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Hydroxyl (OH) radical is one of the important reactive species in the advanced oxidation process. The objective of this study is to present the quantitative analysis of hydroxyl radical generation by using the electron spin resonance (ESR)/spin-trapping technique and to apply this method to evaluation of the enhancement of OH radical generation by several humic substances during ozonation in water. OH radical was trapped with a 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) as a stable adduct, DMPO-OH. The generation of OH radical was demonstrated with mathematical equation of the initial velocity of DMPO-OH generation, and the effect of the compounds, 3-chlorophenol and resorcinol, on OH radical generation expressed with ν0 (10-6 M/s) = {9.7~10.5×[compound (10-9 M)] + 0.0005} exp(57×[ozone (10-9 M)]). The effect of humic substances on OH generation was evaluated with the amount of DMPO-OH with batch system because of high viscosity of humic acid solution. The amount of DMPO-OH reached almost maximum level within 2 min and kept the level till 60 min in most cases. The amount of DMPO-OH depended both of the amounts of ozone and humic acid. The origin of humic acid also affected the amount of DMPO-OH.
Journal of Water and Environment Technology, Vol.1, No.2, 2003 - 209 - ANALYSIS OF HYDROXYL RADICAL GENERATION IN ADVANCED OXIDATION PROCESS -EFFECT OF HUMIC SUBSTANCES DURING OZONATION- Youn-Hee Han and Hideo Utsumi Department of Bio-function science, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, 812-8582 Japan hyh@pch.phar.kyushu-u.ac.jp; utsumi@pch.phar.kyushu-u.ac.jp ABSTRACT Hydroxyl (OH) radical is one of the important reactive species in the advanced oxidation process. The objective of this study is to present the quantitative analysis of hydroxyl radical generation by using the electron spin resonance (ESR)/spin-trapping technique and to apply this method to evaluation of the enhancement of OH radical generation by several humic substances during ozonation in water. OH radical was trapped with a 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) as a stable adduct, DMPO-OH. The generation of OH radical was demonstrated with mathematical equation of the initial velocity of DMPO-OH generation, and the effect of the compounds, 3-chlorophenol and resorcinol, on OH radical generation expressed with ν 0 (10 -6 M/s) = {9.7~10.5×[compound (10 -9 M)] + 0.0005} exp(57×[ozone (10 -9 M)]). The effect of humic substances on OH generation was evaluated with the amount of DMPO-OH with batch system because of high viscosity of humic acid solution. The amount of DMPO-OH reached almost maximum level within 2 min and kept the level till 60 min in most cases. The amount of DMPO-OH depended both of the amounts of ozone and humic acid. The origin of humic acid also affected the amount of DMPO-OH. KEYWORDS Electron spin resonance (ESR); Humic acid; Hydroxyl radical; Ozonation; Spin trapping INTRODUCTION Advanced oxidation process (AOP) is widely used and/or developing for decomposition of pollutants in water and soil, and ozonation is widely employed to decomposition of the substances responsible for musty and odors in water. Hydroxyl (OH) radical is reported as one of the important reactive species in AOP, especially in ozonation (Staehelin et al., 1984, Hoigne et al., 1976). Recently, some kinetic models were provided to explain the efficacy of OH radical in advanced oxidation processes involving ozonation using OH radical scavengers (Beltran et al., 1999; Andreozzi et al., 1999; Westerhoff et al., 1999). However, there are very few papers, which determined the amount and the dynamics of OH radical directly. In order to detect free radicals, electron spin resonance (ESR) spectroscopy is widely utilized because of its sensitivity and selectivity, and ESR/spin-trapping technique has been developed to detect unstable radicals (Janzen and Blackburn, 1969). We have succeeded in direct determination of OH radical generation in water during ozonation using ESR/spin-trapping technique (Utsumi et al., 1994). Recently, we combined ESR/spin-trapping technique with Journal of Water and Environment Technology, Vol.1, No.2, 2003 - 210 - stopped-flow method to determine the rate constant of OH radical generation (Han et al., 1998a), and the enhancing effect of 3-chlorophenol on OH radical generation was mathematically evaluated (Utsumi et al., 2003). OH radical was trapped with a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a stable adduct, DMPO-OH. The initial velocity of DMPO-OH generation was mathematically analyzed and the following equation was obtained, ν 0 (10 -6 M/s) = {9.7×[3-chlorophenol (10 -9 M)] + 0.0005} exp(57×[ozone (10 -9 M)]) . The equation fitted very well with the experimental results, and the correlation coefficient was larger than 0.99. The equation for the enhancing effect by 3-chlorophenol should provide useful information to optimize the condition in ozone treatment process of water containing phenolic pollutants. We also clarified the generation of semiquinone radical as an intermediate of phenol during ozonation (Han et al., 1998b). Humic substances are ubiquitous natural materials occurring in huge amounts in soils, sediments and waters as a product of the chemical and biological transformation of animal and plant residues. A substantial proportion of carbon-containing substances in the environment can be referred to as humic substances - it is estimated that humic substances form 50-90 % of dissolved organic carbon (DOC) in freshwater systems (Preisler et al., 1959). Because of their ability to interact with various components of the environment, humic substances play an important role in soil and aquatic chemistry and therefore have attracted the attention of researchers. Previous studies on the ozone consumption by the humic substances for ozonation process indicated humic substances as radical scavengers (Staehelin et al., 1985). On the other hand, Xiong et al. (1992) and Morioka et al. (1993) reported that small amounts of humic substances might act as promoters, which result in the generation of OH radicals during ozonation. However, the mechanism between humic substances, OH radical and ozone remains unclear. Resorcinol is often used as a model compound of humic acid. Resorcinol also has the enhancing effect on OH radical generation during ozonation (Han et al, unpublished results). In the present paper, we demonstrate the quantitative analysis of OH radical generation by using the electron spin resonance (ESR)/spin-trapping technique and to apply this method to evaluation of the enhancement of OH radical generation by several humic substances during ozonation in water. MATERIALS AND METHODS Materials Humic acids were obtained from the Aldrich, Fluka and ICN companies, and the powdered form of humic acid was suspended in a phosphate buffer (0.1 M, pH 7.4). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotech Co. Ltd. (Tokyo, Japan) and stored at -20 ℃. Other chemicals were of the highest grade commercially available. Phosphate buffer was prepared with pre-ozonated pure water, and all chemicals were dissolved in the buffer just before use, as described previously (Han et al., 1998a; Utsumi et al., 1994). An aqueous ozone solution was prepared by continuously bubbling ozone gas through distilled water using an absorber glass at 20 ℃; ozone was produced from highly pure oxygen (Fukuoka Oxygen Co., Fukuoka, Japan) with an ozone generator (PO-10, Fuji Electric Co., Kanagawa, Japan), as described previously (Han et al., 1998a). Concentration of aqueous ozone was determined using the Indigo method (Bader and Hoigne, Journal of Water and Environment Technology, Vol.1, No.2, 2003 - 211 - 1981). Stopped-flow/ESR measurement The combined technique of rapid data acquisition system with an ESR spectrometer (RE-1X, JEOL, Tokyo, Japan) and a stopped-flow system (Ohtsuka Electric Co. Ltd., Osaka, Japan) was shown in Fig. 1. Fig. 1 Combined system of an ESR spectrometer with a stopped-flow. Trapping of OH radical with DMPO was carried out as described previously (Utsumi et al., 1994), and the ESR signal was recorded with rapid data acquisition systems in order to analyze the initial velocity of OH radical generation more precisely. The DMPO solution containing 3-chlorophenol or resorcinol in phosphate buffer (0.1 M, pH 7.4) was rapidly mixed with various concentration of aqueous ozone at room temperature using stopped-flow system, and then one of the quartet ESR signals was detected with an ESR spectrometer (RE-1X, JEOL, Tokyo, Japan) at 10 mW of microwave (9.44 GHz) and 0.20 mT of field modulation (100 kHz) and acquired with a personal computer (Compaq, Frolinea 4/33s) operated with home-made software. The concentration of DMPO-OH was determined by comparing the signal intensity with that of a standard solution of diphenyl-2-picrylhydrazyl. The condition of ESR measurement was 10 mW of microwave (9.44 GHz) and 0.20 mT of field modulation (100 kHz). The hyperfine splitting constant and g-value were calculated with those of Mn 2+ . Batch system ESR measurements Humic substances were suspended in phosphate buffer (0.1 M, pH 7.4) containing DMPO (100 mM). Immediately after addition of aqueous ozone, 15 µl of the sample solution were transferred into a capillary tube, and then the ESR spectrum was measured at room temperature with an ESR spectrometer under the above conditions. RESULTS AND DISCUSSION OH radical measurement with an ESR spectrometer Fig. 2 shows the typical ESR spectrum obtained 5 min after mixing aqueous ozone with DMPO solution. The spectrum was composed of quartet lines having peak height ratio of 1:2:2:1. The ESR parameters (hyperfine constants a N =1.49 mT, a H =1.49mT and g-value=2.0055) coincided with those of DMPO-OH adduct as demonstrated previously Mixture N S N 2 ozone DMPO+ phenol derivative solution controller ESR measurement Personal Computer Mixture N S N 2 ozone DMPO+ phenol derivative solution controller ESR measurement Personal Computer Journal of Water and Environment Technology, Vol.1, No.2, 2003 - 212 - (Utsumi et al., 1994; Han et al., 1998a), confirming that the quartet signal is DMPO-OH adduct. Fig. 2. Typical ESR spectrum of DMPO-OH adduct generated during ozonation of water. Aqueous ozone (100 µM) was mixed with DMPO (100 mM) at room temperature using stopped-flow system, and then the ESR signals were acquired using a computer-controlled ESR spectrometer operated with homemade software. The condition of ESR measurement was 10 mW of microwave (9.44 GHz) and 0.20 mT of field modulation (100 kHz). Mathematical analysis of OH radical generation enhanced by 3-chlorophenol ESR/stopped-flow experiment was carried out to determine the initial velocity of DMPO-OH generation precisely. The amount of DMPO-OH increased gradually with time after mixing, and the initial velocities of DMPO-OH generation increased in both ozone- and 3-chlorophenol-dose dependent manner (data not shown). In order to obtain the mathematical relation of enhancing parameters of 3-chlorophenol on OH radical generation during ozonation, the initial velocities in the mixture of various amounts of ozone with 3-chlorophenol were curve-fitted with an exponential equation. The exponential curves, ν 0 (10 -6 M/s) = A × exp (B × [ozone (10 -6 M)]), were fitted very well to those of observed data. Table 1 demonstrates the factors of the resulting exponential equations and their correlation coefficients in the plot of DMPO-OH generation versus ozone concentration. Variation of factor B is much smaller than that of the pre-exponential factor A. Thus, using average value (0.057) of factors B, re-curve-fitting was carried out. The re-curve-fitting gave again very high correlation coefficients (Table 1). Table 1. The factors of exponential equation used for curve-fitting of the experimental data. Concentration of experimental calibrated 3-chlorophenol (µM) A B R 2 A’ B’(const.) R’ 2 0 0.0005 0.063 0.99 0.0008 0.057 0.99 0.5 0.0076 0.048 0.99 0.0046 `` 0.98 1.0 0.0054 0.067 0.99 0.0096 `` 0.99 2.0 0.0318 0.049 0.99 0.0204 `` 0.98 The linear relation was obtained as follows, A’ = 9.7×[3-chlorophenol (10 -9 M)] + 0.0005. The present observation strongly confirmed that the enhancing efficiency of 3-chlorophenol on OH radical generation during ozonation is exponentially related to ozone concentration and that the pre-exponential factor depends linearly on 3-chlorophenol concentration. Journal of Water and Environment Technology, Vol.1, No.2, 2003 - 213 - The kinetic equation of enhancing effect on the DMPO-OH generation during ozonation by 3-chlorophenol was expressed as ν 0 (10 -6 M/s) = {9.7×[3-chlorophenol (10 -9 M)] + 0.0005} exp (57×[ozone (10 -9 M)]). In the drinking water treatment, 1-1.5 ppm of ozone, which are corresponding to 20-30 µM, are generally used. The initial velocities of OH radical generation at the 20 µM and 30 µM of ozone concentration in the absence of 3-chlorophenol were calculated to be 0.0016 and 0.0028 µM/s, respectively, using the above equation. Japan Water Works Association reported that more than 11 ppb of phenols were accidentally contaminated in the raw water in 2000. The presence of 10 ppb 3-chlorophenol increases the initial velocities of OH radical generation to 0.0031 and 0.0055 µM/s at the 20 and 30 µM ozone concentrations, respectively, which were corresponding to be 1.9 times faster than those without 3-chlorophenol. On the other hand, the above equation indicates that in the presence of 10 ppb 3-chlorophenol, 1/3 of ozone concentration is enough to produce the same initial velocity of OH radical generation as that without 3-chlorophenol. The kinetic equation for the enhancing effect of 3-chlorophenol obtained in the present paper should provide useful information to optimize the condition in ozone treatment process of water containing phenolic substances. Effect of humic acid on OH radical generation Because of high viscosity of humic acid suspension, batch system was used to evaluate OH radical generation in water containing humic acid during ozonation. 20-60 µM of aqueous ozone was rapidly mixed with DMPO solution containing humic acid at room temperature. After mixing aqueous ozone with DMPO solution, ESR spectrum of quartet lines was observed. The ESR spectrum coincided with those of DMPO-OH adduct as demonstrated previously (Utsumi et al., 1994). Fig. 3. Effect of humic acid on OH radical generation in various ozone, (a) 20 µM, (b) 40 µM, (c) 60 µM. The aqueous ozone was mixed with the solution containing DMPO (100 mM) and different amount of Aldrich humic acid [0 ppm (●), 1 ppm (○), 5 ppm (▼), 10 ppm (▽), 20 ppm (■), 40 ppm (□), 80 ppm (◆), 100 ppm (◇), 150 ppm (▲), 200 ppm (△)], and the concentration of DMPO-OH was determined with ESR measurement. The amount of DMPO-OH increased gradually with time after mixing in ozone and humic acid-dose dependent manner. The amount of DMPO-OH increased gradually and reached plateau almost 60 min after mixing at 20 µM of aqueous ozone (Fig. 3a), although the amount of DMPO-OH reached maximum level within 2 min at 40 µM of ozone (Fig. 3b). At 60 µM of aqueous ozone, the amount of DMPO-OH decreased gradually with time and the decrements were remarkable at high concentration of humic acid (Fig. 3c). Amount of DMPO-OH (µ M) Time (min) (a) (b) 10 20 30 40 50 600 (c) 0.00 0.25 0.50 0.75 1.00 1.25 1.50 10 20 30 40 50 600 1020304050600 Amount of DMPO-OH (µ M) Time (min) (a) (b) 10 20 30 40 50 600 (c) 0.00 0.25 0.50 0.75 1.00 1.25 1.50 10 20 30 40 50 600 0.00 0.25 0.50 0.75 1.00 1.25 1.50 10 20 30 40 50 600 1020304050600 Journal of Water and Environment Technology, Vol.1, No.2, 2003 - 214 - In the previous paper (Utsumi et al., 2003), we demonstrated that the excess generation of OH radical than the trapping capability of DMPO caused disappearance of DMPO-OH signal. The trapping capability is determined with the velocities of OH radical generation, DMPO-OH generation, and reaction between DMPO-OH and OH radical (Kasazaki et al., 2003). If the reaction of DMPO-OH with OH radical precedes the reaction of DMPO with OH radical, the amount of the spin-adduct, DMPO-OH should decrease gradually. In the mixture of 80 ppm humic acid with 60 µM ozone, the velocity of OH radical generation may exceed the velocity of trapping reaction of OH radical with DMPO, causing a decrease of DMPO-OH signal through the reaction of DMPO-OH with OH radical. The enhancing effect of humic acid on OH radical generation was compared with different origin of humic acids from Adrich, Fluka, and ICN (Fig. 4). The amounts of DMPO-OH were almost the same among the three humic acids at 2 min after mixing. In the Aldrich humic acid, the amount of DMPO-OH generation increased gradually and did not decreased till 60 min after mixing (Fig. 4a). However, in the case of Fluka and ICN humic acid, the amount of DMPO-OH decreased gradually at high concentration of humic acids (Fig. 4b, c). The decrements were resembles the result of Fig. 3c. Thus, excess generation of OH radical may occur in the presence of high concentration of Fluka and ICN humic acids, causing a decrease of DMPO-OH signal during incubation. Fig. 4. Effect of origin of humic acids (a; Aldrich, b; Fluka, and c; ICN) on OH radical generation during ozonation. The amounts of humic acids were 0 ppm (●), 1 ppm (○), 5 ppm (▼), 10 ppm (▽), 20 ppm (■), 40 ppm (□), 80 ppm (◆), 100 ppm (◇), 150 ppm (▲), 200 ppm (△). The condition of ESR measurement was described in the legend of Fig 3. According to elemental analysis reported by the companies, Aldrich humic acid consisted of 39.03 % of carbon, 4.43 % hydrogen, 0.68 % of nitrogen. Fluka and ICN humic acids were consisted of over 49 % of carbon, and the other elements were as same as that of Aldrich humic acid. These facts imply the possibility that the OH radical generation might depend on a mount of carbon in humic acids. CONCLUSION The present paper demonstrates the mathematical analysis of OH radical generation in water Amount of DMPO-OH (µM) Time (min) (a) (b) 0.00 0.25 0.50 0.75 1.00 1.25 1.50 (c) 10 20 30 40 50 60010 20 30 40 50 600 1020304050600 Amount of DMPO-OH (µM) Time (min) (a) (b) 0.00 0.25 0.50 0.75 1.00 1.25 1.50 (c) 10 20 30 40 50 60010 20 30 40 50 600 1020304050600 (a) (b)(b) 0.00 0.25 0.50 0.75 1.00 1.25 1.50 (c) 10 20 30 40 50 60010 20 30 40 50 600 1020304050600 Journal of Water and Environment Technology, Vol.1, No.2, 2003 - 215 - containing micropollutants during ozonation. OH radical was trapped with a 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) as a stable adduct, DMPO-OH. The generation of OH radical was demonstrated with mathematical equation of the initial velocity of DMPO-OH generation, and the effect of the compounds, 3-chlorophenol and resorcinol, on OH radical generation expressed with ν 0 (10 -6 M/s) = {9.7~10.5×[compound (10 -9 M)] + 0.0005} exp(57×[ozone (10 -9 M)]). The effect of humic acids on OH radical generation during ozonation was also evaluated with the spin-adduct formation using an ESR spectrometer in batch system. OH radical generated was converted into stable DMPO-OH as a spin-adduct, and the amounts of DMPO-OH were analyzed as a function of the concentration of ozone and humic acids. The amount of DMPO-OH depended both of the amounts of ozone and humic acid. The origin of humic acid also affected the amount of DMPO-OH. ACKNOWLEDGEMENTS This work was supported in part by Grants-in-Aid of Scientific Research, for Research in Priority Areas, for Cooperative Research, for General Science Research, and for Developmental Scientific Research from the Ministry of Education, Science, Sports and Culture, by the Fundamental Research Fund for the Environmental Future from Environmental Agency, Government of Japan, and by a grant from the Takeda Science Foundation. REFERENCE Andreozzi R., Caprio V., Insola A. and Marotta R. (1999). 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Res., 33(10), 2265-2276. . 50 600 (c) 0.00 0.25 0.50 0.75 1. 00 1. 25 1. 50 10 20 30 40 50 600 0.00 0.25 0.50 0.75 1. 00 1. 25 1. 50 10 20 30 40 50 600 10 20304050600 Journal of Water. 0.75 1. 00 1. 25 1. 50 (c) 10 20 30 40 50 60 010 20 30 40 50 600 10 20304050600 Amount of DMPO-OH (µM) Time (min) (a) (b) 0.00 0.25 0.50 0.75 1. 00 1. 25 1. 50