Article pubs.acs.org/IECR
Chloramphenicol Degradation in Fenton and Photo-Fenton: Formation of Fe2+-Chloramphenicol Chelate and Reaction Pathways Ardhendu Sekhar Giri and Animes Kumar Golder* Department of Chemical Engineering, Indian Institute of Technology, Guwahati, Assam 781039, India S Supporting Information *
ABSTRACT: The aim of the present study was to investigate the formation of the Fe2+-chloramphenicol complex (FeCHPLCOM) and to compare the performances of Fenton processes for the degradation of CHPL and the FeCHPLCOM mixture at high concentration. LC-MS spectra evidenced for formation of charge transfer FeCHPLCOM at a 1:1 CHPL-to-Fe2+ ratio. CHPL acted as a bidented ligand coordinated with Fe2+. The IR study uncovered that ligand-to-metal bonding weakened the stretching frequency of both the O−H bond and the −CO group through coordination with Fe2+. The enhanced FeCHPLCOM degradation (13.4% more) was found in a photoassisted Fenton reaction. The asymmetry centers of CHPL with weakly bonded hydrogen atoms of high nucleophilic character were the most preferred sites of HO• attack. CHPL was oxidized through segmentation of the aliphatic chain attached to the benzene ring under acidic conditions (pH 3). The toxicity of CHPL to Escherichia coli (E. coli) XL 10GOLD decreased significantly after 45 min of decomposition.
1. INTRODUCTION The incidences of antibiotic resistant bacteria have been increased primarily due to high drug administration without proper supervision.1 Several studies have reported the occurrences of pharmaceutical residues especially in the last two decades.2,3 The contamination typically in the range from ng/L to μg/L is considered as a potential threat to the environment.4 Chloramphenicol (CHPL) is a broad-spectrum antibiotic exhibiting the activity against both Gram-positive and Gram-negative bacteria. It imparts the action through inhibition of microorganism protein synthesis. Antibiotics act as a ligand for the formation of a ligand-tometal charge transfer (LMCT) complex.5 3d-transition metals could coordinate through carbonyl and hydroxyl groups of ligands such as CHPL. Wahed et al. (2008) observed formation of a purple complex on reaction of Cu2+ with CHPL in an alkaline aqueous solution (pH 13).6 It is actually a complex anion with a metal-to-ligand ratio of 1:2. Fazakerley et al. (1973) found that the Cu2+-CHPL complex is quite stable in both aqueous and methanol solutions.7 The complexation of Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+ with CHPL occurred at around pH 7. It is prudent from the earlier studies that CHPL can act as a bidentate chelating ligand for selective coordination with Fe2+ in an aqueous solution. The redox reaction in the formation of iron complexes promoted by LMCT is an important source of Fe2+ in the Fenton process.8 Hence, faster Fe2+ to Fe3+ interconversion has a significant role on the degradation of the contaminants.9 Puttaswamy et al. (2009) suggested that the improvement of degradation depends mainly on the following: (i) higher absorption coefficient of iron organic complexes than free iron ions, (ii) high quantum yield of Fe2+ generation, and (iii) generation of the CO2•− radical.10 Aqueous effluent laden with CHPL gives relatively higher COD in comparison to its BOD values i.e. it shows up a lower BOD5/COD ratio.11 The efficiency of the biological processes © 2014 American Chemical Society
for the treatment of wastewater laden with CHPL is ordinarily inhibited.12 Likewise, an investigation was performed to uncover the Fe2+-CHPL complex (FeCHPLCOM) formation in terms of UV−vis spectroscopy, HPLC, FTIR, and LC-MS analyses. The performance of Fenton (FP) and photo-Fenton processes (PFP) for the degradation of CHPL and FeCHPLCOM was investigated. To identify the degradation products, LC-MS spectra were acquired when CHPL decomposition was found to be almost invariant. The proposed mechanism showed the routes of CHPL decomposition with low errors (g/moL) determined from the difference between the proposed and extract masses. Furthermore, the antimicrobial activity of CHPL and its degradation products were evaluated using E. coli.
2. MATERIAL AND METHODS 2.1. Materials. CHPL (purity >99% w/w) was obtained from Sigma-Aldrich (China). The chemical structure is shown in Figure S1 of the Supporting Information. Oxalic acid (98% w/w purity) and acetonitrile (99% v/v purity) of HPLC grade were procured Merck (India). Ferrous ammonium sulfate heptahydrate (99% w/w purity), sulfuric acid (98% v/v purity), H2O2 (50% v/v purity), NaOH (purity >98% w/w), and ethanol (purity >99.9 v/v %) were purchased from Merck (India). Escherichia coli (E. coli) XL 10GOLD (purity >98% w/ w) was collected from the Department of Biotechnology, Indian Institute of Technology Guwahati. Yeast (99% w/v purity) and tryptone (98% v/v purity) were from Himedia (India). Mili-Q water (model: Elix 3, M/s Millipore, USA) was used to prepare all reagent and solutions. Received: Revised: Accepted: Published: 16196
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2.2. Analytical Procedure. UV−vis spectra were acquired by employing a spectrophotometer (model: UV-2300) of Thermo Scientific (India) with an optical path length of 1 cm. High performance liquid chromatography (HPLC) was used for the determination of CHPL concentration. A C18 column with 4.6 i.d. mm and 25 mm in length (5 μm particle size) was employed. An HPLC instrument (model: LC-20AD) of Simadzu (Japan) equipped with an UV−vis detector was used. The mobile phase was a mixture of oxalic acid (0.01 M) and acetonitrile (60:40 v/v) at a flow rate of 0.4 mL/min. The identification of CHPL fragments was carried out in a liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) system (Waters Q-Tof Premier & Aquity UPLC). The characteristic stretching frequencies of CHPL and the Fe2+CHPL mixture were recorded using Fourier Transformed Infrared Spectroscopy (FTIR) (model: IR affinity-1, Shimadzu, Japan) in the range from 4500 to 500 cm−1. A total organic carbon (TOC) analyzer (model: 1030C Aurora) of O.I. Analytical (USA) was employed for measurement of TOC by a nondispersive infrared method. Solution pH was read using a pH meter (model: pH/ion 510) of Eutech Instruments (Malaysia). The titanium sulfate method was used to determine the residual concentration of H2O2.13 Ferrous iron was measured by the 1,10-phenanthroline method.14 A calibration curve was prepared for the determination of concentration of FeCHPLCOM with 100 mg/L CHPL at different concentrations of Fe2+ ranging from 0.5 to 2.0 mM. The complexation reaction was allowed for 10 min. The concentration of free CHPL was determined from its calibration curve without iron in solution corresponding to the retention time of 7.8 min. The concentration of FeCHPLCOM was found out from the mass balance, and a calibration equation was generated against a peak area at 4.3 min of retention time. The antimicrobial activity of CHPL and its degradation products were evaluated by recording the number of colony forming units (CFU) of E. coli bacteria. It was inoculated in Luria−Bertani (LB) media (yeast extract 5, tryptone 10, and NaCl 10 are in g/L). The media pH was adjusted to 7.2 using 1 M NaOH and autoclaved (model: 7407PAD, Medica Instrument Mfg. Co., India) for 20 min at 15 lbs pressure (121 °C). 50 μL of initial cultured E. coli was added to 5 mL of media and incubated for 24 h at 37 °C on an orbital shaker at 180 rpm (model: ORBITEK, Scigenics Biotech, India). The growth media with harvested cells analyzed into 3 parts. One mL of diluted growth media was added on the agar plate,15 and the number of CFU was counted after 24 h of incubation at 37 °C. The second sample was diluted at different proportions, and the absorption intensity was recorded at 621 nm. A calibration curve was prepared by plotting the number of CFU/mL against the absorption intensity. The third sample was centrifuged at 2000 rpm for 15 min. The supernatant was discarded, and the harvested bacterial cells were washed in deionized water. It was then suspended in 50 mL of LB media and further diluted to have CFU/mL of ∼2 × 108. One mL of media and 5 mL of a CHPL sample withdrawn at different times was mixed and incubated for 24 h at 37 °C.16 The corresponding absorbance was recorded and converted to CFU/mL from the calibration curve. 2.3. Experimental Procedure. Iron is susceptible to complex with a lot of compounds. It is necessary to uncover the performance of PFP over FP when such iron-organic complexation is significant. This is one of the reasons that initial
CHPL concentration of 100 mg/L (TOC 31.3 mg/L) was taken to have higher concentration of FeCHPLCOM. Usually, such high CHPL concentration is not found in municipal wastewater treatment plan. Trovo et al. (2013) worked with CHPL concentration of 214 mg/L to evaluate the degradation kinetics, antimicrobial activity, and its toxicity.17 Batch experiment was conducted for the degradation study with continuous stirring. A 1000 mL capacity cylindrical borosilicate vessel (Ø 10.5 cm) was used as the reactor. 0.05 N H2SO4 was used for the adjustment of solution pH prior to addition of catalyst. Fe2+ ([Fe(NH4)2(SO4)]·7H2O) was added and mixed for about 10 min at 260 rpm on a magnetic stirrer (model: Spinot 6020; stirring bar: Ø 0.8 mm, length 40 mm) of Tarson (India), and solution pH was recorded. After that H2O2 was added in a desired amount to the CHPL/Fe2+ solution to obtain the final volume of 400 mL. Ten mL of sample was withdrawn, and 1 mL of 0.1 (N) NaOH was added instantaneously to stop the reaction. NaOH addition increased pH at around 12.4. Conversion of Fe2+ to Fe3+ is extremely fast at such high pH.18 Sludge was separated out by centrifugation at 2000 rpm for 30 min, and clear supernatant was heated at 70 °C to destroy residual H2O2, if any.8 80 μL and 5 mL of clear liquid were taken out for the determination of CHPL and TOC concentrations, respectively. The remaining liquid was further filtered using 0.45 μm cellulose filter. About a 2 mL sample was required for the determination mass spectra, and just one drop was used for FTIR analyses. PFP was performed in a similar manner under UV light irradiation. An UV lamp (wavelength: 362 nm, intensity: 12 W/m2) obtained from Hong Kong Jie Meng International Lighting Ltd. Company (China) was employed for this work. The intensity of UV light was selected based on the earlier studies.3,18 The UV lamp was fixed just on the top of solution at about 5 cm above. The solution temperature was controlled with proper cooling arrangement (25 ± 2 °C).
3. RESULTS AND DISCUSSION 3.1. Formation of the Fe2+-CHPL Complex (FeCHPLCOM). 3.1.1. Evidences of FeCHPLCOM from UV−Vis Spectra. The absorption spectra of CHPL and FeCHPLCOM showed three necessary maximum peaks (λmax) at 213, 263, and 389 nm (Figure S2 of the Supporting Information). The band at 213 nm may be attributed to π-π* transition. While the other two bands at 263 and 382 nm can be assigned to n-π* transitions of O−H and CO groups of free CHPL ligand.6 These bands slightly shifted to lower absorbance and shorter wavelength (blue shift). It signifies that O−H and CO groups were involved in complexation with Fe2+.19 There was small shifting of both intensities and wavelength with the extent of complexation i.e. with different Fe2+ concentration. 3.1.2. High Performance Liquid Chromatograph and FeCHPLCOM. Pure CHPL appeared in the chromatogram at 7.8 min (Figure S3 of the Supporting Information). A new peak was found at 4.3 min of retention time in the presence of Fe2+. It evidenced for formation of a new compound i.e. FeCHPLCOM. It could easily eliminate proton (H+) due to complexation and is more polar compared to free CHPL molecule. The chromatograph was recorded up to 45 min, but no significant peak was noted. It used 6 orbitals from 4s, 4p, and 4d by accepting electrons from carbonyl oxygen and−OH group attached to C2 in complexation reaction. Both lone electron pairs and one negative ion of bidentate ligands could bond to the central Fe2+ ion. Fe(II) species react with H2O2, 16197
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and HO• is formed via Haber-Weiss reaction mechanism (eq 1).20
experiments were performed at different pH values from 2 to 4 with an arbitrarily selected value of Fe 2+ and H 2 O 2 concentration but with prior experience. After that, the doses of Fe2+ and H2O2 were tuned at the pH value obtained in the trial run. Further, pH was varied 2 to 4 with the best setting of Fe2+ and H2O2 concentration obtained before. Fortunately, pH was the same for both the experiments at varying pH. The results are shown in Figures S8−S10 of the Supporting Information. CHPL decomposition reached a maxima and then fell down when one of the operating parameters was increased keeping other two constant. The highest decomposition obtained is presented in Figure 1. The maximum CHPL
[Fe(II) − Ln] + H 2O2 → [Fe(III) − Ln − 1]+ + OH· + OH‐
(1) +
Fe(III)-ligand complexes and [Fe(III)-Ln‑1] could be photochemically reduced to Fe(II) (eq 2). [Fe(III) − Ln − 1]+ + hν → [Fe(II) − Ln − 2]+ + L‐
(2)
It is clearly evident from the chromatograph that the peak area corresponding to CHPL gradually decreased with an increase in Fe2+ dose at a fixed CHPL concentration (Figure S3). The peak area representing FeCHPLCOM increased at the same time. A linear relation between FeCHPLCOM concentration and peak area was noted at 4.3 min of retention time. Two experiments were conducted to study the possibility of the Fe3+-CHPL complex at pH 3 and 5. The Fe3+ and CHPL mixture was contacted for 10 min, and the mass spectra were obtained. It shows that Fe3+ could not form a complex with CHPL (Figures S4 and S5 of the Supporting Information). 3.1.3. Evidences of FeCHPLCOM from IR Spectra. Infrared (IR) spectra could provide important information for the formation of a chelate complex of CHPL and Fe2+ ions by differentiating the characteristic stretching frequencies. The complexation reaction between Fe2+ and CHPL took place through binding of an oxygen atom of carbonyl and C1 of hydroxyl groups.7 IR spectra recorded are shown in Figure S6 of the Supporting Information. The wave numbers corresponding to different functional groups are summarized in Table S1 of the Supporting Information. The most interesting feature of the IR spectra of the Fe2+-CHPL mixture was the shifting and reduction in the frequencies of the bands. The notable differences were in the regions from 1700 to 1850 and 3200 to 3500 cm−1 for −CO and −OH groups, respectively. The stretching frequencies of both O−H and CO groups in the Fe2+-CHPL mixture gradually reduced with an increase in Fe2+ concentration. The band at 1710 cm−1 in the free ligand can be assigned to CO vibration, and it significantly shifted to 1686 cm−1 in the presence of Fe2+. CHPL behaves as a dibasic acid having two −OH groups, and they lie out of the plane. It seems that probably FeCHPLCOM was formed through attachment of one hydroxyl and one amide CO groups. A free O−H bond increased the stretching absorption at about 3600 cm−1. Fe2+ coordination lengthened and weakened the O−H bond, and so it lowered the vibrational frequency.21 The same explanation is also valid for Fe2+ coordination with the CO group. Kemp et al. (1991) suggested that the presence of any hydrogen-bonding also weakens the stretching frequency of both the O−H bond and the −CO group.21 It created strain on the O−H bond and made a single bond character in the −CO group. 3.1.4. Evidences of FeCHPLCOM from LC-MS Spectra. LCMS spectra of pure CHPL and the CHPL-Fe2+ mixture are shown in Figure S7 of the Supporting Information. Unreacted and protonated CHPL are designated with m/z of 325.26 and 326.37. It is also clear that FeCHPLCOM (m/z 381.2) was formed at a 1:1 Fe2+-to-ligand ratio. Fe(II) can form chelate complexes at Fe(II) to the CHPL ratio of 1:1, 1:2, and 1:3.22 3.2. CHPL and FeCHPLCOM Degradation in FP and PFP. pH, Fe2+, and H2O2 concentrations were varied from 2 to 4, 0.5 to 2 mM, and 5 to 25 mM, respectively, to determine the maximum amount of CHPL degradation. The initial trial
Figure 1. CHPL and TOC removal with reaction time in FP and PFP. Photoreaction with an UV lamp of 9 W. FP and PFP conducted at pH 3.0 with [CHPL]0 = 100 mg/L, Fe2+ = 1.75 mM, H2O2 = 20 mM, and temperature 25 °C.
degradation obtained in PFP under the experimental conditions was selected for Fenton experiments without UV light illumination. PFP gave slightly higher CHPL and TOC reductions. CHPL and TOC removal took place in two distinct rate periods (Figure 1). The transition in both processes were from 2.5 to 10 min for both CHPL and TOC reduction. CHPL removal of 70.1% at 2.5 min increased to 88.4% in 10 min in the case of FP. The same were 80.3 and 93.8% for PFP. H2O2 (20 mM) alone gave around 7.2% CHPL decomposition. Only UV irradiation did not show a notable effect on CHPL degradation. The use of Fe(II) as initial specie together with high initial concentrations of iron and hydrogen peroxide implies a high initial radicals generation. Consequently, the production of additional radicals is not important and that is why Fenton and photo-Fenton exhibited similar decomposition behavior. The irradiation level used (12 W/m2) was medium favoring Fenton’s importance. Moreover, the role of UV was insignificant because some intermediates of CHPL could reduce Fe3+.23,24 There was a slight higher removal in PFP which was confirmed from the repetition of experiments. TOC reduction of around 56−60 and 67−69% were determined at 2.5 and 10 min of oxidation in PFP and FP. The faster initial rate of TOC removal was due to mineralization of amide chain. The second stage was related to the opening and mineralization of substituted benzene ring. 16198
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FeCHPLCOM formation should gradually decrease with increasing pH. 3.3. Degradation Pathways of CHPL. LC-MS spectra were recorded to identify the reaction products (Figures 3a and
Usually, the large molecular weight intermediates are either mineralized or broken to lower molecular weight fragments like oxalic and acetic acids. Kavitha et al. (2004) indicated that carboxylic acids are eliminated quickly in PFP during the first stage of oxidation.25 The faster CHPL and TOC removal also can be corroborated from the residual concentration of H2O2 and Fe2+. It was 1.3 and 45.2% for H2O2 and Fe2+, respectively, after 45 min of Fenton reaction. The results are in well accordance with the literature.25,26 Iron-chelation played an important role on CHPL mineralization. PFP exhibited higher degradation ability of such complexes under UV luminesces. Oxalic acid is formed during Fenton reactions. An iron(III)-oxalate complex gets stability through conjugation with Fe(III) due to availability of a vacant 3d orbital, but it could lose its stability because of UV light absorption.21,25 We did not visualize any sludge formation in PFP. Sludge formed in FP was separated by filtration using a 0.45 μm cellulose ester filter at the end of the experimental run with 1.75 mM Fe2+, 20 mM H2O2, and pH 3. It was washed with distilled water and dissolved in concentrated H2SO4. CHPL and TOC reduction of 3.9 and 1.8% were observed due to adsorption on iron-sludge. About 12.9 mg/L of CHPL was found to form FeCHPLCOM with 1.75 mM Fe2+ at pH. It gave an initial FeCHPLCOM (MW 381.2 g/mol) of 14.9 mg/L. H2O2 was added after 10 min of chelation time, and the variation of CHPL and FeCHPLCOM concentrations is shown in Figure S11 (Supporting Information). The decrease in peak intensities of CHPL and FeCHPLCOM is shown in Figure 2. In FP,
Figure 3. (a) Daughter-ion spectrum of CHPL obtained at 10 min of PFP. Photoreaction with an UV lamp of 9 W. Experimental conditions: [CHPL]0 = 100 mg/L, Fe2+/H2O2 molar ratio = 0.088, pH = 3.0. and temperature = 25 °C. (b) Daughter-ion spectrum of CHPL obtained at 10 min of FP. Experimental conditions: [CHPL]0 = 100 mg/L, Fe2+/H2O2 molar ratio = 0.088, pH = 3.0, and temperature = 25 °C.
3b). The mechanisms of CHPL degradation are proposed and supported from the fragments obtained in mass spectra. CHPL has two asymmetry centers i.e. C1 and C2 with weakly bonded hydrogen atoms. It can be easily abstracted by HO•.12 The presence of an amide group attached to the C2 center increased neucleophilicity of both the centers.22 There were 21 fragments (D1-D21) in mass spectra of FP and PFP. The errors in masses were calculated from the difference of the proposed and exact structural masses of protonated ions. Reasonably low errors were obtained for most of the compounds (Table S2 of the Supporting Information). The degradation mechanisms are presented in Figure 4. D1 was identified in PFP formed with low error (0.06 g/mol) by direct hydroxylation of HO• at the C2 asymmetric center. There were two most possible pathways for the cleavage of D1 as shown in Figure 4a. Path #1 shows the route of formation of D2 and D3 with a proposed mass-tocharge ratio (m/z) of 239.30 and 171.13. Both fragments were
Figure 2. Chromatogram of residual CHPL and FeCHPLCOM found in FP and PFP at 10 min of reaction. Experimental conditions: pH = 3, Fe2+ = 1.75 mM, H2O2 = 20 mM, temperature = 25 °C, and [CHPL]0 = 100 mg/L. Photoreaction with an UV lamp of 9 W.
around 92.7 and 80.7% decomposition of CHPL and FeCHPLCOM was noted in 45 min. It was 95.4 and 94.1% in PFP. Around 2.7 and 13.4% more CHPL and FeCHPLCOM decomposition took place in PFP. Only 2.3% more TOC reduction was found in PFP when both the reagents (Fe2+ and H2O2) were added together in CHPL solution. FeCHPLCOM concentration was around 14.9, 7.3, and 0.63 mg/L at pH 3, 7, and 10, respectively. It is possible as the lone electron pair could be free at acidic pH. However, at neutral or alkaline medium, the hydroxyl group (−OH) attached to the C1 position gets deprotonated. Therefore, the extent of 16199
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Figure 4. continued
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Figure 4. Mechanism of CHPL degradation. (a) Decomposition pathways of D1 molecule with m/z 323.13. (b) Continuation of pathways of D1 cleavage. (c) Decomposition route continued from D10 with m/z 269.13. (d) Pathways of FeCHPLCOM degradation.
identified in PFP. Path #2 presents the formation route of D4 to D7 with m/z of 330.17, 164.97, 111.04, and 279.12, respectively. D2 was yielded by hydroxylation followed by decarboxylation of D1 with formation of dichloromethane. In the presence of two chlorine atoms at the geminal position of the side chain, the carbonyl carbon became slightly neucleophilic in character. There was a tendency for shifting of the lone electron of the −Cl group toward the carbonyl carbon. The attack at the C1 asymmetric center by HO• yielded D3 with m/z 171.13. The appearance of D4 in PFP was via an unstable intermediate with m/z 333.12 from the D1 molecule. D4 exists as a disodium salt in acidic medium (pH < 3). p-Nitrobenzoic acid (D5) was detected in PFP. It was formed from the same intermediate by an electrophilic attack of HO• at carbonyl carbon. D5 could form a stable iron complex (Figure S12 of the Supporting Information) which would inhibit the reaction with peroxide.27 The susceptibility of oxidation of the primary alcoholic group (−CH2OH) of the intermediate with m/z 92.08 originated D6.25 This compound also could form a chelate complex of Fe2+ with two coordination sites i.e. −COOH and −OH (Figure S12). D6 was found only in FP indicating its degradability under UV-luminance.28 Nitrobenzene (m/z = 122.09) was originated from the D5 daughter ion on decarboxylation. D7 was originated from the same daughter ion by intermolecular hydrogen bonding of p-nitrophenol which was yielded by direct hydroxylation at the p-position with respect to the −NO2 group. Intermolecular hydrogen bonding of p-nitrophenol is a weak bonding21 resulting from partial charge separation on oxygen and the H atom in −NO2 and −OH groups. The D7
dimer was only identified in FP. Such a weak H-bond could be easily broken down under UV irradiation.29 CHPL also could be fragmented through the routes as shown in Figure 4b. Sodium salts of oxalic acids could be originated upon degradation of CHPL (Figure 4b). Oxalate has the tendency to form iron-complexes with both Fe(II) and Fe(III) at the pH range of 2−3.30 D8 was formed by homolytic cleavage of the H−Cl bond at the geminal position with release of hypochlorous acid (path #3). The product of the cyclization reaction at the 2,4-position of the aliphatic chain of D8 molecule was D9 (m/z of 221.16). Enhanced cyclization occurred due to an increase in electrophilicity of the geminal carbon atom. Path #4 shows the formation pathways of D10 and D11. The D10 molecule was originated by dehydrogenation from an intermediate product with m/z 119.14. It could chelate through an extensive conjugation with Fe2+ due to a vacant 3dorbital (Figure S12). The stability of such complexes could disappear in the presence of UV light. D11 was yielded by direct hydroxylation at a less substituted double bond center. According to Hoffman’s rule, electrophiles are added at a less substituted double bond of alkene type molecules.31 The formation route of D12 to D16 is presented through path #5. D12 identified in FP was originated by homolytic cleavage of CHPL along with D13 on further hydroxylation of an intermediate with m/z of 161.08. p-Aminobenzyl alcohol (m/ z 122.17) appeared on −NO2 reduction by an aqueous electron.32 It was sequentially hydroxylated to D14 and D15. The D14 fragment was found both in FP and PFP. Ortho (o-) and para (p-) positions became more electron rich centers due to the mesmeric effect of the −NH2 group.33 One additional 16201
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cleaved by an HO• attack at the C2 center. The growth of E. coli was completely inhibited in the presence of 100 mg/L CHPL, and the susceptibility of toxicity was reduced significantly in PFP.
−OH was introduced at the o-position with respect to the −NH2 group of D15. The D16 molecule was in equilibrium with D15. D8 was subfragmented as in path #6 (Figure 4c). An electrophilic attack at the carbonyl carbon of the amide group evidence the appearance of D17. The activity of carbonyl carbon is already discussed. Decarboxylation of the −COOH group of D17 originated the D18 fragment due to the presence of an electron withdrawing group (−NO2). D19 was formed on further dehydrogenation of D18. D20 and D21 were yielded on reduction of −NO2 and substitution of −OH groups with removal of −NH3 (Figure 4c). Figure 4d shows the degradation pathway of FeCHPLCOM. D2 and sodium oxalate were formed by hydroxylation probably during pH adjustment for the termination of the Fenton reaction. Fe(III)-oxalate was mineralized to CO2 in the presence of UV-light.34 Fe(III) to Fe(II) introversion could occur due to redoxidation as supported by the literature.23 Only D11 and D14 were detected in both the oxidation processes even though they could not chelate with iron. The adjacent two −OH groups of these fragments are out of the plane.6 3.4. Antimicrobial Activity of CHPL and Its Decomposition Products. Figure S13 (Supporting Information) shows the toxicity of CHPL and its degradation products during FP and PFP to E. coli in LB media after 24 h of exposure. The growth of E. coli was inhibited almost completely in the presence of 100 mg/L CHPL (0.23 × 107 CFU/mL). The growth of E. coli increased to 8 × 107 CFU/mL in control toxicity assay (LB+deionized water). Liang et. al (2013) showed that the growth of E. coli strain more or less withdrawn in the presence of 32 mg/L CHPL after 24 h of exposure.4 The susceptibility of toxicity to E. coli was more in FP even though there was no significant difference between CHPL and TOC values in FP and PFP. The concentration of unreacted CHPL was 8 mg/L (TOC 9.4 mg/L) in FP, and it was 5.5 mg/L in PFP (TOC 8.8 mg/L). About 55.8 and 35.2% cell death were observed upon 24 h exposure of E. coli in the reaction mixture collected after 45 min of FP and PFP in comparison to the control condition. p-Nitrophenol35 and D15, a benzyl alcohol derivative intermediate,36 are toxic in nature. p-Nitrophenol was identified both in FP and PFP, whereas D15 was originated only in FP.
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ASSOCIATED CONTENT
* Supporting Information S
Tables S1 and S2 and Figures S1−S13 containing additional information on IR frequencies, mass-to-charge ratio of reaction products, CHPL structure, identification of iron-CHPL complex, experimental optimization of CHPL decomposition, formation of iron-chelate complexes, and mass spectra of CHPL as outlined in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +91-361-2582269. Fax: +91-361-258-2291. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully thank the Indian Institute of Guwahati (India), for providing the research fellowship to Mr. Ardhendu Sekhar Giri and the necessary research facilities to the Department of Chemical Engineering and Central Instruments Facility (CIF). We also thank the Department of Biotechnology for supplying the bacteria (E. coli) for the toxicity assay.
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REFERENCES
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4. CONCLUSIONS The reactivity of CHPL toward Fe2+ and its decomposition behavior in Fenton and light assisted Fenton reactions were studied. FeCHPLCOM was formed at a 1:1 CHPL-to-Fe2+ ratio. Chelation took place through binding of an oxygen atom attached to C2 and the carbonyl oxygen of an amide group. Concentration of FeCHPLCOM was calculated by high performance liquid chromatography. Oxidation and reduction of CHPL as well as its degradation products were dependent on the redox cycle of Fe2+ and Fe3+. However, enhanced FeCHPLCOM decomposition was found in PFP. Maximum CHPL removal of 95.4 and 92.7% were noted against mineralization efficiency of 69 and 71% in FP and PFP. Decomposition of FeCHPLCOM was more in PFP with 10.7% higher in comparison to CHPL. A total of 21 intermediates were detected in mass spectra within the mass-to-charge ratio of 100 to 400. The proposed mechanism implies that most of the intermediates were formed by the degradation of a side chain of a CHPL molecule. The aliphatic amide chain was 16202
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Industrial & Engineering Chemistry Research
Article
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