Anal. Chem. 1990, 62, 2381-2384 2381

Nonenzymatic Method for the Determination of Hydrogen Peroxide in Atmospheric Samples

Jai H. Lee* a n d Igna t ius N. Tang Environmental Chemistry Division, Brookhaven National Laboratory, Upton, New York 11973

J u d i t h B. Weinstein-Lloyd

ChemistrylPhysics Program, State University of New YorklOld Westbury, Old Westbury, New York 11568

A nonenzymatic method is developed and compared with the well-known enzymatic ( p -hydroxyphenyl)acetic acid (pOH- PAA) method for the determination of hydrogen peroxide in aqueous atmospherlc samples. The new method is based on the Fe(I1)-catalyzed oxidation of benzoic acid by H,O, to form hydroxylated products (OHBA), whkh are analyzed by fluorescence detectlon. The limit of detection and linear re- sponse range of the new method are comparable to those of the pOHPAA technique. I n addition, the new Fenton-OHBA method has the advantage of using inexpensive, stable, easily available chemical reagents that do not require refrigeration. The new method is Insensitive to moderate transition-metal concentrations often found in atmospherlc samples.

INTRODUCTION As a principal oxidant of atmospheric S(IV), hydrogen

peroxide is believed to play an important role in acid de- position (1-4), and its availability may control the aqueous- phase conversion of SOz to sulfate in the Eastern United States (5). In recent years, numerous field measurements have mapped the temporal, seasonal, and geographical concentra- tions of atmospheric hydrogen peroxide in gas and aqueous phases. H202 concentrations averaging several micromolar, but occasionally exceeding 100 WM in precipitation (6-8) and cloudwater (5,8, 9), 200 ppb in ice ( lo) , and several ppbv in air (11, 12), have been reported.

Techniques for the determination of atmospheric hydrogen peroxide have been the subject of substantial research efforts. Early measurements employing the absorbance of the violet complex formed in a solution of Ti(IV), HzOz, and 8-quinolinol (13, 14) suffered from low sensitivity and substantial inter- ference from organic hydroperoxides and ozone. A similar technique, involving absorbance measurements of the oxo- dioxo complex formed when V(V) reacts with dioxo- pyridine-2,6-dicarboxylate and Hz02, exhibited comparable sensitivity (15). Kok et al. made use of the chemiluminescent oxidation of luminol (1,4-phthalazinedione) by H202 in the presence of copper as a catalyst (16). In addition to requiring purification of the luminol, this technique exhibited negative interference from transition metals (1 7). A modification of the technique, using hemin as a catalyst, led to improved sensitivity but still suffered from interferences (18). Klockow and Jacob developed a fluorescence method based on the oxidation of bis(trichloropheny1) oxalate by HzOz in the presence of perylene (19). The authors note that an Fe(II1) impurity interferes a t M. The horseradish peroxidase (HRP) catalyzed destruction of scopoletin fluorescence upon reaction with H202 was used as the basis of an analytical technique by Zika et al. (20, 21). Because it makes use of fluorescence quenching, the scopoletin method is not as convenient or sensitive as direct fluorescence methods.

Among the more widely used methods is a fluorescence technique originated by Guilbault (22) and further developed

by Lazrus et al. (23). This method is based upon the reaction between peroxide and (p-hydroxypheny1)acetic acid (pOH- PAA), catalyzed by HRP, to generate a fluorescent dimer. Although the technique is highly sensitive and relatively free from interferences, it suffers the disadvantages common to many enzyme assays, notably reagent instability and high cost.

Several investigators have compared HzOz analysis methods by simultaneous determination of samples by two or more techniques. In one comparison, Lazrus (23) found that luminol consistently gave a higher reading for Hz02 concentration than pOHPAA. The luminol technique did, however, produce reasonable agreement with the Ti(IV)-H202-quinolinol me- thod (24). Analysis by pOHPAA agreed well with the per- oxyoxalate chemiluminescence technique (25). Measurement of gaseous HzOz by scrubbing air on a common manifold revealed the pOHPAA technique to be 1 order of magnitude more sensitive than the luminol technique and less subject to gas-phase interferences (26).

We have developed an analytical scheme based upon the well-known Fenton reaction, in which ferrous ion reacts with hydrogen peroxide to yield the hydroxyl radical (27). The hydroxyl radical is scavenged by benzoic acid (BA) to form isomeric hydroxybenzoic acids (OHBA), which fluoresce strongly (28). In this work, the validity of the nonenzymatic method is established by simultaneous analysis of H,02-spiked atmospheric precipitation samples by the Fenton-OHBA and pOHPAA methods.

EXPERIMENTAL SECTION Chemicals. Ferrous sulfate, sulfuric acid, and hydrochloric

acid (Mallinckrodt AR), 0- (Aldrich ACS reagent), m- (Aldrich 99%), and p-hydroxybenzoic acid (Aldrich 99+% gold label), and benzoic acid (Baker 99.8%) were used without further purification. All reagents were prepared by using high-purity water from a Millipore ultrapurification system.

pOHPAA reagent contained 4.5 mM pOHPAA, 0.20 mM EDTA, and 1.8 units/mL horseradish peroxidase. The solution pH was adjusted to 8.5 with Tris buffer and HC1. (p-Hydroxy- pheny1)acetic acid ( Eastman 98 % minimum), hydrochloric acid (Mallinckrodt AR), EDTA (Sigma 99.5%), Tris buffer (Sigma reagent), and horseradish peroxidase (Sigma Type VI, P8375) were used without further purification.

Apparatus and Procedures. Fenton-OHBA analysis was carried out by using a single-channel modification of the apparatus developed by Lazrus (23), represented schematically in Figure 1. Three inlet ports are used, one each for the H20p sample, ferrous sulfate/benzoic acid solution, and NaOH. The air- stripping coil was retained for future application of the technique to the measurement of gaseous HzOz, and aqueous HzOz samples were introduced through the stripping solution port. Room air entering the stripping coil at a rate of 1.05 L min-' was freed of gaseous H202 by a Hopcalite column. After separation of air from the liquid phase in a vertical tube, the HzOz stream joined the liquid stream containing ferrous sulfate and benzoic acid. Hy- droxyl radicals produced from the H2O2-Fe(II) reaction were scavenged by benzoic acid in a reaction coil. The solution con- taining hydroxylated products was made alkaline with 0.18 M NaOH to enhance fluorescence intensity (28). Air bubbles were

0003-2700/90/0362-2381$02.50/0 0 1990 American Chemical Society

2382 ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990

Room Air

Stripping Soiution or ~ ~ ~ L f ; ~ ~ ) H202 Sample (0 32mL / min) t

\ n

Waste

Waste

Waste

Waste

Flgure 1. Schematic diagram of the Fenton-OHBA analytical system.

introduced into the flow stream to enhance mixing and were removed by a debubbler before analysis. Fluorescence mea- surements were made with a Kratos 950 filter fluorometer equipped with a 0.1-cm flow-through cell, mercury lamp, Schott UGll wide-band excitation filter (Aex = 313 nm), and an L399 cutoff emission filter (Aem > 399 nm).

Ferrous sulfate/benzoic acid solution contained 1.5 mM ferrous sulfate and 3.0 mM benzoic acid dissolved in 0.016 M sulfuric acid. Reagents generally contained a low concentration of fluorescent impurities; the signal reported in this work is the difference between the fluorescence intensity with and without HzOz.

The system used for hydrogen peroxide analysis by the pOH- PAA technique has been described elsewhere (8). Briefly, each peroxide sample was mixed with an equal volume of pOHPAA reagent and introduced through an injection valve into a flow of deionized water. The formation of a fluorescent dimer from the HRP-catalyzed reaction between pOHPAA and H202 is complete before injection, and the dimer is stable for several hours. The flow stream passed through a 1.0-cm flow-through cell, and fluorescence was monitored by a Perkin-Elmer Model 201 spec- trofluorometer. The excitation and emission monochromators were set at 321 and 401 nm, respectively.

Calibration curves for both methods were obtained daily, by using hydrogen peroxide standards prepared from 3% H202, which had been titrated against standardized potassium permanganate.

Precipitation samples for peroxide analysis were collected at ground level in polyethylene containers and refrigerated in rig- orously cleaned Pyrex vessels. These samples were spiked with microliter quantities of H202 stock solution for the comparison study.

RESULTS AND DISCUSSION

nique is summarized by the following scheme: Reaction Scheme. The Fenton-OHBA analytical tech-

(1)

m-OHC6H5COOH, and p-OHC6H5COOH (2)

Fe(I1) + HzOz - Fe(II1) + OH- + OH OH + CGH~COOH ---* o-OHC~H~COOH,

OH + Fe(I1) - Fe(II1) + OH- (3) The rate constant for eq 1, known as the Fenton reaction, is 57.8 M-' s-l (29) in solutions containing ferrous sulfate in sulfuric acid. Under our experimental conditions, complex- ation of Fe(I1) with benzoate may alter the rate of reaction 1. Reaction 2 is nearly diffusion-controlled, a t 4.3 X lo9 M-l s-l (30). To minimize scavenging of OH radicals by reaction 3, which is characterized by a second-order rate constant of 4.3 X lo8 M-' s-l (31), we maintained the BA and Fe(I1) concentration in a t least a 2:1 ratio.

Reaction Conditions. The optimum conditions for pro- duction of OHBA were determined by varying the pH for reaction 1, Fe(I1) concentration, reaction time, and pH of the fluorescent products.

Solutions of benzoic acid and ferrous sulfate are stable to air oxidation for several days as long as the pH is kept below 2.5. At a higher pH, one observes a large, nonreproducible background fluorescence signal. This effect can be attributed to ferrous ion catalyzed air oxidation of benzoic acid, as no

30 0 0.5 1 .o 1.5 2.0 2 5

[ Fe ( n ) 1 (mM)

Flgure 2. Dependence of the fluorescence signal on Fe(1I) concen- tration. Experimental conditions: pH = 1.8, [H202] = 4.1 X M, 2-min reaction time, 3.0 > [BA]/[Fe(II)] > 2.0.

significant fluorescence is observed in neutral air-saturated benzoic acid solutions in the absence of Fe(I1) or in such solutions containing Fe(I1) if they are deoxygenated.

The fluorescence signal is virtually insensitive to changes in pH when reaction 1 is carried out below pH 2.5. In these experiments, OHBA isomers were produced in the reaction coil at pH 1.8. Before detection, the solution pH was rapidly raised to 11.6 to optimize the fluorescence intensity of OHBA (see below). However, a t this pH, the ferrous ion is rapidly oxidized to the ferric ion. Fe(II1) is unstable in the resulting solution and slowly precipitates on standing, presumably as the hydroxide and/or oxide. The presence of colloidal iron decreases the signal, an interference that worsens as the Fe(II1) concentration increases. However, it is desirable to keep the Fe(I1) concentration as high as possible to accelerate the rate of reaction 1. This reasoning prompted us to seek an Fe(I1) concentration that would maximize the rate of formation of OHBA while minimizing the quenching of the fluorescence signal by Fe(II1). The dependence of the fluorescence signal on Fe(I1) concentration, shown in Figure 2 for a single peroxide concentration and a 2-min reaction time, indicates that the optimum ferrous ion concentration lies between 1 and 2 mM.

The dependence of fluorescence signal on reaction time for reaction 1 is shown in Figure 3 for several concentrations of Fe(I1). As expected from kinetic considerations, the signal reaches a plateau at earlier times for higher Fe(I1) concen- trations. The figure also illustrates the fluorescence quenching described earlier, which diminishes the ultimate plateau signal for solutions with higher iron concentrations. I t is evident from Figure 3 that the highest signal is produced by using the lowest possible concentration of Fe(I1). However, this in- creased sensitivity occurs at the expense of an unacceptably long analysis time. In these experiments, we utilized a 1.5 mM Fe(I1) solution, which produced the highest fluorescence signal under the constraint of a 2-min reaction time.

The pH dependence of the fluorescence intensities of isomeric hydroxybenzoic acids has been studied (28). The fluorescence signal increases dramatically a t the pK of the OHBA isomers and reaches a plateau at pH 5 and 11.6 for the ortho and meta isomers, respectively. In calibration studies using our apparatus, the fluorescence of the meta isomer was approximately twice that of the ortho at pH 11.6 (Figure 4). However, upon increasing the pH of the OHBA product flow stream from 5 to 11.6 in actual analyses, we observed an increase in fluorescence intensity by a factor of 2.2 . This suggests that 0- and m-OHBA are produced in a

ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990 2383

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1 0 2 4 6 8 10

REACTION TIME (mm)

Figure 3. Dependence of fluorescence signal on reaction time: (A) 0.55 mM; (+) 0.75 mM; (0) 1.49 mM; (0) 1.80 mM; (0) 2.09 mM. Experimental conditions: pH = 1.8, [H202] = 4.1 X IO-' M, 3.0 > [BA]/[Fe(II)] > 2.0, [Fe(II)]. Data for 1.49 mM has reached 90% of the plateau value at 2 min.

150

z 8

" 0 0.8 1.60 2.40 3.20 4.00

[ OHBA 1 (PM) Figure 4. Dependence of fluorescence signal on concentrations of 0- and m-OHBA at pH 11.6.

2:l ratio. The para isomer does not fluoresce under the conditions employed.

Calibration and Sensitivity. The calibration curve for Fenton-OHBA analysis under our experimental conditions is linear for H202 concentrations between lo4 and lo4 M. The detection limit of this technique is 2 X M, based on a signal to noise ratio of 1. However, as shown in Figure 5, the fluorescence intensity begins to deviate from a linear response to H202 concentrations above 5 X loa M. Regression analysis of the linear portion of the calibration curve gives a standard deviation of 1% for the slope and a correlation coefficient of 0.999.

It has been reported that various EDTA complexes of Fe(I1) produce OH via reaction 1 but at significantly faster rates (32). Such a rate increase would enhance the sensitivity of the Fenton-OHBA technique by decreasing the concentration of Fe(I1) required for analysis and thereby decreasing the extent of fluorescence quenching by Fe(II1). Our experiments uti- lizing Fe(I1) complexes in reaction 1 resulted in a large,

80 I /'I

I H , 0,l (lw Figure 5. Calibration curve for Fenton-OHBA analysis. Experimental Conditions: pH = 1.8, 2.0-min reaction time, [BA]/[Fe(II)] = 2.0.

unstable background reading, apparently due to Fe-com- plex-catalyzed air oxidation of benzoic acid, and were therefore discontinued.

Interferences. Chemical analysis of precipitation samples from the Northeastern United States has shown the presence of the following species: Hz02, HCHO, H+, NOs-, SO4%, NH4+, Na+, C1- (6). In our experiments, the presence of 0.4 mM NH4N03, 0.4 mM NaC1, and 0.08 mM HCHO did not affect H202 concentrations measured by the Fenton-OHBA tech- nique. To address the possibility of a spurious signal due to reaction of S(1V) with Fe(III), we observed the fluorescence signal of a sample containing 10 r M S(1V) (33). The absence of a signal under these conditions demonstrates that S(1V) does not interfere.

Field investigations have shown that organic hydroperoxides can comprise a significant fraction of the total peroxides in atmospheric samples (8,34-37). Unlike the Fe(II)-H202 re- action (l), the Fe(I1)-ROOH reaction does not give rise to an OH radical, and is therefore not expected to produce OHBA (38,39). Because of the presence of millimolar levels of the ferrous ion, this technique is also insensitive to the micromolar levels of transition metals often found in atmospheric samples.

Comparison of Fenton and pOHPAA Techniques. Precipitation samples collected on Long Island, NY, were spiked with H202 and analyzed simultaneously by the Fen- ton-OHBA and the pOHPAA methods. Linear regression analysis of H202 concentration determined by the Fenton- OHBA method versus that determined by the pOHPAA method gives a slope of 0.96 and a correlation coefficient of 0.997 (Figure 6). The comparison showing good agreement over a wide range of H202 concentration clearly indicates the utility of the Fenton-OHBA method for analysis of atmos- pheric samples.

CONCLUSION We have developed and demonstrated the use of the Fenton

reaction as an analytical tool for the measurement of H202 and have shown it to be sensitive, linear, and to give results consistent with that of the frequently used pOHPAA method. Moreover, the present technique has the advantage of using inexpensive, easily available nonenzymatic reagents that do not require refrigeration and of being insensitive to the moderate concentration of inorganic ions and transition metals

2384 ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990

c

t A 1 0 5 /"i

L

E - - 1 - N C

t

t t

4

POHPAA[ H,O,] (M) Flgure 8. Comparison of Fenton-OHBA and pOHPAA technique. Measurements are made on precipitation samples containing added H,O,; the solid line represents a slope of 1.

often found in atmospheric samples. Because of its high sensitivity and lack of interferences, the Fenton-OHBA me- thod can easily be adopted for gaseous HzOz measurement at ambient concentration levels.

ACKNOWLEDGMENT Helpful discussions with B. H. J. Bielski, D. Cabelli, J. Fajer,

S. E. Schwartz, and L. Newman during this study are ap- preciated. We are indebted to Y.-N. Lee and S. Springston for providing instruments for this work and to D. Leahy for technical assistance.

Registry No. FeSO,, 7720-78-7; benzoic acid, 65-85-0; water, 7732-18-5; HZOB, 7722-84-1.

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RECEIVED for review April 27, 1990. Accepted July 30,1990. This material is based upon work supported in part by Na- tional Science Foundation Grants ATM-8808480 and ATM- 8911296 to J.W.-L. and performed under the auspices of the United States Department of Energy under Contract No. DE-AC02-76CH00016.