Chemosphere 52 (2003) 17971805 www.elsevier.

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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 aecting 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 buer using N-tert-butyl-alpha-phenylnitron as the spin trap. The concentrations of Fe2 , H2 O2 , and phosphate buer are the parameters studied. The Taguchi method and the orthogonal experiment design were used to evaluate the eects 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 purication technologies using hydroxyl free radical as a green oxidant. 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: hrsccky@hku.hk (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 eects compared to other oxidants like chlorine or ozone. The most well known generation of hydroxyl radicals in aqueous solution is the Fentons 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 diculty 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 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 denitive 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

1 Even with the use of a spin trap to extend the time frame for experimental detection, the ESR detection of OH is much inuenced by many factors. Besides the factors aecting its generation, scavengers in the media can strongly aect 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 eciency of the hydroxyl radical production is suciently high for some practical applications, but is aected by pH, ferrous ion concentration, hydrogen peroxide concentration, iron chelate such as ethylenediaminetetraacetate (EDTA), and other scavengers and impurities. Several studies have reported the eects 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 eciency of OH generation decreases with increasing Fe2 concentration over 1 lM when the concentration of hydrogen peroxide was maintained constant at 90 lM. Mizuta et al. (1997) also observed

that the eciency for generation of DMPO-OH gradually decreased when the Fe2 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) buer 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 Fe2 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 Fe2 and H2 O2 concentrations are warranted. In this paper, we report ESR detection of OH produced by Fenton reaction under an extended range of Fe2 and H2 O2 concentrations. Eects of experimental conditions including the phosphate buer 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 purication. 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 buer (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 Fe2 solution of the required concentration with or without EDTA and phosphate buer in xed 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 , Fe2 , EDTA and phosphate buer were changed to the required nal concentrations. A 0.1 ml volume of the mixed solution

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was rapidly injected into the ESR quartz sample tube and ESR measurements were performed immediately. 2.2. ESR measurement

formation. All experiments are conducted at room temperature.

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 aected by many parameters such as concentrations of additives, pH, and temperature, the main parameters must be the concentration of Fe2 and the concentration of H2 O2 . Since EDTA is added to form a chelate complex with Fe2 , and that phosphate buer aects the pH of the solution, we decide to study the eects of four parameters: concentration of Fe2 , concentration of H2 O2 , concentration of EDTA, and concentration of phosphate buer on the hydroxyl ESR signal. Firstly, we study the eect of EDTA concentration at constant concentrations of Fe2 , H2 O2 and phosphate buer. Considering that the eect of EDTA should be incorporated in the Fe2 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 Fe2 , the concentration of H2 O2 , and the concentration of buer solution, respectively. The signal peak height of ESR, related to the relative amount of the hydroxyl radicals formed, is used to evaluate eects of the experimental conditions on the hydroxyl radical 3.1. Eect 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 hyperne splitting constants aN 16:23 G, aH 2:54 G. They give the characteristic nger print of the PBNOH adduct. The dierence 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 hydroxylPBN adduct. Concentrations of iron (II) salt, hydrogen peroxide, EDTA and phosphate buer 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 EDTAFe2 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 Fe3 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]/ [Fe2 ] ratio must be optimized for maximum hydroxyl radical production detection. So, our preliminary experiment was conducted under xed concentrations of iron (II) salt, hydrogen peroxide but with dierent EDTA concentrations to determine the optimum [EDTA]/ [Fe2 ] ratio with or without phosphate buer 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 buer, 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. 1 2 3 4 5 6 7 8 9 A ([Fe2 EDTA]) (mM) 0.1 0.1 0.1 0.3 0.3 0.3 2 2 2 B ([H2 O2 ]) (mM) 0.1 1 11 0.1 1 11 0.1 1 11 C ([phosphate]) (mM) 0 0.8 4 0.8 4 0 4 0 0.8 Signal intensity 1299 1365 2274 3553 8609 3074 1528 6174 10 689 S/N ratio 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 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 dierent meaning from that in analytical chemistry. The term signal represents the desirable value for an output characteristic and the term noise 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-lowerthe-better, the-higher the-better, and the-normalthe 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 signicant. More details about Taguchi method can be available in the literature (Yang and Tarng, 1998). 3.3. Analysis of the S/N ratio 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 Fe2 s 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 10 log n1 gi 2 2
i1

3440

3460

3480 Value G

3500

3520

Fig. 1. A typical ESR spectrum of hydroxyl radical generated from Fenton reaction and trapped by PBN.

Fig. 2. The eect of EDTA concentration on the ESR signal intensity in 0.33 mM iron (II) salt, 0.12 mM hydrogen peroxide solution (N) without phosphate buer, ( ) with 0.8 mM phosphate buer. The connecting lines are used to show the trend of data.

hydrogen peroxide solution with 0.8 mM phosphate buer. 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]/[Fe2 ] 11.2 for both cases. So, the [EDTA]/ [Fe2 ] 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 Fe2 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.

where n and gi are the degree of freedom (n 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 eect of each parameter at dierent levels. For example, the mean S/N ratio for the Fe2 concentration at levels 1, 2 and 3 can be calculated by averaging the S/N ratios for the experiments 13, 46 and 79, 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 dierence between the maximal mean S/N ratio and the minimal mean S/N ratio for each level of the designed parameters Symbol A B C Parameters [Fe2 EDTA] [H2 O2 ] Phosphate buer Mean S/N ratio Level 1 64.0 65.7 69.3 Level 2 73.2 72.4 71.4 Level 3 73.4 72.5 69.8 Maxmin 9.4 6.8 2.1

The total mean S/N ratio 70.2.

Pt SSp

j1 sgj

" #2 m 1 X g m i1 i

Fig. 3. Variation of mean S/N ratio with the parameter level for each designed parameter. (N) Parameter A (Fe2 EDTA), ( ) parameter B (H2 O2 ), (j) parameter C (phosphate buer). 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.

where p represents one of the tested parameters, j the level number of this parameter p, t the number of levels for each parameter (here, t 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 m 1, while the degree of freedom of the tested parameter is DP t 1. The variance of the tested parameter is VP 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 VP =Ve ). The corrected sum of squares Sp can be calculated as: Sp SSp DP Ve The percentage contribution u can be calculated as: uP Sp =SST 6 5

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 Fe2 and H2 O2 are insignicantly to aect the ESR signal in the high concentration range. 3.3.1. Analysis of variance To investigate which design parameter signicantly aect 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 gi g 3 m i1 i i1 where m is the number of the experiment. The sum of squares from the tested parameter SSp can be calculated as:

The parameter with the bigger uP gives the more signicant eect on the ESR signal. Table 3 shows the result of ANOVA. It is found that Fe2 concentration is the most signicant parameter aecting the ESR signal. The change of the phosphate buer has an insignicant eect on the ESR signal. From the divergence of mean S/N ratios (maxmin value in Table 2), it is also observed that the order of the three factors eect 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: Fe2 H2 O2 ! Fe3 OH OH Fe
II

7
2

OH2 2 H2 O2 6

! Fe OH2 5 OH

III

OH OH 8

1802 Table 3 Results of the ANOVA analysis Symbol A B C Error Total Parameter [Fe EDTA] [H2 O2 ] Phosphate buer
2

S.-A. Cheng et al. / Chemosphere 52 (2003) 17971805

d.f. 2 2 2 2 8

SSp 170.0 92.3 7.5 41.7 311.5

VP 85.0 46.2 3.7 20.9

FP 4.1 2.2 0.18

uP (%) 54.6 29.6 2.4 13.4 100

Another one is BrayGorin reaction mechanism that hydroxyl radical is formed via the formation of an intermediate Fe (IV): Fe2 H2 O2 ! FeIV O2 H2 O FeIV O2 H2 O ! Fe3 OH OH 9 10

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 Fe2 or other similar catalysts, the decomposition rate of H2 O2 is very low. When Fe2 is added, H2 O2 can be easily converted to hydroxyl radical (k 63). Therefore, Fe2 is the most signicant parameter aecting the ESR signal. The kinetics of OH formation can be schematized as follows (with addition of EDTA): Fe2 EDTA H2 O2 ! OH
ka

R11, HO2 in reaction R13, O in reaction R14 as listed 2 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 POH 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: m ka kc Fe2 EDTAH2 O2 P kb S kc P 14

11 12 13

OH S ! D OH P ! POH
kc

kb

where S represents the scavenging reagents such as H2 O2 in reaction R3, Fe2 in reaction R6, OH in reaction
Table 4 The reaction steps involved in the Fenton mechanism Reaction R1. Fe H2 O2 ! Fe OH OH R2. Fe3 H2 O2 ! Fe2 H HO2 R3. OH H2 O2 ! HO2 H2 O R4. HO2 ! O H 2 R5. O H ! HO2 2 R6. OH Fe2 ! Fe3 OH R7. HO2 Fe2 H ! Fe3 H2 O2 R8. HO2 Fe3 ! Fe2 H O2 R9. O Fe2 2H ! Fe3 H2 O2 2 R10. O Fe3 ! Fe2 O2 2 R11. OH OH ! H2 O2 R12. HO2 HO2 ! O2 H2 O2 R13. HO2 OH ! O2 H2 O R14. OH O ! O2 OH 2 R15. HO2 O H ! O2 H2 O2 2
2 3

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 eect of reaction R11 can be also neglected due to the low concentration of OH. Thus the main scavenging reactions are R3 and R6. The eect of the concentration of Fe2 EDTA and H2 O2 on the ESR signal can be deduced from Eq. (14). For example, when the concentration of either Fe2 EDTA or H2 O2 is low compared to [P] or

k (M1 s1 ) 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

References 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 Fe2 EDTA or H2 O2 and proportional to the concentration of Fe2 EDTA or H2 O2 . At high concentrations of Fe2 EDTA, S % Fe2 EDTA, kb S ) kc P and Eq. (14) becomes: m ka kc Fe2 EDTAH2 O2 P k 0 H2 O2 P kb Fe2 EDTA 15

The rate (m) of the adduct formation is independent of Fe2 EDTA concentration. The same result can be deduced for high concentration of H2 O2 . So, in the high concentration, changing concentration of Fe2 EDTA and H2 O2 has no signicant eect on the hydroxyl radical formation, as evident in Fig. 3 and Table 2. However, in the case that both [Fe2 EDTA] and H2 O2 becoming the dominant scavengers, [S] could be proportional to [Fe2 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 Fe2 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 Fe2 . 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 OHPBN adduct reducing the ESR signal detected (Janzen et al., 1992). Generally, a phosphate buer 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 Fe2 EDTA to form a ternary complex, hence decreasing the activity of Fe2 (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:

Fig. 4. Eects of the parameter concentration on the ESR signal intensity. (a) In 2 mM Fe2 , 2 mM EDTA, 33 mM PBN and 0.8 mM phosphate buer solutions with dierent H2 O2 concentrations; (b) in 11 mM H2 O2 , 33 mM PBN and 0.8 mM phosphate buer solutions with dierent Fe2 EDTA concentrations (the concentration of EDTA is the same as that of Fe2 ).

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 Fe2 and H2 O2 . The following conclusions can be drawn: 1. A Fe2 /EDTA ratio 1:1 is more suitable for the formation of hydroxyl radical. 2. The order of importance for the several factors studied are Fe2 > H2 O2 > phosphate buer. 3. At suciently high concentration or either Fe2 or H2 O2 , the ESR signal has attained the maximum level and further increase in concentration of Fe2

OH HPO2 ! OH HPO 4 4

16

At low concentrations of Fe2 and H2 O2 , the concentration of EDTA is generally low and a substantial inhibitory eect on the hydroxyl radical formation is observed (Fig. 2 and references: Yoshimura et al., 1992; Vella and Munder, 1993). At high concentration of Fe2 EDTA, the phosphate does not aect the hydroxyl radical formation in the concentration range studied (Figs. 2 and 3 and Table 3). These results may be due to

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S.-A. Cheng et al. / Chemosphere 52 (2003) 17971805 Fenton, H.J.H., 1894. Oxidation of tartaric acid in presence of iron. J. Chem. Soc. 65, 899910. 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, 187193. 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 + UVa comparative study. Ozone Sci. Eng. 12, 7394. 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 dierent stagesimplications for the redox states of copper and manganese. Biochem. J. 234, 225228. 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, 41694176. 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 euents. Water Sci. Technol., 38,179186. 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, 169173. 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, 24532464. 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, 17691782. 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, 409417. Lipczynska-Kochany, E., Sprah, G., Harms, S., 1995. Inuence of some groundwater and surface waters constituents on the degradation of 4-chlorophenol by Fenton reaction. Chemosphere 30, 920. 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, 563580. 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, 11071120.

or H2 O2 has no eect on the ESR signal of the hydroxyl adduct. 4. The optimal condition for the hydroxyl radical formation by the Fenton reaction is: 2 mM Fe2 , 2 mM EDTA, 11 mM H2 O2 and 0.8 mM phosphate buer 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
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