employed a polycation, poly(vinyl-N-hexylpyridinium bromide). The successful attachment of these two types of quaternary ammonium was indicated by X-ray photoelectron spectroscopy (XPS) analysis. Both types of functionalized ACs show highly effective antibacterial activities against Escherichia coli and Staphylococcus aureus. Furthermore, the functionalized ACs can be used in repeated antibacterial applications with little loss in efficacy. Using phenol as a model compound, the adsorption capacity of the different ACs was also investigated. The different degrees of decrease in the adsorptive capacity of the two types of functionalized ACs can be related to the changes in the surface area and pore size distribution arising from the different functionalization routes. Introduction Activated carbon (AC) has been widely used in air pollution control and wastewater treatment to remove various pollutants because of its large surface area and high adsorption capacity.1,2 It has diverse applications ranging from filters in gas masks and big ventilation systems3 to water treatment systems of hospital renal hemodialysis care units.4 It has been reported that bacteria which attach to carbon particles are highly resistant to disinfection processes due to biofilm formation, which causes the carbon itself to become a source of bacterial contamination.4,5 Hence, it would be advantageous if the AC also possessed antibacterial activity to kill air- or waterborne bacteria. The preparation of antibacterial ACs has been attempted, and much effort has been devoted to the impregnation of silver or metal oxides in ACs.6-10 Though silver and metal oxides have attractive antibacterial activities, their primary shortcoming is that the particles are easily washed out since they are just deposited on the surface of the AC.8,9 Furthermore, with increasing silver content the specific surface area of the carbon decreases greatly, resulting in reduced adsorption capability.9 A number of quaternary ammoniums are known to exhibit good bactericidal properties.11-17 At the same time, we and other groups have reported that antibacterial properties can be conferred on surfaces of substrates by the covalent attachment of quaternary ammonium with resultant bactericidal activities similar to that of free quaternary ammoniums.12-14,17,18 In this approach, the antibacterial agents will not leach out from the surface, hence providing long-term effectiveness. In this paper, we describe how two types of quaternary ammoniums can be covalently attached to the surface of an AC. The surface chemical compositions of the modified ACs were analyzed by X-ray photoelectron spectroscopy (XPS), and the characteristics * To whom correspondence should be addressed. Tel.: +65 65162176. Fax: +65 67791936. E-mail:
[email protected].
and morphologies of the AC were investigated using surface area and pore size analysis, and scanning electron microscopy (SEM). The antibacterial properties of the functionalized AC against Gram-negative Escherichia coli (E. coli) and Grampositive Staphylococcus aureus (S. aureus) were evaluated. Since the surface functionalization process employed entails changing the nature of the surface of the AC, the issue of whether this results in compromising the adsorption capacity of the AC needs to be addressed. As such, phenol adsorption by the AC before and after the functionalization process was also assessed. Experimental Section Materials. A granular AC (20-40 mesh) purchased from Aldrich was used as the starting material. 3-(Trimethoxysilyl)propyldimethyloctadecylammonium chloride (QAS) was obtained from Dow Corning. Poly(4-vinylpyridine) (PVP) (Mw ) 160 000 g/mol), hexyl bromide, phenol, and all other chemicals and solvents were from Aldrich. Peptone, yeast extract, agar, and beef extract were purchased from Oxoid. E. coli DH5R and S. aureus 25923 were obtained from American Type Culture Collection. Ultrapure water (>18.2 MΩ cm, Millipore Milli-Q system) was used in the experiments. Preparation of Functionalized ACs. The AC was thoroughly washed with water until the washing liquid attained a constant pH. It was then dried at 120 °C for 12 h before being subjected to the functionalization process. An overall scheme of the functionalization process is shown in Figure 1. In a typical experiment, 5.0 g of AC was added to concentrated HNO3 (69 wt % HNO3) at a ratio of 1 g/10 mL to achieve oxidation of its surface. The mixture was stirred for 10 h under reflux. The oxidized carbon was washed with water until no further change in pH could be detected and then dried under vacuum for 12 h at 60 °C, resulting in 4.4 g of oxidized AC, AC-COOH. In the next step, the surface carboxylic acid groups were allowed to
10.1021/ie0608096 CCC: $37.00 © 2007 American Chemical Society Published on Web 12/16/2006
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Figure 1. Schematic representation of the two routes for the functionalization of AC with quaternary ammonium groups.
react with thionyl chloride to generate acid chloride groups. In a typical experiment, 3 g of dry AC-COOH was dispersed in 20 mL of thionyl chloride and the mixture was stirred at room temperature for 4 h. The solid was then separated by filtration and washed with anhydrous CHCl3. Subsequently it was dried under vacuum at room temperature for 6 h, to obtain AC-COCl (2.8 g). Two types of quaternary ammoniums, QAS and poly(vinylN-hexylpyridinium bromide), respectively were used to functionalize the as-prepared carbon surface. For the attachment of QAS, 2 g of dry AC-COCl was allowed to react with 20 mL of glycol in the presence of triethylamine. The suspension was stirred at room temperature for 10 h, and the resulting solid was separated and washed with dry tetrahydrofuran and CH2Cl2. After repeated washings, the solid was dried overnight under vacuum, to obtain AC-OH (1.8 g). Subsequently 1.5 g of AC-OH was reacted with 40 mL of 10 wt % QAS in water at 80 °C for 24 h. QAS possesses a silyl group, which can be covalently bound to the AC-OH surface.13 The resulting solid (denoted as Q-AC) was washed with water and dried under vacuum. For the binding of hexyl-PVP, 2 g of dry AC-COCl and 1 g of 3-bromopropylamine hydrobromide were reacted in 20 mL of dry CH2Cl2 in the presence of triethylamine. The resulting solid (AC-Br) was washed and dried in the same manner mentioned above. Subsequently, 1.5 g of dry AC-Br was placed in a solution of 6 g of PVP in 60 mL of nitromethane/hexyl bromide (10:1, v/v). The reaction mixture was stirred at 75 °C for 24 h, and the resulting solid was washed with acetone and methanol followed by drying under vacuum. Under the conditions used, only a few pyridine groups of the PVP chain are alkylated by the Br of 3-bromopropylamine, with the majority being alkylated by hexyl bromide.19 This functionalized AC will be denoted as P-AC in the subsequent discussion.
Materials and Surface Characterizations. The AC or AC-COOH was mixed with KBr to make test pellet specimens for Fourier transform infrared (FTIR) analysis using a Bio-Rad FTS 135 spectrophotometer. Surface compositions were measured using XPS on an AXIS HSi spectrometer (Kratos Analytical Ltd.) with an Al KR X-ray source (1486.6 eV photons) at a constant dwell time of 100 ms and a pass energy of 40 eV. The anode voltage was 15 kV, and the anode current was 10 mA. The pressure in the analysis chamber was maintained at 7.0 × 10-6 Pa or lower during each measurement. The substrates were mounted on the standard sample studs by means of double-sided adhesive tape. The core-level signals were obtained at a photoelectron takeoff angle of 90° (with respect to the sample surface). All binding energies (BEs) were referenced to the C 1s hydrocarbon peak at 284.6 eV. In the peak synthesis, the line width (full width at half-maximum) of the Gaussian peaks was maintained constant for all components in a particular spectrum. Surface elemental components were determined from peak area ratios corrected with the experimentally determined sensitivity factors and were reliable to within (10%. The elemental sensitivity factors were determined using stable binary compounds of well-established stoichiometries. The surface morphology of the carbons was visualized using a field emission scanning electron microscope (FE-SEM; JEOL, JSM-6700F). The specific surface area (SBET) was determined using a NOVA 3000 BET Analyzer (QuantaChrome) according to the Brunauer-Emmett-Teller (BET) method. Before the adsorption isotherms were obtained, the activated carbon samples were purged with pure nitrogen gas overnight at a temperature of 80 °C to remove any contaminant and moisture that may have been present in the samples. The pore volumes (V’s) were obtained from the volumes of nitrogen adsorbed at a relative pressure (p/p0, where p0 is the saturation
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pressure) of 0.99. The pore size distribution was obtained by applying the density-functional-theory (DFT) method. The amount of N+ immobilized on the ACs was determined by the modified dye interaction method.14,16,17 A 0.1 g sample of Q-AC or P-AC was immersed in a 1 wt % fluorescein (Na salt) solution in water for 10 min with constant shaking, followed by thorough rinsing with water. The ACs were recovered with centrifugation. The stained AC particles were then placed in an aqueous solution of 0.25 wt % cetyltrimethylammonium chloride, and the mixture was shaken for 10 min to desorb the dye. A 0.1 M aqueous phosphate buffer, pH 8.0, was then added in a ratio of 1 part buffer to 9 parts cetyltrimethylammonium chloride solution, and the absorbance of the resultant solution was measured at 501 nm. The amount of dye bound to N+ was calculated on the basis of a standard calibration. The corresponding N+ concentration was then calculated based on the assumption of one dye molecule per seven N+ units.17 This dye was reported to bind to quaternary amino groups only and not tertiary or primary ones.20 The longterm stability of the quaternary groups on Q-AC and P-AC was assessed using the water abrasion test. A 1 g sample of the functionalized AC was stirred in 30 mL of water at 300 rpm and at room temperature. After specific time intervals, the AC was filtered. About 0.1 g of the AC was removed each time, dried overnight under vacuum, and the N+ concentration was determined as described above. The water abrasion experiment was continued using the remaining AC under the same conditions. Determination of Antibacterial Properties of Functionalized ACs. E. coli and S. aureus were cultivated in 50 mL of a 3.1% yeast-dextrose broth (containing 10 g/L peptone, 8 g/L beef extract, 5 g/L sodium chloride, 5 g/L glucose, and 3 g/L yeast extract at a pH 6.8) at 37 °C. All glassware and carbon samples were sterilized in an autoclave at 120 °C for 20 min or with UV irradiation before use. The bacteria-containing broth was centrifuged at 2700 rpm for 10 min, and after the removal of the supernatant, the cells were washed twice with sterile phosphate-buffered solution (PBS), and resuspended in PBS at a concentration of 107 cells/ mL. A 0.1 g sample of ACs (either pristine or functionalized) was immersed in 30 mL of this suspension in an Erlenmeyer flask and shaken at 200 rpm at 37 °C. The control experiment was carried out without AC. The viable cell counts of the bacteria were measured by the surface spread-plate method. At the predetermined time, 1 mL of bacteria culture was taken from the flask and decimal serial dilutions with PBS were repeated with each initial sample. A 0.1 mL drop of the diluted sample was then spread onto triplicate solid growth agar plates. After incubation of the plates at 37 °C for 24 h, the number of viable cells (colonies) was counted manually and the results after multiplication with the dilution factor were expressed as mean colony-forming units (CFU) per milliliter.18,21,22 Antibacterial efficacy in repeated applications was investigated using the same 100 mg of functionalized ACs in consecutive antibacterial assays against E. coli. After one batch, the particles were recovered using a filter. The particles were shaken in water for 30 min and then used for the next batch of assay. Adsorption of Phenol in Aqueous Solution. Experiments for the determination of the adsorption isotherms of phenol were carried out as follows. A known mass of the AC was mixed with aqueous solutions of phenol of different initial concentrations without adding any chemical to control the pH. The suspensions were shaken at 30 °C for 3 days, and the phenol
Figure 2. FTIR spectra of (a) AC and (b) AC-COOH.
solution was then filtered.23 Preliminary kinetic experiments indicated that adsorption equilibrium was reached in less than 3 days for the ACs. The concentration of phenol was analyzed using a UV-visible spectrophotometer (UV-1601, Shimadzu, Japan) at a wavelength of 270 nm. In order to reduce measurement errors, the UV absorption intensity of each equilibrium solution sample was measured three times and the average value was used to calculate the equilibrium concentration based on a standard calibration curve. The adsorbed quantity was determined as the difference between the initial amount of phenol in the solution and the remaining amount after equilibration. The amount adsorbed at equilibrium, qe (mg/g), was calculated using the following equation:
qe )
(C0 - Ce)V W
where C0 and Ce are the initial and equilibrium concentrations of phenol (mg/L), respectively, V is the volume of the solution, W (g) is the mass of the carbon, and qe (mg/g) is the amount adsorbed at equilibrium concentration Ce (mg/L). The adsorption equilibrium data were fitted to the Langmuir equation:
qe )
qmaxbCe 1 + bCe
where qmax is the maximum adsorption capacity and b is the Langmuir constant. qmax can be obtained by linear regression of (Ce/qe) against Ce. Results and Discussion Functionalization of AC. The first step in the functionalization process involves the treatment of the AC with concentrated HNO3 to prepare the AC-COOH sample. The FTIR spectrum of AC-COOH shows a significant increase in the absorbance in the range of 3200-3600 cm-1 compared to that of AC (Figure 2), indicating an increase in hydroxy groups, which may be due to surface carboxylic groups. Furthermore, the appearance of a sharp absorption band at 1380 cm-1 indicates an abundance of carboxyl-carbonate structures.24-27 These features clearly show that the oxidation of the AC by HNO3 generates a large number of carboxyl groups. The carboxyl groups were then activated by thionyl chloride for the subsequent reaction to attach the quaternary ammonium groups. The success of the covalent attachment of the two types of quaternary ammonium on AC can be ascertained by comparing
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Figure 3. XPS wide scans of AC (a), Q-AC (b), and P-AC (c); N 1s core-level spectra of AC (d), Q-AC (e), and P-AC (f).
the XPS spectra before and after the functionalization. Parts a, b, and c of Figure 3 show the XPS wide scans of AC, Q-AC, and P-AC, respectively. The corresponding N 1s core-level spectra of these ACs are shown in Figure 3d-f. The N 1s peak component is not discernible in the wide scan and core-level spectra of AC (Figure 3a,d). In the case of Q-AC (Figure 3b) the appearance of a strong N 1s signal at a binding energy of 400 eV, Si 2p signal at 100 eV, and Cl 2p signal at 200 eV is consistent with the presence of the QAS (structure given in Figure 1). The N 1s core-level spectrum of Q-AC (Figure 3e) has a predominant peak at 401.7 eV attributable to the positively charged nitrogen (N+). In the case of P-AC, the wide scan indicates the presence of N and Br (at 70 eV). However, the wide scan does not offer conclusive evidence for the presence of the desired poly(vinylN-pyridinium bromide) since the reaction of AC-COCl with 3-bromopropylamine would also result in the presence of N and Br on the surface of the AC (see Figure 1). This issue was further clarified from the N 1s and Br 3d core-level spectra. The N 1s core-level spectrum (Figure 3f) can be deconvoluted into a predominant N+ peak at 401.7 eV and an additional peak at a binding energy of 398.5 eV due to )NH- of the pyridine groups which were not quaternized. As the probing depth of the XPS technique is
