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Optimizing electron spin resonance detection of hydroxyl radical in water
Chemosphere 52 (2003) 1797–1805 www.elsevier.com/locate/chemosphere
Optimizing electron spin resonance detection of hydroxyl radical in water Shao-An Cheng 1, Wai-Kit Fung, Kwong-Yu Chan *, P.K. Shen Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China Received 3 July 2002; received in revised form 28 March 2003; accepted 1 April 2003
Abstract The parameters affecting the electron spin resonance (ESR) detection of hydroxyl free radical in water are studied and optimized. The hydroxyl radical is generated by the Fenton reaction with iron (II) ammonium sulfate and hydrogen peroxide reacting in a phosphate buffer using N-tert-butyl-alpha-phenylnitron as the spin trap. The concentrations of Fe2þ , H2 O2 , and phosphate buffer are the parameters studied. The Taguchi method and the orthogonal experiment design were used to evaluate the effects of these parameters on the ESR signal intensity. By the analysis of the signal-tonoise ratio and the analysis of variance, the order of importance of the various parameters on the hydroxyl radical formation is determined for optimal ESR detection of hydroxyl radical. The results will help the development of water purification technologies using hydroxyl free radical as a green oxidant. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Hydroxyl radical; Fenton reaction; Orthogonal experiment design; Electron spin resonance
1. Introduction The hydroxyl radical OH is perhaps the most important free radical in chemistry and biology. There have been much interests for using antioxidants to reduce the occurrence of hydroxyl radical in biological systems. On the other hand, hydroxyl radical has been considered in advanced green oxidation for treating drinking water and wastewater. The hydroxyl radical has an exceptional oxidizing power (Ruppert et al., 1994; Ku et al., 1996; Ito et al., 1998; Andreozzi et al., 1999) and almost all polluting organics and bacteria can be destroyed (Buxton et al., 1988; Ince and Apikyan, 2000). The additional attractive feature for drinking
*
Corresponding author. Tel.: +852-2859-7919; fax: +8522857-1586. E-mail address: [email protected] (K.-Y. Chan). 1 Permanent address: Department of Chemistry, Zhejiang University, Hangzhou 310027, China.
water treatment is that the short life time of the radicals leaves no residue effects compared to other oxidants like chlorine or ozone. The most well known generation of hydroxyl radicals in aqueous solution is the FentonÕs reaction (Fenton, 1894; Puppo, 1992; Yoshiki et al., 1995) or the Fenton-like reaction (Pignatello, 1992). Productions of hydroxyl radicals from ozone or hydrogen peroxide with ultraviolet radiation, and from mixing ozone with hydrogen peroxide have also been reported (Guittonneau et al., 1990; Ito et al., 1998). Investigations to maximize the production of hydroxyl radical for advanced green oxidation have been hindered by the technical difficulty of detecting and quantifying the elusive and short lived free radical. The detection of hydroxyl radical in biological experiments is not a routine technique and has also been a focus of many recent studies. The life time of a hydroxyl radical is ca.109 s (Janzen et al., 1992). It can only be detected indirectly. The detection methods include the electron spin resonance (ESR)-spin trapping method (Yamazaki and Piette, 1990; Yamazaki et al., 1990; Stadtman and
0045-6535/03/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00369-2
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Berlett, 1991; Ueda et al., 1996; Mizuta et al., 1997; Ma et al., 1999 and references therein), chemical probe method (Lindsey and Tarr, 2000), high-performance liquid chromatography (HPLC) (Fukui et al., 1993; Brillas et al., 1998; Ito et al., 1998), Fourier transform infrared spectroscopy (FTIR) (Carr and Baird, 2000), gas chromatography (GC) (Bremner et al., 2000) and mass spectroscopy (MS) (Oturan et al., 2000). In addition, more indirect methods of estimating the relative amount of OH by monitoring the concentrations of other reactants and products have been used (Kochany and Lipczynska-Kochany, 1992). Electron spin spectroscopy is the most definitive method to detect free radicals (Janzen et al., 1992; Utsumi et al., 1994; Han et al., 1998) since the characteristic unpaired electron is probed. Spin traps such as nitrones or nitroso compound such as 5,5-dimethyl-1pyrroline-1-oxide (DMPO) and phenyl-tert-butylnitrone (PBN) are used to react with OH to generate a long lived nitroxide free radical (spin adduct) which is then detected by ESR spectroscopy. The reaction of PBN to trap a hydroxyl radical is given as (Janzen et al., 1992): OH C N H O
+
CMe3
+
H O
C N CMe3 H O
ð1Þ Even with the use of a spin trap to extend the time frame for experimental detection, the ESR detection of OH is much influenced by many factors. Besides the factors affecting its generation, scavengers in the media can strongly affect the detection of OH. It will be useful to analyze and optimize the conditions of ESR detection of OH in standard systems to help deploying the technique for various applications. The Fenton reaction is the obvious candidate for such optimization studies. In the Fenton reaction, hydrogen peroxide is converted to hydroxyl radical in the presence of iron (II) ion. The efficiency of the hydroxyl radical production is sufficiently high for some practical applications, but is affected by pH, ferrous ion concentration, hydrogen peroxide concentration, iron chelate such as ethylenediaminetetraacetate (EDTA), and other scavengers and impurities. Several studies have reported the effects of these parameters on the Fenton reaction with ESR or other detection methods (Yamazaki and Piette, 1990; Yamazaki et al., 1990; Stadtman and Berlett, 1991; Sawer et al., 1993; Zhao et al., 1994; Ueda et al., 1996; Mizuta et al., 1997; Ma et al., 1999; Lindsey and Tarr, 2000). Using ESR detection, Yamazaki and Piette (1990) reported that the efficiency of OH generation decreases with increasing Fe2þ concentration over 1 lM when the concentration of hydrogen peroxide was maintained constant at 90 lM. Mizuta et al. (1997) also observed
that the efficiency for generation of DMPO-OH gradually decreased when the Fe2þ concentration increased to 2.5 lM. Ma et al. (1999) studied the generation of the hydroxyl radicals by the Fenton reaction in the PBS (KH2 PO4 –NaOH) buffer solution in the presence of ascorbic acid. However, almost all studies focus on the low substrate concentrations (mostly below 1 mM). The OH generation and consumption involve complex mechanisms with parallel and serial reaction steps. In the Fenton reaction, while more OH may be generated in high concentrations of Fe2þ and H2 O2 , the reactants also serve as scavengers to eliminate OH produced. It will be of practical interests, e.g. degradation of pollutants, whether high level of OH can be generated and detected at high concentration of reactants. Investigations of ESR detection of OH with higher Fe2þ and H2 O2 concentrations are warranted. In this paper, we report ESR detection of OH produced by Fenton reaction under an extended range of Fe2þ and H2 O2 concentrations. Effects of experimental conditions including the phosphate buffer concentration and EDTA chelate concentration on the hydroxyl radical detection are investigated with the help of the Taguchi method and the orthogonal design of experimental parameters. In addition, the complex mechanism of OH generation and consumption is discussed in light of the results of the orthogonal design of ESR detection.
2. Experimental 2.1. Materials and methods Ferrous ammonium sulfate (ACS reagent), PBN (98%), hydrogen peroxide 50% w/w solution, EDTA disodium salt (LR), potassium dihydrogen phosphate (ACS reagent) and disodium hydrogen phosphate (AR) are from Aldrich or Sigma Co. and used directly without further purification. All other chemicals are of analytical quality. All solutions were prepared using 18.2 MX deionized water produced with the Millipore MilliQ system. PBN, hydrogen peroxide and iron (II) solutions were freshly prepared in each experiment. A phosphate buffer (K2 HPO4 + NaH2 PO4 , pH 6.865 at 25 °C) was prepared according to the British standard. In each experiment, solution A was prepared by mixing 1 ml 0.1 M PBN with 1 ml H2 O2 of the required concentration, and solution B was prepared by mixing Fe2þ solution of the required concentration with or without EDTA and phosphate buffer in fixed concentrations. The Fenton reaction was initiated by adding 1 ml of solution B to solution A. After mixing, the concentration of PBN was 33.3 mM and the concentration of H2 O2 , Fe2þ , EDTA and phosphate buffer were changed to the required final concentrations. A 0.1 ml volume of the mixed solution
S.-A. Cheng et al. / Chemosphere 52 (2003) 1797–1805
was rapidly injected into the ESR quartz sample tube and ESR measurements were performed immediately.
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formation. All experiments are conducted at room temperature.
2.2. ESR measurement 3. Results ESR measurements were performed on a Bruker EMX100 ESR spectrometer with 100 kHz modulation at room temperature. ESR spectrometer settings were as follow: modulation amplitude, 2.00 G; receiver gain, 5.02 104 ; time constant, 5 ms; sweep time, 84 s; scan cycle: seven cycles; microwave with frequency, 9.76 GHz and power, 20 mW. The concentration of PBN was 33.3 mM for all experiments. Under these conditions, a stable ESR signal can be obtained for all experiments. Though the hydroxyl radical formation through Fenton reaction is affected by many parameters such as concentrations of additives, pH, and temperature, the main parameters must be the concentration of Fe2þ and the concentration of H2 O2 . Since EDTA is added to form a chelate complex with Fe2þ , and that phosphate buffer affects the pH of the solution, we decide to study the effects of four parameters: concentration of Fe2þ , concentration of H2 O2 , concentration of EDTA, and concentration of phosphate buffer on the hydroxyl ESR signal. Firstly, we study the effect of EDTA concentration at constant concentrations of Fe2þ , H2 O2 and phosphate buffer. Considering that the effect of EDTA should be incorporated in the Fe2þ concentration, an orthogonal array experiment is constructed to assess the importance of various parameters. An orthogonal array experiment (Taguchi experiment) (Hicks, 1993) with variation of three experimental parameters is constructed. Each parameter has three selected levels and an L9 (34 ) orthogonal array comprised of three columns and nine rows constructed are shown in Table 1. In Table 1, A, B, C indicate the concentration of Fe2þ , the concentration of H2 O2 , and the concentration of buffer solution, respectively. The signal peak height of ESR, related to the relative amount of the hydroxyl radicals formed, is used to evaluate effects of the experimental conditions on the hydroxyl radical
3.1. Effect of EDTA A typical measured ESR signal of the hydroxyl adduct created in the Fenton reaction with PBN spin trap is shown in Fig. 1. The spectrum is composed of three pairs of peaks with the hyperfine splitting constants aN ¼ 16:23 G, aH ¼ 2:54 G. They give the characteristic finger print of the PBN–OH adduct. The difference between the signal intensity at 3456.25 G and that at 3460.35 G was taken to represent the signal height and the concentration of the hydroxyl–PBN adduct. Concentrations of iron (II) salt, hydrogen peroxide, EDTA and phosphate buffer have been varied. The parameters of ESR scanning are kept constant for all measurements. Generally, EDTA is added to form a complex with iron (II) and iron (III). The EDTA–Fe2þ complex prevents iron (II) from binding to the hydroxyl radical and hence promote the H2 O2 decomposition and hydroxyl radical production. On the other hand, adding EDTA increases the solubility of iron (III) oxide and promotes the iron redox cycling (Blesa et al., 1994). The EDTA– Fe3þ can also react directly with H2 O2 to form OH (Gutteridge and Bannister, 1986; Aruoma et al., 1989). Excess EDTA will also scavenge the hydroxyl radical (Walling et al., 1975; Zhao et al., 1994). The [EDTA]/ [Fe2þ ] ratio must be optimized for maximum hydroxyl radical production detection. So, our preliminary experiment was conducted under fixed concentrations of iron (II) salt, hydrogen peroxide but with different EDTA concentrations to determine the optimum [EDTA]/ [Fe2þ ] ratio with or without phosphate buffer solutions. Fig. 2 shows the variation of signal intensity of ESR with the concentration of EDTA in 0.33 mM iron (II) salt, 0.12 mM hydrogen peroxide solution without phosphate buffer, and in 0.33 mM iron (II) salt, 0.12 mM
Table 1 The measured ESR signal height and the calculated S/N ratio of each designed experiment Experiment no.
A ([Fe2þ –EDTA]) (mM)
B ([H2 O2 ]) (mM)
C ([phosphate]) (mM)
Signal intensity
S/N ratio
1 2 3 4 5 6 7 8 9
0.1 0.1 0.1 0.3 0.3 0.3 2 2 2
0.1 1 11 0.1 1 11 0.1 1 11
0 0.8 4 0.8 4 0 4 0 0.8
1299 1365 2274 3553 8609 3074 1528 6174 10 689
62.3 62.7 67.1 71.0 78.7 69.8 63.7 75.8 80.6
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3.2. Analysis of the Taguchi experiment
3440
3460
3480 Value G
3500
3520
Fig. 1. A typical ESR spectrum of hydroxyl radical generated from Fenton reaction and trapped by PBN.
The Taguchi method, developed by Taguchi (1990), is generally used to optimize a process or product by a special design of orthogonal arrays of a small number of experiments. The experimental results are analyzed using the signal-to-noise (S/N) ratio and ANOVA analyses to determine the optimal design parameters and estimate the contribution of each design parameter to a certain characteristic. The S/N ratio, has a different meaning from that in analytical chemistry. The term ÔsignalÕ represents the desirable value for an output characteristic and the term ÔnoiseÕ represents the undesirable value for the output characteristic. Usually, there are three categories of the characteristic in the analysis of the S/N ratio, viz. the-lower–the-better, the-higher– the-better, and the-normal–the better. For all categories, a greater S/N ratio is better. The optimal level of the design parameters is the level with the greatest S/N ratio. ANOVA is a statistical analysis of the variance and gives information about which process parameter is statistically significant. More details about Taguchi method can be available in the literature (Yang and Tarng, 1998). 3.3. Analysis of the S/N ratio
Fig. 2. The effect of EDTA concentration on the ESR signal intensity in 0.33 mM iron (II) salt, 0.12 mM hydrogen peroxide solution (N) without phosphate buffer, ( ) with 0.8 mM phosphate buffer. The connecting lines are used to show the trend of data.
hydrogen peroxide solution with 0.8 mM phosphate buffer. It can be observed that as the EDTA concentration increases the signal intensity of ESR increases in the low concentration range and then decreases in the high concentration range with a sharp maximum at [EDTA]/[Fe2þ ] ¼ 1–1.2 for both cases. So, the [EDTA]/ [Fe2þ ] ratio for maximum hydroxyl radical production detection should have above value. Similar results of a linear relationship are reported (Zhao et al., 1994) for low concentrations of hydrogen peroxide and Fe2þ – EDTA in the present of deoxyribose. In there (Zhao et al., 1994), however, the amount of hydroxyl radical was only estimated indirectly by analyzing the oxidation product of deoxyribose and ESR experiments were not conducted.
Nine experiments were conducted according to the Taguchi method (Hicks, 1993) based on the arrangement of the orthogonal array in Table 1. The EDTA concentration is always kept the same as Fe2þ Õs in the orthogonal array of experiments. The experimental signal intensities obtained, having the higher the better properties, were converted to a S/N ratio according to the following equation: " # n X S=N ¼ 10 log n1 ðgi Þ2 ð2Þ i¼1
where n and gi are the degree of freedom (n ¼ 9) and the ESR signal intensity, respectively. The values of S/N ratio are listed in Table 1. Since the experimental design is orthogonal, it is then possible to separate out the effect of each parameter at different levels. For example, the mean S/N ratio for the Fe2þ concentration at levels 1, 2 and 3 can be calculated by averaging the S/N ratios for the experiments 1–3, 4–6 and 7–9, respectively. The mean S/N ratio for each level of the other parameters can be computed in the similar manner. Table 2 shows the mean S/N ratios and Fig. 3 shows the variation of mean S/N ratio with the parameter level for each designed parameter. The total mean S/N ratio for nine experiments is also calculated and shown in Table 2 and Fig. 3. S/N ratio can gives us the optimal levels of design parameters with the greatest S/N ratio. From Table 2 and Fig. 3, it can be seen that the A3, B3 and C2 is the
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Table 2 The mean S/N ratio and the difference between the maximal mean S/N ratio and the minimal mean S/N ratio for each level of the designed parameters Symbol
Parameters
A B C
[Fe2þ –EDTA] [H2 O2 ] Phosphate buffer
Mean S/N ratio Level 1
Level 2
Level 3
Max–min
64.0 65.7 69.3
73.2 72.4 71.4
73.4 72.5 69.8
9.4 6.8 2.1
The total mean S/N ratio ¼ 70.2.
Pt SSp ¼
Fig. 3. Variation of mean S/N ratio with the parameter level for each designed parameter. (N) Parameter A (Fe2þ –EDTA), ( ) parameter B (H2 O2 ), (j) parameter C (phosphate buffer). The subscript represents the level of the parameter, such as A1, A2, and A3 mean the level 1 (0.1 mM), level 2 (0.3 mM), and level 3 (2 mM) of the parameter A, respectively. The broken line represents the total mean S/N ratio.
j¼1 ðsgj Þ
t
2
" #2 m 1 X g m i¼1 i
ð4Þ
where ÔpÕ represents one of the tested parameters, j the level number of this parameter p, t the number of levels for each parameter (here, t ¼ 3), sgj the sum of the S/N ratio involving this parameter and level j, m the number of the experiments. The total degree of freedom (d.f.) is DT ¼ m 1, while the degree of freedom of the tested parameter is DP ¼ t 1. The variance of the tested parameter is VP ¼ SSp =DP . Then the F -value for each design parameter is simply the ratio of mean-of-square deviations to the mean of the squared error (FP ¼ VP =Ve ). The corrected sum of squares Sp can be calculated as: Sp ¼ SSp DP Ve
ð5Þ
The percentage contribution u can be calculated as: uP ¼ Sp =SST
optimal combination of the parameter levels. However, the mean S/N ratio at level 2 is very close to that at level 3 for both parameter A and parameter B, indicating that changes in concentration of Fe2þ and H2 O2 are insignificantly to affect the ESR signal in the high concentration range. 3.3.1. Analysis of variance To investigate which design parameter significantly affect the ESR signal of OH adduct, an analysis of variance (ANOVA) is introduced. This is to accomplished by separating the total variability of the S/N ratios, which is measured by the sum of the squared deviations from the total mean S/N ratio, into contributions by each of the design parameters and the error. The total sum of square SST from the S/N ratio g can be calculated as (Yang and Tarng, 1998): " #2 m m X 1 X 2 SST ¼ gi g ð3Þ m i¼1 i i¼1 where m is the number of the experiment. The sum of squares from the tested parameter SSp can be calculated as:
ð6Þ
The parameter with the bigger uP gives the more significant effect on the ESR signal. Table 3 shows the result of ANOVA. It is found that Fe2þ concentration is the most significant parameter affecting the ESR signal. The change of the phosphate buffer has an insignificant effect on the ESR signal. From the divergence of mean S/N ratios (max–min value in Table 2), it is also observed that the order of the three factorsÕ effect on ESR signal is A > B > C.
4. Discussion Two major mechanisms of Fenton reaction have been suggested in literature. One is the reaction mechanism that hydrogen peroxide is reduced at the iron center with the generation of free hydroxyl radical as follow: Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH II
Fe
ðOH2 Þ2þ 6 þ H2 O2
III
! ½Fe ðOH2 Þ5 OH
ð7Þ 2þ
þ OH þ OH ð8Þ
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Table 3 Results of the ANOVA analysis Symbol
Parameter 2þ
A B C Error
[Fe –EDTA] [H2 O2 ] Phosphate buffer
Total
d.f.
SSp
VP
FP
2 2 2 2
170.0 92.3 7.5 41.7
85.0 46.2 3.7 20.9
4.1 2.2 0.18
8
311.5
Another one is Bray–Gorin reaction mechanism that hydroxyl radical is formed via the formation of an intermediate Fe (IV): Fe2þ þ H2 O2 ! ½FeIV ¼ O 2þ þ H2 O
ð9Þ
½FeIV ¼ O 2þ þ H2 O ! Fe3þ þ OH þ OH
ð10Þ
Other reactions involved in the Fenton system are listed in Table 4. It can be seen from Table 4 that the reactions R1 and R2 are the limiting steps for producing hydroxyl radical. Without Fe2þ or other similar catalysts, the decomposition rate of H2 O2 is very low. When Fe2þ is added, H2 O2 can be easily converted to hydroxyl radical (k ¼ 63). Therefore, Fe2þ is the most significant parameter affecting the ESR signal. The kinetics of OH formation can be schematized as follows (with addition of EDTA): ka
Fe2þ –EDTA þ H2 O2 ! OH
ð11Þ
kb
OH þ S ! D
OH þ P ! P–OH
ð12Þ
kc
ð13Þ
where S represents the scavenging reagents such as H2 O2 in reaction R3, Fe2þ in reaction R6, OH in reaction
uP (%) 54.6 29.6 2.4 13.4 100
R11, HO2 in reaction R13, O 2 in reaction R14 as listed in Table 4, and others such as EDTA and phosphate; ka and kb are the corresponding rate constants; D is the scavenging reaction products; P is the PBN spin trap; and P–OH is the OH adduct. Reaction (13) is the same as Eq. (1). The rate constant (kc ) of the trapping reaction (Eq. (13)) is 6.1 109 M s1 (Sridhar et al., 1984). Using a steady-state approximation commonly applied to transient intermediates such as free radicals (Laidler, 1979), the rate of change of concentration of OH is zero. The rate (m) of the hydroxyl radical adduct formation (Eq. (13)) can be expressed as: m¼
ka kc ½Fe2þ –EDTA ½H2 O2 ½P
kb ½S þ kc ½P
In the scavenging reactions R3, R6, R11, R13 and R14, the rates of reactions R13 and R14 is negligible since the reactions are subsequent reactions of R3 with trace concentrations of radical reactant species. The effect of reaction R11 can be also neglected due to the low concentration of OH. Thus the main scavenging reactions are R3 and R6. The effect of the concentration of Fe2þ – EDTA and H2 O2 on the ESR signal can be deduced from Eq. (14). For example, when the concentration of either Fe2þ –EDTA or H2 O2 is low compared to [P] or
Table 4 The reaction steps involved in the Fenton mechanism Reaction 2þ
3þ
R1. Fe þ H2 O2 ! Fe þ OH þ OH R2. Fe3þ þ H2 O2 ! Fe2þ þ Hþ þ HO2 R3. OH þ H2 O2 ! HO2 þ H2 O þ R4. HO2 ! O 2 þH þ þ H ! HO R5. O 2 2 2þ R6. OH þ Fe ! Fe3þ þ OH R7. HO2 þ Fe2þ ðþHþ Þ ! Fe3þ þ H2 O2 R8. HO2 þ Fe3þ ! Fe2þ þ Hþ þ O2 2þ þ 3þ þ H2 O2 R9. O 2 þ Fe ðþ2H Þ ! Fe 3þ 2þ R10. O2 þ Fe ! Fe þ O2 R11. OH þ OH ! H2 O2 R12. HO2 þ HO2 ! O2 þ H2 O2 R13. HO2 þ OH ! O2 þ H2 O R14. OH þ O 2 ! O2 þ OH þ R15. HO2 þ O ðþH Þ ! O 2 þ H2 O2 2
ð14Þ
k (M1 s1 )
References
63 0.01 2.7 107 1.58 105 1 1010 3.2 108 1.2 106 3.1 105 1 107 5 107 4.2 109 8.3 105 1 1010 1 1010 9.7 107
Metelitsa (1971) Walling and Goosen (1973) Christensen et al. (1982) Bielski et al. (1985) Bielski et al. (1985) Stuglik and Zagorski (1981) Jayson et al. (1969) Rush and Bielski (1985) Rush and Bielski (1985) Rothschild and Allen (1958) Sehested et al. (1969) Bielski et al. (1985) Sehested et al. (1969) Sehested et al. (1969) Bielski et al. (1985)
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other species, the rate (m) of the adduct formation and hence the ESR signal intensity are limited by Fe2þ – EDTA or H2 O2 and proportional to the concentration of Fe2þ –EDTA or H2 O2 . At high concentrations of Fe2þ –EDTA, ½S ½Fe2þ –EDTA , kb ½S kc ½P and Eq. (14) becomes: m¼
ka kc ½Fe2þ –EDTA ½H2 O2 ½P
¼ k 0 ½H2 O2 ½P
kb ½Fe2þ –EDTA
ð15Þ
The rate (m) of the adduct formation is independent of Fe2þ –EDTA concentration. The same result can be deduced for high concentration of H2 O2 . So, in the high concentration, changing concentration of Fe2þ –EDTA and H2 O2 has no significant effect on the hydroxyl radical formation, as evident in Fig. 3 and Table 2. However, in the case that both [Fe2þ –EDTA] and H2 O2 becoming the dominant scavengers, [S] could be proportional to [Fe2þ –EDTA]*[H2 O2 ]. The ESR signal intensity should reach a saturation value. To test the above conclusions derived from the analytical results of orthogonal experiment, further ESR experiments were conducted in the solutions with extended range of H2 O2 and Fe2þ concentrations varied separately. It can be seen from the results in Fig. 4 that the two parameters show a similar trend with signal increasing at low concentrations and saturated at ac. 11 mM for H2 O2 and 2 mM for Fe2þ . The same saturation value was observed. According to the reaction R1 in Table 4, the pH of reaction medium increases as the hydroxyl radical formation proceeds. High OH may hinder the hydroxyl radical formation by equilibrium consideration. Moreover, hydroxide ions can break down the OH–PBN adduct reducing the ESR signal detected (Janzen et al., 1992). Generally, a phosphate buffer was used to maintain the pH at a low level. However, the phosphate concentration must be within a suitable range. Phosphate may not only bind with Fe2þ –EDTA to form a ternary complex, hence decreasing the activity of Fe2þ (Yoshimura et al., 1992; Vella and Munder, 1993), but it can also be a scavenger of hydroxyl radicals (Lipczynska-Kochany et al., 1995) as described in the following reaction:
OH þ HPO2 4 ! OH þ HPO4
ð16Þ
At low concentrations of Fe2þ and H2 O2 , the concentration of EDTA is generally low and a substantial inhibitory effect on the hydroxyl radical formation is observed (Fig. 2 and references: Yoshimura et al., 1992; Vella and Munder, 1993). At high concentration of Fe2þ –EDTA, the phosphate does not affect the hydroxyl radical formation in the concentration range studied (Figs. 2 and 3 and Table 3). These results may be due to
Fig. 4. Effects of the parameter concentration on the ESR signal intensity. (a) In 2 mM Fe2þ , 2 mM EDTA, 33 mM PBN and 0.8 mM phosphate buffer solutions with different H2 O2 concentrations; (b) in 11 mM H2 O2 , 33 mM PBN and 0.8 mM phosphate buffer solutions with different Fe2þ –EDTA concentrations (the concentration of EDTA is the same as that of Fe2þ ).
larger contribution of EDTA to the formation of hydroxyl radical.
5. Conclusions The factors on the ESR detection of hydroxyl radical were studied using the Taguchi method and orthogonal experiment design on the Fenton reaction with a large concentration range of Fe2þ and H2 O2 . The following conclusions can be drawn: 1. A Fe2þ /EDTA ratio 1:1 is more suitable for the formation of hydroxyl radical. 2. The order of importance for the several factors studied are Fe2þ > H2 O2 > phosphate buffer. 3. At sufficiently high concentration or either Fe2þ or H2 O2 , the ESR signal has attained the maximum level and further increase in concentration of Fe2þ
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or H2 O2 has no effect on the ESR signal of the hydroxyl adduct. 4. The optimal condition for the hydroxyl radical formation by the Fenton reaction is: 2 mM Fe2þ , 2 mM EDTA, 11 mM H2 O2 and 0.8 mM phosphate buffer using a PBN concentration of 33.3 mM in this study. The method and the results of this study are useful for ESR study of hydroxyl free radical in various systems, especially for high concentrations of reactants as in the advanced oxidation of drinking water.
Acknowledgements This project was funded by HKU for AoE on Water Environment Engineering. S.A. Cheng acknowledges the support of his PDF on Green Oxidation by University of Hong Kong. References Andreozzi, R., Caprio, V., Insola, A., Marotta, R., 1999. Advanced oxidation processes (AOP) for water purification and recovery. Catal. Today 53, 51–59. Aruoma, O.I., Halliwell, B., Gajewaski, E., Dizdaroglu, M., 1989. Damage to the bases in DNA induced by hydrogen peroxide and ferric ion chelates. J. Biol. Chem. 264, 20509– 20512. Bielski, B.H., Cabelli, D.E., Aruda, R.L., Ross, A.B., 1985. Reactivity of HO2 /O2 radicals in aqueous solution. J. Phys. Chem. Ref. Data 14, 1041–1077. Blesa, M.A., Morando, P.J., Regazzoni, A.E., 1994. Chemical Dissolution of Metal of Oxides. CRC Press, Boca Raton, FL. Bremner, D.H., Burgess, A.E., Li, F.B., 2000. Coupling of chemical, electrochemical and ultrasonic energies for controlled generation of hydroxyl radicals––Direct synthesis of phenol by benzene hydroxylation. Appl. Catal. A: Gen. 203, 111–120. Brillas, E., Mur, E., Sauleda, R., Sanchez, L., Peral, J., Domenech, X., Casado, J., 1998. Aniline mineralization by AOPÕs: anodic oxidation, photocatalysis, electro-Fenton and photoelectro-Fenton processes. Appl. Catal. B: Environ. 16, 31–42. Buxton, G.V., Greenstock, C.L., Helman, W.P., Ross, A.B., 1988. Critical-review of rate constants for reactions of hydrated electrons, hydrogen-atoms and hydroxyl radicals ( OH/ O ) in aqueous-solution. J. Phys. Chem. Ref. Data 17, 513–886. Carr, S.A., Baird, R.B., 2000. Mineralization as a mechanism for TOC removal: study of ozone/ozone-peroxide oxidation using FT-IR. Water Res. 34, 4036–4048. Christensen, H., Sehested, K., Corfitzen, H., 1982. Reactions of hydroxyl radicals with hydrogen peroxide at ambient and elevated temperatures. J. Phys. Chem. 86, 1588–1590.
Fenton, H.J.H., 1894. Oxidation of tartaric acid in presence of iron. J. Chem. Soc. 65, 899–910. Fukui, S., Hanasaki, Y., Ogawa, S., 1993. High-performance liquid chromatographic determination of methanesulphinic acid as a method for the determination of hydroxyl radicals. J. Chromatogr. 630, 187–193. Guittonneau, S., Delaat, J., Duguet, J.P., Bonnel, C., Dore, M., 1990. Oxidation of parachloronitrobenzene in dilute aqueous solution by O3 + UV and H2 O2 + UV––a comparative study. Ozone Sci. Eng. 12, 73–94. Gutteridge, J.M.C., Bannister, J.V., 1986. Copper and zinc and manganese superoxide dismutases inhibit deoxyribose degradation by the superoxide-driven tenton reaction at 2 different stages––implications for the redox states of copper and manganese. Biochem. J. 234, 225–228. Han, S.K., Ichikawa, K., Utsumi, H., 1998. Quantitative analysis for the enhancement of hydroxyl radical generation by phenols during ozonation of water. Water Res. 32, 3261– 3266. Hicks, C.R., 1993. Fundamental Concepts in the Design of Experiments. Oxford University Press, New York. Ince, N.H., Apikyan, I.G., 2000. Combination of activated carbon adsorption with light-enhanced chemical oxidation via hydrogen peroxide. Water Res. 34, 4169–4176. Ito, K., Jian, W., Nishijima, W., Baes, A.U., Shoto, E., Okada, M., 1998. Comparison of ozonation and AOPs combined with biodegradation for removal of THM precursors in treated sewage effluents. Water Sci. Technol., 38,179–186. Janzen, E.G., Kotake, Y., Hinton, R.D., 1992. Stabilities of hydroxyl radical spin adducts of PBN type spin traps. Free Radical Biol. Med. 12, 169–173. Jayson, G.G., Keene, J.P., Stirling, D.A., Swallow, A.J., 1969. Pulse-radiolysis study of some unstable complexes of iron. Trans. Faraday. Soc. 65, 2453–2464. Kochany, J., Lipczynska-Kochany, E., 1992. Application of the EPR spin-trapping technique for the investigation of the reactions of carbonate, bicarbonate, and phosphate anions with hydoxyl radicals generated by the photolysis of H2 O2 . Chemosphere 25, 1769–1782. Ku, Y., Su, W.J., Shen, Y.S., 1996. Decomposition kinetics of ozone in aqueous solution. Ind. Eng. Chem. Res. 35, 3369– 3374. Laidler, K.J., 1979. Chemical Kinetics. Tata McGraw-Hill, New Delhi, p. 327. Lindsey, M.E., Tarr, M.A., 2000. Quantitation of hydroxyl radical during Fenton oxidation following a single addition of iron and peroxide. Chemosphere 41, 409–417. Lipczynska-Kochany, E., Sprah, G., Harms, S., 1995. Influence of some groundwater and surface waters constituents on the degradation of 4-chlorophenol by Fenton reaction. Chemosphere 30, 9–20. Ma, Z.R., Zhao, B.L., Yuan, Z.B., 1999. Application of electrochemical and spin trapping techniques in the investigation of hydroxyl radicals. Anal. Chim. Acta 389, 213– 218. Metelitsa, D.I., 1971. Mechanisms of hydroxylation of aromatic compounds. Russ. Chem. Rev. 40, 563–580. Mizuta, Y., Masumizu, T., Kohno, M., Mori, A., Packer, L., 1997. Kinetic analysis of the Fenton reaction by ESR-spin trapping. Biochem. Mol. Biol. Int. 43, 1107–1120.
S.-A. Cheng et al. / Chemosphere 52 (2003) 1797–1805 Oturan, M.A., Peiroten, J., Chartrin, P., Acher, A.J., 2000. Complete destruction of p-nitrophenol in aqueous medium by electro-Fenton method. Environ. Sci. Technol. 34, 3474– 3479. Pignatello, J.J., 1992. Dark and photoassisted Fe3þ catalyzed degradation of chlorophenoxy herbicides by hydrogen peroxide. Environ. Sci. Technol. 26, 944–951. Puppo, A., 1992. Effect of flavonoids on hydroxyl radical formation by Fenton-type reactions––influence of the iron chelator. Phytochemistry 31, 85–88. Rothschild, W.G., Allen, A.Q., 1958. Studies in radiolysis of ferrous sulfate solutions, III. Air-free solutions at higher pH. Radiat. Res. 8, 101–110. Ruppert, G., Bauer, R., Heisler, G., 1994. UV–O3 , UV–H2 O2 , UV–TiO2 and the photo-Fenton reaction––comparison of advanced oxidation processes for waste water treatment. Chemosphere 28, 1447–1454. Rush, J.D., Bielski, B.H., 1985. Pulse radiolytic studies of the reactions of HO2 /O2 with Fe(II)/Fe(III) ion. The reactivity of HO2 /O2 with ferric ions and its implication on the occurrence of the Haber–Wiss reaction. J. Phys. Chem. 89, 5062–5066. Sawer, D.T., Kang, C., Llobet, A., Redman, C., 1993. Fenton reagents (1:1 FeII LX /HOOH) react via [LX FeII OOH(BHþ )] (1) as Hydroxylases RH ! ROH, not as generators of free hydroxyl radicals (HO ). J. Am. Chem. Soc. 115,, 5817–5818. Sehested, K., Bjergbakke, E., Rasmussen, O.L., 1969. Reactions of H2 O2 in the pulse-irradiated Fe(II)–O2 system. J. Chem. Phys. 51, 3159–3166. Sridhar, R., Beaumont, P.C., Powers, E.L., 1984. In: Bors, W., Saran, M., Tait, D. (Eds.), Oxygen Radicals in Chemistry and Biology. de Gruyter, Berlin, pp. 101–107. Stadtman, E.R., Berlett, B.S., 1991. Fenton chemistry: amino acid oxidation. J. Biol. Chem. 266, 17201–17211. Stuglik, Z., Zagorski, Z.P., 1981. Pulse radiolysis of neutral iron(II) solutions––oxidation of ferrous-ions by OH radicals. Radiat. Phys. Chem. 17, 229–233. Taguchi, G., 1990. Introduction to Quality Engineering. Asian Productivity Organization, Tokyo. Ueda, J.I., Saito, N., Shimazu, Y., Ozawa, T., 1996. A comparison of scavenging abilities of antioxidants against hydroxyl radicals. Arch. Biochem. Biophys. 333, 377–384.
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Utsumi, H., Hakoda, M., Shimbara, S., Nagaoka, H., Chung, Y.S., Hamada, A., 1994. Active oxygen species generated during chlorination and ozonation. Water Sci. Technol. 30, 91–99. Vella, P.A., Munder, J.A., 1993. In: Technologies in Hazardous Waste Management IIITedder, D.W., Pohland, F.G. (Eds.), ACS Symposium Series, vol. 518, pp. 85–105 (Chapter 5). Walling, C., Goosen, A., 1973. Mechanisms of the ferric ion catalyzed decomposition of hydrogen peroxide, effect of organic substrates. J. Am. Chem. Soc. 95, 2987– 2991. Walling, C., Partch, R.E., Weil, T., 1975. Kinetics of the decomposition of hydrogen peroxide catalysed by ferric ethylenediaminetetraacetate complex. Proc. Natl. Acad. Sci. USA 72, 140–142. Yamazaki, I., Piette, L.H., 1990. ESR spin-trapping studies on the reaction of Fe2þ ions with H2 O2 -reactive species in oxygen toxicity in biology. J. Biol. Chem. 265, 13589– 13594. Yamazaki, I., Piette, L.H., Grover, T.A., 1990. Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems: effect of iron chelates. J. Biol. Chem. 265, 652–659. Yang, W.H., Tarng, Y.S., 1998. Design optimization of cutting parameters for turning operations based on the Taguchi method. J. Mater. Process. Technol. 84, 122–129. Yoshiki, Y., Okubo, K., Onuma, M., Igarashi, K., 1995. Chemiluminescence of benzoic and cinnamic-acids, and flavonoids in the presence of aldehyde and hydrogenperoxide or hydroxyl radical by Fenton reaction. Phytochemistry 39, 225–229. Yoshimura, Y., Matsuaki, Y., Watanabe, T., Uchiyama, K., Ohsawa, K., Imaeda, K., 1992. Effects of buffer solutions and chelators on the generation of hydroxyl radical and lipid peroxidation in the Fenton reaction system. J. Clin. Biochem. Nutr. 13, 147–154. Zhao, M.J., Jung, L., Tanielian, C., Mechin, R., 1994. Kinetics of the competitive degradation of deoxyribose and other biomolecules by hydroxyl radicals produced by the Fenton reaction. Free Radical Res. 20, 345–363.
Optimizing electron spin resonance detection of hydroxyl radical in water Shao-An Cheng 1, Wai-Kit Fung, Kwong-Yu Chan *, P.K. Shen Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China Received 3 July 2002; received in revised form 28 March 2003; accepted 1 April 2003
Abstract The parameters affecting the electron spin resonance (ESR) detection of hydroxyl free radical in water are studied and optimized. The hydroxyl radical is generated by the Fenton reaction with iron (II) ammonium sulfate and hydrogen peroxide reacting in a phosphate buffer using N-tert-butyl-alpha-phenylnitron as the spin trap. The concentrations of Fe2þ , H2 O2 , and phosphate buffer are the parameters studied. The Taguchi method and the orthogonal experiment design were used to evaluate the effects of these parameters on the ESR signal intensity. By the analysis of the signal-tonoise ratio and the analysis of variance, the order of importance of the various parameters on the hydroxyl radical formation is determined for optimal ESR detection of hydroxyl radical. The results will help the development of water purification technologies using hydroxyl free radical as a green oxidant. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Hydroxyl radical; Fenton reaction; Orthogonal experiment design; Electron spin resonance
1. Introduction The hydroxyl radical OH is perhaps the most important free radical in chemistry and biology. There have been much interests for using antioxidants to reduce the occurrence of hydroxyl radical in biological systems. On the other hand, hydroxyl radical has been considered in advanced green oxidation for treating drinking water and wastewater. The hydroxyl radical has an exceptional oxidizing power (Ruppert et al., 1994; Ku et al., 1996; Ito et al., 1998; Andreozzi et al., 1999) and almost all polluting organics and bacteria can be destroyed (Buxton et al., 1988; Ince and Apikyan, 2000). The additional attractive feature for drinking
*
Corresponding author. Tel.: +852-2859-7919; fax: +8522857-1586. E-mail address: [email protected] (K.-Y. Chan). 1 Permanent address: Department of Chemistry, Zhejiang University, Hangzhou 310027, China.
water treatment is that the short life time of the radicals leaves no residue effects compared to other oxidants like chlorine or ozone. The most well known generation of hydroxyl radicals in aqueous solution is the FentonÕs reaction (Fenton, 1894; Puppo, 1992; Yoshiki et al., 1995) or the Fenton-like reaction (Pignatello, 1992). Productions of hydroxyl radicals from ozone or hydrogen peroxide with ultraviolet radiation, and from mixing ozone with hydrogen peroxide have also been reported (Guittonneau et al., 1990; Ito et al., 1998). Investigations to maximize the production of hydroxyl radical for advanced green oxidation have been hindered by the technical difficulty of detecting and quantifying the elusive and short lived free radical. The detection of hydroxyl radical in biological experiments is not a routine technique and has also been a focus of many recent studies. The life time of a hydroxyl radical is ca.109 s (Janzen et al., 1992). It can only be detected indirectly. The detection methods include the electron spin resonance (ESR)-spin trapping method (Yamazaki and Piette, 1990; Yamazaki et al., 1990; Stadtman and
0045-6535/03/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00369-2
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Berlett, 1991; Ueda et al., 1996; Mizuta et al., 1997; Ma et al., 1999 and references therein), chemical probe method (Lindsey and Tarr, 2000), high-performance liquid chromatography (HPLC) (Fukui et al., 1993; Brillas et al., 1998; Ito et al., 1998), Fourier transform infrared spectroscopy (FTIR) (Carr and Baird, 2000), gas chromatography (GC) (Bremner et al., 2000) and mass spectroscopy (MS) (Oturan et al., 2000). In addition, more indirect methods of estimating the relative amount of OH by monitoring the concentrations of other reactants and products have been used (Kochany and Lipczynska-Kochany, 1992). Electron spin spectroscopy is the most definitive method to detect free radicals (Janzen et al., 1992; Utsumi et al., 1994; Han et al., 1998) since the characteristic unpaired electron is probed. Spin traps such as nitrones or nitroso compound such as 5,5-dimethyl-1pyrroline-1-oxide (DMPO) and phenyl-tert-butylnitrone (PBN) are used to react with OH to generate a long lived nitroxide free radical (spin adduct) which is then detected by ESR spectroscopy. The reaction of PBN to trap a hydroxyl radical is given as (Janzen et al., 1992): OH C N H O
+
CMe3
+
H O
C N CMe3 H O
ð1Þ Even with the use of a spin trap to extend the time frame for experimental detection, the ESR detection of OH is much influenced by many factors. Besides the factors affecting its generation, scavengers in the media can strongly affect the detection of OH. It will be useful to analyze and optimize the conditions of ESR detection of OH in standard systems to help deploying the technique for various applications. The Fenton reaction is the obvious candidate for such optimization studies. In the Fenton reaction, hydrogen peroxide is converted to hydroxyl radical in the presence of iron (II) ion. The efficiency of the hydroxyl radical production is sufficiently high for some practical applications, but is affected by pH, ferrous ion concentration, hydrogen peroxide concentration, iron chelate such as ethylenediaminetetraacetate (EDTA), and other scavengers and impurities. Several studies have reported the effects of these parameters on the Fenton reaction with ESR or other detection methods (Yamazaki and Piette, 1990; Yamazaki et al., 1990; Stadtman and Berlett, 1991; Sawer et al., 1993; Zhao et al., 1994; Ueda et al., 1996; Mizuta et al., 1997; Ma et al., 1999; Lindsey and Tarr, 2000). Using ESR detection, Yamazaki and Piette (1990) reported that the efficiency of OH generation decreases with increasing Fe2þ concentration over 1 lM when the concentration of hydrogen peroxide was maintained constant at 90 lM. Mizuta et al. (1997) also observed
that the efficiency for generation of DMPO-OH gradually decreased when the Fe2þ concentration increased to 2.5 lM. Ma et al. (1999) studied the generation of the hydroxyl radicals by the Fenton reaction in the PBS (KH2 PO4 –NaOH) buffer solution in the presence of ascorbic acid. However, almost all studies focus on the low substrate concentrations (mostly below 1 mM). The OH generation and consumption involve complex mechanisms with parallel and serial reaction steps. In the Fenton reaction, while more OH may be generated in high concentrations of Fe2þ and H2 O2 , the reactants also serve as scavengers to eliminate OH produced. It will be of practical interests, e.g. degradation of pollutants, whether high level of OH can be generated and detected at high concentration of reactants. Investigations of ESR detection of OH with higher Fe2þ and H2 O2 concentrations are warranted. In this paper, we report ESR detection of OH produced by Fenton reaction under an extended range of Fe2þ and H2 O2 concentrations. Effects of experimental conditions including the phosphate buffer concentration and EDTA chelate concentration on the hydroxyl radical detection are investigated with the help of the Taguchi method and the orthogonal design of experimental parameters. In addition, the complex mechanism of OH generation and consumption is discussed in light of the results of the orthogonal design of ESR detection.
2. Experimental 2.1. Materials and methods Ferrous ammonium sulfate (ACS reagent), PBN (98%), hydrogen peroxide 50% w/w solution, EDTA disodium salt (LR), potassium dihydrogen phosphate (ACS reagent) and disodium hydrogen phosphate (AR) are from Aldrich or Sigma Co. and used directly without further purification. All other chemicals are of analytical quality. All solutions were prepared using 18.2 MX deionized water produced with the Millipore MilliQ system. PBN, hydrogen peroxide and iron (II) solutions were freshly prepared in each experiment. A phosphate buffer (K2 HPO4 + NaH2 PO4 , pH 6.865 at 25 °C) was prepared according to the British standard. In each experiment, solution A was prepared by mixing 1 ml 0.1 M PBN with 1 ml H2 O2 of the required concentration, and solution B was prepared by mixing Fe2þ solution of the required concentration with or without EDTA and phosphate buffer in fixed concentrations. The Fenton reaction was initiated by adding 1 ml of solution B to solution A. After mixing, the concentration of PBN was 33.3 mM and the concentration of H2 O2 , Fe2þ , EDTA and phosphate buffer were changed to the required final concentrations. A 0.1 ml volume of the mixed solution
S.-A. Cheng et al. / Chemosphere 52 (2003) 1797–1805
was rapidly injected into the ESR quartz sample tube and ESR measurements were performed immediately.
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formation. All experiments are conducted at room temperature.
2.2. ESR measurement 3. Results ESR measurements were performed on a Bruker EMX100 ESR spectrometer with 100 kHz modulation at room temperature. ESR spectrometer settings were as follow: modulation amplitude, 2.00 G; receiver gain, 5.02 104 ; time constant, 5 ms; sweep time, 84 s; scan cycle: seven cycles; microwave with frequency, 9.76 GHz and power, 20 mW. The concentration of PBN was 33.3 mM for all experiments. Under these conditions, a stable ESR signal can be obtained for all experiments. Though the hydroxyl radical formation through Fenton reaction is affected by many parameters such as concentrations of additives, pH, and temperature, the main parameters must be the concentration of Fe2þ and the concentration of H2 O2 . Since EDTA is added to form a chelate complex with Fe2þ , and that phosphate buffer affects the pH of the solution, we decide to study the effects of four parameters: concentration of Fe2þ , concentration of H2 O2 , concentration of EDTA, and concentration of phosphate buffer on the hydroxyl ESR signal. Firstly, we study the effect of EDTA concentration at constant concentrations of Fe2þ , H2 O2 and phosphate buffer. Considering that the effect of EDTA should be incorporated in the Fe2þ concentration, an orthogonal array experiment is constructed to assess the importance of various parameters. An orthogonal array experiment (Taguchi experiment) (Hicks, 1993) with variation of three experimental parameters is constructed. Each parameter has three selected levels and an L9 (34 ) orthogonal array comprised of three columns and nine rows constructed are shown in Table 1. In Table 1, A, B, C indicate the concentration of Fe2þ , the concentration of H2 O2 , and the concentration of buffer solution, respectively. The signal peak height of ESR, related to the relative amount of the hydroxyl radicals formed, is used to evaluate effects of the experimental conditions on the hydroxyl radical
3.1. Effect of EDTA A typical measured ESR signal of the hydroxyl adduct created in the Fenton reaction with PBN spin trap is shown in Fig. 1. The spectrum is composed of three pairs of peaks with the hyperfine splitting constants aN ¼ 16:23 G, aH ¼ 2:54 G. They give the characteristic finger print of the PBN–OH adduct. The difference between the signal intensity at 3456.25 G and that at 3460.35 G was taken to represent the signal height and the concentration of the hydroxyl–PBN adduct. Concentrations of iron (II) salt, hydrogen peroxide, EDTA and phosphate buffer have been varied. The parameters of ESR scanning are kept constant for all measurements. Generally, EDTA is added to form a complex with iron (II) and iron (III). The EDTA–Fe2þ complex prevents iron (II) from binding to the hydroxyl radical and hence promote the H2 O2 decomposition and hydroxyl radical production. On the other hand, adding EDTA increases the solubility of iron (III) oxide and promotes the iron redox cycling (Blesa et al., 1994). The EDTA– Fe3þ can also react directly with H2 O2 to form OH (Gutteridge and Bannister, 1986; Aruoma et al., 1989). Excess EDTA will also scavenge the hydroxyl radical (Walling et al., 1975; Zhao et al., 1994). The [EDTA]/ [Fe2þ ] ratio must be optimized for maximum hydroxyl radical production detection. So, our preliminary experiment was conducted under fixed concentrations of iron (II) salt, hydrogen peroxide but with different EDTA concentrations to determine the optimum [EDTA]/ [Fe2þ ] ratio with or without phosphate buffer solutions. Fig. 2 shows the variation of signal intensity of ESR with the concentration of EDTA in 0.33 mM iron (II) salt, 0.12 mM hydrogen peroxide solution without phosphate buffer, and in 0.33 mM iron (II) salt, 0.12 mM
Table 1 The measured ESR signal height and the calculated S/N ratio of each designed experiment Experiment no.
A ([Fe2þ –EDTA]) (mM)
B ([H2 O2 ]) (mM)
C ([phosphate]) (mM)
Signal intensity
S/N ratio
1 2 3 4 5 6 7 8 9
0.1 0.1 0.1 0.3 0.3 0.3 2 2 2
0.1 1 11 0.1 1 11 0.1 1 11
0 0.8 4 0.8 4 0 4 0 0.8
1299 1365 2274 3553 8609 3074 1528 6174 10 689
62.3 62.7 67.1 71.0 78.7 69.8 63.7 75.8 80.6
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3.2. Analysis of the Taguchi experiment
3440
3460
3480 Value G
3500
3520
Fig. 1. A typical ESR spectrum of hydroxyl radical generated from Fenton reaction and trapped by PBN.
The Taguchi method, developed by Taguchi (1990), is generally used to optimize a process or product by a special design of orthogonal arrays of a small number of experiments. The experimental results are analyzed using the signal-to-noise (S/N) ratio and ANOVA analyses to determine the optimal design parameters and estimate the contribution of each design parameter to a certain characteristic. The S/N ratio, has a different meaning from that in analytical chemistry. The term ÔsignalÕ represents the desirable value for an output characteristic and the term ÔnoiseÕ represents the undesirable value for the output characteristic. Usually, there are three categories of the characteristic in the analysis of the S/N ratio, viz. the-lower–the-better, the-higher– the-better, and the-normal–the better. For all categories, a greater S/N ratio is better. The optimal level of the design parameters is the level with the greatest S/N ratio. ANOVA is a statistical analysis of the variance and gives information about which process parameter is statistically significant. More details about Taguchi method can be available in the literature (Yang and Tarng, 1998). 3.3. Analysis of the S/N ratio
Fig. 2. The effect of EDTA concentration on the ESR signal intensity in 0.33 mM iron (II) salt, 0.12 mM hydrogen peroxide solution (N) without phosphate buffer, ( ) with 0.8 mM phosphate buffer. The connecting lines are used to show the trend of data.
hydrogen peroxide solution with 0.8 mM phosphate buffer. It can be observed that as the EDTA concentration increases the signal intensity of ESR increases in the low concentration range and then decreases in the high concentration range with a sharp maximum at [EDTA]/[Fe2þ ] ¼ 1–1.2 for both cases. So, the [EDTA]/ [Fe2þ ] ratio for maximum hydroxyl radical production detection should have above value. Similar results of a linear relationship are reported (Zhao et al., 1994) for low concentrations of hydrogen peroxide and Fe2þ – EDTA in the present of deoxyribose. In there (Zhao et al., 1994), however, the amount of hydroxyl radical was only estimated indirectly by analyzing the oxidation product of deoxyribose and ESR experiments were not conducted.
Nine experiments were conducted according to the Taguchi method (Hicks, 1993) based on the arrangement of the orthogonal array in Table 1. The EDTA concentration is always kept the same as Fe2þ Õs in the orthogonal array of experiments. The experimental signal intensities obtained, having the higher the better properties, were converted to a S/N ratio according to the following equation: " # n X S=N ¼ 10 log n1 ðgi Þ2 ð2Þ i¼1
where n and gi are the degree of freedom (n ¼ 9) and the ESR signal intensity, respectively. The values of S/N ratio are listed in Table 1. Since the experimental design is orthogonal, it is then possible to separate out the effect of each parameter at different levels. For example, the mean S/N ratio for the Fe2þ concentration at levels 1, 2 and 3 can be calculated by averaging the S/N ratios for the experiments 1–3, 4–6 and 7–9, respectively. The mean S/N ratio for each level of the other parameters can be computed in the similar manner. Table 2 shows the mean S/N ratios and Fig. 3 shows the variation of mean S/N ratio with the parameter level for each designed parameter. The total mean S/N ratio for nine experiments is also calculated and shown in Table 2 and Fig. 3. S/N ratio can gives us the optimal levels of design parameters with the greatest S/N ratio. From Table 2 and Fig. 3, it can be seen that the A3, B3 and C2 is the
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Table 2 The mean S/N ratio and the difference between the maximal mean S/N ratio and the minimal mean S/N ratio for each level of the designed parameters Symbol
Parameters
A B C
[Fe2þ –EDTA] [H2 O2 ] Phosphate buffer
Mean S/N ratio Level 1
Level 2
Level 3
Max–min
64.0 65.7 69.3
73.2 72.4 71.4
73.4 72.5 69.8
9.4 6.8 2.1
The total mean S/N ratio ¼ 70.2.
Pt SSp ¼
Fig. 3. Variation of mean S/N ratio with the parameter level for each designed parameter. (N) Parameter A (Fe2þ –EDTA), ( ) parameter B (H2 O2 ), (j) parameter C (phosphate buffer). The subscript represents the level of the parameter, such as A1, A2, and A3 mean the level 1 (0.1 mM), level 2 (0.3 mM), and level 3 (2 mM) of the parameter A, respectively. The broken line represents the total mean S/N ratio.
j¼1 ðsgj Þ
t
2
" #2 m 1 X g m i¼1 i
ð4Þ
where ÔpÕ represents one of the tested parameters, j the level number of this parameter p, t the number of levels for each parameter (here, t ¼ 3), sgj the sum of the S/N ratio involving this parameter and level j, m the number of the experiments. The total degree of freedom (d.f.) is DT ¼ m 1, while the degree of freedom of the tested parameter is DP ¼ t 1. The variance of the tested parameter is VP ¼ SSp =DP . Then the F -value for each design parameter is simply the ratio of mean-of-square deviations to the mean of the squared error (FP ¼ VP =Ve ). The corrected sum of squares Sp can be calculated as: Sp ¼ SSp DP Ve
ð5Þ
The percentage contribution u can be calculated as: uP ¼ Sp =SST
optimal combination of the parameter levels. However, the mean S/N ratio at level 2 is very close to that at level 3 for both parameter A and parameter B, indicating that changes in concentration of Fe2þ and H2 O2 are insignificantly to affect the ESR signal in the high concentration range. 3.3.1. Analysis of variance To investigate which design parameter significantly affect the ESR signal of OH adduct, an analysis of variance (ANOVA) is introduced. This is to accomplished by separating the total variability of the S/N ratios, which is measured by the sum of the squared deviations from the total mean S/N ratio, into contributions by each of the design parameters and the error. The total sum of square SST from the S/N ratio g can be calculated as (Yang and Tarng, 1998): " #2 m m X 1 X 2 SST ¼ gi g ð3Þ m i¼1 i i¼1 where m is the number of the experiment. The sum of squares from the tested parameter SSp can be calculated as:
ð6Þ
The parameter with the bigger uP gives the more significant effect on the ESR signal. Table 3 shows the result of ANOVA. It is found that Fe2þ concentration is the most significant parameter affecting the ESR signal. The change of the phosphate buffer has an insignificant effect on the ESR signal. From the divergence of mean S/N ratios (max–min value in Table 2), it is also observed that the order of the three factorsÕ effect on ESR signal is A > B > C.
4. Discussion Two major mechanisms of Fenton reaction have been suggested in literature. One is the reaction mechanism that hydrogen peroxide is reduced at the iron center with the generation of free hydroxyl radical as follow: Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH II
Fe
ðOH2 Þ2þ 6 þ H2 O2
III
! ½Fe ðOH2 Þ5 OH
ð7Þ 2þ
þ OH þ OH ð8Þ
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Table 3 Results of the ANOVA analysis Symbol
Parameter 2þ
A B C Error
[Fe –EDTA] [H2 O2 ] Phosphate buffer
Total
d.f.
SSp
VP
FP
2 2 2 2
170.0 92.3 7.5 41.7
85.0 46.2 3.7 20.9
4.1 2.2 0.18
8
311.5
Another one is Bray–Gorin reaction mechanism that hydroxyl radical is formed via the formation of an intermediate Fe (IV): Fe2þ þ H2 O2 ! ½FeIV ¼ O 2þ þ H2 O
ð9Þ
½FeIV ¼ O 2þ þ H2 O ! Fe3þ þ OH þ OH
ð10Þ
Other reactions involved in the Fenton system are listed in Table 4. It can be seen from Table 4 that the reactions R1 and R2 are the limiting steps for producing hydroxyl radical. Without Fe2þ or other similar catalysts, the decomposition rate of H2 O2 is very low. When Fe2þ is added, H2 O2 can be easily converted to hydroxyl radical (k ¼ 63). Therefore, Fe2þ is the most significant parameter affecting the ESR signal. The kinetics of OH formation can be schematized as follows (with addition of EDTA): ka
Fe2þ –EDTA þ H2 O2 ! OH
ð11Þ
kb
OH þ S ! D
OH þ P ! P–OH
ð12Þ
kc
ð13Þ
where S represents the scavenging reagents such as H2 O2 in reaction R3, Fe2þ in reaction R6, OH in reaction
uP (%) 54.6 29.6 2.4 13.4 100
R11, HO2 in reaction R13, O 2 in reaction R14 as listed in Table 4, and others such as EDTA and phosphate; ka and kb are the corresponding rate constants; D is the scavenging reaction products; P is the PBN spin trap; and P–OH is the OH adduct. Reaction (13) is the same as Eq. (1). The rate constant (kc ) of the trapping reaction (Eq. (13)) is 6.1 109 M s1 (Sridhar et al., 1984). Using a steady-state approximation commonly applied to transient intermediates such as free radicals (Laidler, 1979), the rate of change of concentration of OH is zero. The rate (m) of the hydroxyl radical adduct formation (Eq. (13)) can be expressed as: m¼
ka kc ½Fe2þ –EDTA ½H2 O2 ½P
kb ½S þ kc ½P
In the scavenging reactions R3, R6, R11, R13 and R14, the rates of reactions R13 and R14 is negligible since the reactions are subsequent reactions of R3 with trace concentrations of radical reactant species. The effect of reaction R11 can be also neglected due to the low concentration of OH. Thus the main scavenging reactions are R3 and R6. The effect of the concentration of Fe2þ – EDTA and H2 O2 on the ESR signal can be deduced from Eq. (14). For example, when the concentration of either Fe2þ –EDTA or H2 O2 is low compared to [P] or
Table 4 The reaction steps involved in the Fenton mechanism Reaction 2þ
3þ
R1. Fe þ H2 O2 ! Fe þ OH þ OH R2. Fe3þ þ H2 O2 ! Fe2þ þ Hþ þ HO2 R3. OH þ H2 O2 ! HO2 þ H2 O þ R4. HO2 ! O 2 þH þ þ H ! HO R5. O 2 2 2þ R6. OH þ Fe ! Fe3þ þ OH R7. HO2 þ Fe2þ ðþHþ Þ ! Fe3þ þ H2 O2 R8. HO2 þ Fe3þ ! Fe2þ þ Hþ þ O2 2þ þ 3þ þ H2 O2 R9. O 2 þ Fe ðþ2H Þ ! Fe 3þ 2þ R10. O2 þ Fe ! Fe þ O2 R11. OH þ OH ! H2 O2 R12. HO2 þ HO2 ! O2 þ H2 O2 R13. HO2 þ OH ! O2 þ H2 O R14. OH þ O 2 ! O2 þ OH þ R15. HO2 þ O ðþH Þ ! O 2 þ H2 O2 2
ð14Þ
k (M1 s1 )
References
63 0.01 2.7 107 1.58 105 1 1010 3.2 108 1.2 106 3.1 105 1 107 5 107 4.2 109 8.3 105 1 1010 1 1010 9.7 107
Metelitsa (1971) Walling and Goosen (1973) Christensen et al. (1982) Bielski et al. (1985) Bielski et al. (1985) Stuglik and Zagorski (1981) Jayson et al. (1969) Rush and Bielski (1985) Rush and Bielski (1985) Rothschild and Allen (1958) Sehested et al. (1969) Bielski et al. (1985) Sehested et al. (1969) Sehested et al. (1969) Bielski et al. (1985)
S.-A. Cheng et al. / Chemosphere 52 (2003) 1797–1805
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other species, the rate (m) of the adduct formation and hence the ESR signal intensity are limited by Fe2þ – EDTA or H2 O2 and proportional to the concentration of Fe2þ –EDTA or H2 O2 . At high concentrations of Fe2þ –EDTA, ½S ½Fe2þ –EDTA , kb ½S kc ½P and Eq. (14) becomes: m¼
ka kc ½Fe2þ –EDTA ½H2 O2 ½P
¼ k 0 ½H2 O2 ½P
kb ½Fe2þ –EDTA
ð15Þ
The rate (m) of the adduct formation is independent of Fe2þ –EDTA concentration. The same result can be deduced for high concentration of H2 O2 . So, in the high concentration, changing concentration of Fe2þ –EDTA and H2 O2 has no significant effect on the hydroxyl radical formation, as evident in Fig. 3 and Table 2. However, in the case that both [Fe2þ –EDTA] and H2 O2 becoming the dominant scavengers, [S] could be proportional to [Fe2þ –EDTA]*[H2 O2 ]. The ESR signal intensity should reach a saturation value. To test the above conclusions derived from the analytical results of orthogonal experiment, further ESR experiments were conducted in the solutions with extended range of H2 O2 and Fe2þ concentrations varied separately. It can be seen from the results in Fig. 4 that the two parameters show a similar trend with signal increasing at low concentrations and saturated at ac. 11 mM for H2 O2 and 2 mM for Fe2þ . The same saturation value was observed. According to the reaction R1 in Table 4, the pH of reaction medium increases as the hydroxyl radical formation proceeds. High OH may hinder the hydroxyl radical formation by equilibrium consideration. Moreover, hydroxide ions can break down the OH–PBN adduct reducing the ESR signal detected (Janzen et al., 1992). Generally, a phosphate buffer was used to maintain the pH at a low level. However, the phosphate concentration must be within a suitable range. Phosphate may not only bind with Fe2þ –EDTA to form a ternary complex, hence decreasing the activity of Fe2þ (Yoshimura et al., 1992; Vella and Munder, 1993), but it can also be a scavenger of hydroxyl radicals (Lipczynska-Kochany et al., 1995) as described in the following reaction:
OH þ HPO2 4 ! OH þ HPO4
ð16Þ
At low concentrations of Fe2þ and H2 O2 , the concentration of EDTA is generally low and a substantial inhibitory effect on the hydroxyl radical formation is observed (Fig. 2 and references: Yoshimura et al., 1992; Vella and Munder, 1993). At high concentration of Fe2þ –EDTA, the phosphate does not affect the hydroxyl radical formation in the concentration range studied (Figs. 2 and 3 and Table 3). These results may be due to
Fig. 4. Effects of the parameter concentration on the ESR signal intensity. (a) In 2 mM Fe2þ , 2 mM EDTA, 33 mM PBN and 0.8 mM phosphate buffer solutions with different H2 O2 concentrations; (b) in 11 mM H2 O2 , 33 mM PBN and 0.8 mM phosphate buffer solutions with different Fe2þ –EDTA concentrations (the concentration of EDTA is the same as that of Fe2þ ).
larger contribution of EDTA to the formation of hydroxyl radical.
5. Conclusions The factors on the ESR detection of hydroxyl radical were studied using the Taguchi method and orthogonal experiment design on the Fenton reaction with a large concentration range of Fe2þ and H2 O2 . The following conclusions can be drawn: 1. A Fe2þ /EDTA ratio 1:1 is more suitable for the formation of hydroxyl radical. 2. The order of importance for the several factors studied are Fe2þ > H2 O2 > phosphate buffer. 3. At sufficiently high concentration or either Fe2þ or H2 O2 , the ESR signal has attained the maximum level and further increase in concentration of Fe2þ
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or H2 O2 has no effect on the ESR signal of the hydroxyl adduct. 4. The optimal condition for the hydroxyl radical formation by the Fenton reaction is: 2 mM Fe2þ , 2 mM EDTA, 11 mM H2 O2 and 0.8 mM phosphate buffer using a PBN concentration of 33.3 mM in this study. The method and the results of this study are useful for ESR study of hydroxyl free radical in various systems, especially for high concentrations of reactants as in the advanced oxidation of drinking water.
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