Anal. Chem. 1984, 56, 2849-2850
ythrosine, 16423-68-0;pyridoxamine, 85-87-0;pyridoxol, 65-23-6; phenol, 108-95-2; 15-crown-5, 33100-27-5; hematein, 475-25-2; saccharose, 57-50-1;acetate, 64-19-7; salicyclic acid, 69-72-7;toluol-4-sulfonicacid, 104-15-4;citrate calcium complex, 7693-13-2; Tiron, 149-45-1;alizarin-S, 130-22-3; calmagite, 3147-14-6.
LITERATURE CITED (1) Buffle, J.; Staub, C. Anal. Chem., preceding paper in this issue. (2) Macko. C.;Maler, J.; Eisenrelch, S. J.; Hoffman, M. R . AIChf Symp. Ser. 1978, 75, 163. (3) Buffle, J.; Deladoey, P.; Haerdi, W. Anal. Chlm. Acta 1978, 101, 339. (4) O'Neil, T. L.; Fisette, G. R.; Lindsey, E. E. AIChf Symp. Ser. 1975, 7 1 , 100. (5) Bulbula, A,; Lindsey, E. E.; Archer, R. D.; Lenz, R. W. AIChf Symp. Ser. 1975, 7 1 , 105. (6) Amicon publication no. 426V. Ultrafiltration systems and equipment. Selection guide and catalogue.
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(7) Nuclepore, Catalog Lab 50; Filtratlon Products for the Laboratory, 1980. (8) Good, N. E.; Wlnget, G. D.; Winter, W.; Conally, T. N.; Isawa, S.; Singh, R. M. M. 8iochemlstry 1966, 5, 467. (9) Martell, A. E.; Smith, R. M. "Critical Stablllty Constants"; Plenum Press: New York, 1977; Vol. 1. (IO) Conway, B. E. "Ionic Hydration in Chemistry and Biophysics"; Elsevier: Oxford NY, 1981. (11) Dytnerskll, Y. N.; Polyakof, G. V.; Likavyi, L. S. THeor. Found. Chem. f n g . (fngl. Trans/.) 1972, 6 (4), 565. (12) Buffle, J. I n "Circulation of Metals in the Environment"; Slgel, H., Ed.; Marcel Decker: New York, 1984; Metal Ions in Biological Systems, Vol. 18, Chapter 6.
RECEIVED April 27,1984. Accepted July 24,1984. This work was supported by Project No. 2.413-0.82 of the Swiss National Foundation.
Flow Injection and Photometric Determination of Hydrogen Peroxide in Rainwater with N-Ethyl-N-(sulfopropy1)aniline Sodium Salt Brooks C. Madsen* and Mark S. Kromis Department of Chemistry, University of Central Florida, Orlando, Florida 32816
Flow injection wRh photometric detection is used to determine hydrogen peroxide at environmental concentralions In rainwater. Reagent composition and physical variables were evaluated and optifhired. Prominent features of the method include a hear calibration curve from 1.5 X lo-' to 4 X lob M and a sampling rate of 80 h-'. The method is not susceptlble to interference by Mn( I I ) and Fe( I I I ) which limit utilization of a commonly used iumlnol-hydrogen peroxide chemiluminescence method.
The determination of hydrogen peroxide a t environmental levels in rainwater and in the atmosphere after capture in an aqueous phase is commonly accomplished by a chemiluminescence method which is based on the Cu(I1)-catalyzed reaction of luminol (5-amino-2,3-dihydro-1,4phthalazinedione) with hydrogen peroxide ( I ) . It has recently been shown (2) that the presence of Mn(I1) and Fe(II1) a t concentrations typically found in rainwater interferes and significantly reduces the chemiluminescence signal. Flow injection analysis (FIA) has been shown to be a versatile and valuable tool for both research applications and routine chemical determinations (3). Several applications which use continuous flow analysis (1,2)or FIA (4)have been reported for the determination of hydrogen peroxide. These applications focus upon chemiluminescence detection. A recent communication has described an improved spectrophotometric method for the determination of hydrogen peroxide with new water-soluble hydrogen donors (5). The series of chromophores studied were formed by oxidative condensation of the hydrogen donor with 4-aminoantipyrene (4-AAP) in the presence of hydrogen peroxide and peroxidase. The reported molar absorptivity of 41 300 for the chromophore which results when the hydrogen donor is the N-ethyl-N(sulfopropy1)aniline sodium salt (ALPS) suggests that concentrations of hydrogen peroxide below the reported 6 x IO4 M level can be measured. This communication describes
adaptation of this spectrophotometric method to a FIA procedure for determination of hydrogen peroxide in rainwater. Reagent dependence, temperature, and FIA system variables are considered. The selected operating conditions permit up to 80 Pamples h-l to be processed. A detection limit of about 1.4 X lom7M can be achieved. A linear calibration curve is obtained, and results are compared with those obtained by continuous flow chemiluminescence.
EXPERIMENTAL SECTION Apparatus. Two flow systems were used during method development. The flow system illustrated in Figure 1represents the final configuration. Preliminary experiments were performed with a flow system where the single mixed reagent line as illustrated in Figure 1was replaced with three separate reagent lines. The latter system facilitated evaluation of individual reagent concentrations when all but one reagent concentration is held constant. Teflon tubing (0.81-mm i.d.) was used for manifold construction except for pump tubing which was short sections of Tygon tubing. A Buchlkr 2-6100 polystatic pump and Altex 202-00 manual rotary injection valve with a 0.58-mL sample loop were used. Absorbance measurements were made at 560 nm with a Perkin-Elmer 552 spectrophotometer equipped with a 1-cm flow-through cuvette (Helma Type 178.12, volume 0.018 mL). Absorbance was determined from peak height measurements recorded on a Perkin-Elmer Hitabhi Model 57 X-Y recorder. Lower concentrations were measured at 0.05 AUFS scale expansion. Base-line noise was approximately 0.001 AU. Reagents. The 4-aminoantipyrene (Aldrich) and peroxidase (90 U mg-'; Sigma, P-1825) were used as received. The ALPS was synthesizedfrom N-ethylaniline and 1,3-propanesultone (both Aldrich) as previously described (5). Buffer solutions were prepared from reagent grade cbemicals. Hydrogen peroxide, 30%, standardized with permanganate (6) was used to prepare calibration standards. All solutions were prepared with deionized water obtained from a Cidligan SR polishing system (Culligan). Metal ion solutions were prepared from reagent grade MnS04.H20, Fe(NH4)(S0,),.12Hz0, Co(NH4),(SO4),.l2H20, Ni(NH,)%(S04)2.6820,ZnSO4.7Hz0, and CuS04.5H20. The FIA mixed reagent used to obtain analytical results consisted of 2.0 mM ALPS, 0.8 mM 4-AAP, pH 5.83 phosphate buffer (56 mM H2P04-, 8.0 mM HPOA2-),and 23 mg L-' peroxidase.
0003-2700/84/0356-2849$01.50/0 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56,NO. 14, DECEMBER 1984 Flow Rate
Table I. Determination of Hydrogen Peroxide in Rainwater Samples
mL min"
H20
4.5
-
sample
FIA, M (XIOs)
1 2
3.15 9.35
-12 -51
3 4
0.15 6.91
60 -21
5 6 7 8
1.15 2.32 16.5 22.9
-18 -5.1 -55 -50
560 nm
INJECTOR
CL
percent difference vs. FIA with with Mn(I1)" Fe(III)b FIA CL FIA CL FIA
PUMP
Flgure 1.
Components of the flow injection system.
The continuous flow chemiluminescence determinations of hydrogen peroxide were performed as described (2) except a Waters Model 420 fluorescence detector with excitation source removed was used to measure the chemiluminescence signal. A Tygon flow cell similar to that described previously was constructed and used (7).
RESULTS AND DISCUSSION Flow Injection System Variables. Design features of the FIA manifold affect operating characteristics of the system. The proposed manual method for H202determination (5) includes color development for 10 min a t 37 "C. These conditions suggest that FIA would not be appropriate as an alternative to the manual method because one characteristic of most FIA systems is rapid sample throughput. Preliminary measurements showed that color development at elevated temperatures was nearly instantaneous. Color development observed a t ambient temperature in the FIA system where sample residence time was at least 15 s matched that observed a t elevated temperatures. Selection of the FIA system illustrated in Figure 1was based upon evaluation of flow rates, manifold mixing coil length, and injection loop size. When flow rates are changed from 2.0 to 6.5 mL min-l, a decrease in maximum absorbance of less than 10% is observed. Mixing coil length changes from 1.4 to 3.5 m result in a decrease of about 20% in maximum absorbance. The absorbance signal increases in proportion to sample loop size up to 0.6 mL. Chemical Variables. Those reagents which directly influence formation of the absorbing species were evaluated to determine optimum concentrations that provide high absorbance without utilization of a large excess of reagents. Various concentrations of ALPS, 4-AAP and peroxidase and pH were evaluated. Although the flow system with separate reagent lines are used to evaluate these effects, results have been presented as they relate to the system illustrated in Figure 1. When ALPS, 4-AAP, and peroxidase concentrations are increased, the absorbance signal also increases. Dramatic changes are observed initially followed by more gradual increases. Peroxidase gave no increase above 23 mg L-' while concentration increases for ALPS from 0.86 to 1.7 mM and for 4-AAP from 0.46 to 0.92 mM gave increases of 15% and 1070,respectively. Phosphate, acetate, and succinate buffers were utilized to evaluate the optimum pH range. The three buffer systems gave comparable results over the range pH 4.4-6.9 with maximum color development occurring from pH 5.0 to 6.0. Previous work (5) had not evaluated pH dependence of the reaction below pH 5.5. A mixed reagent which includes ALPS, 4-AAP, peroxidase, and buffer when prepared daily yields results comparable to those obtained with the multiple reagent line FIA system. Analytical Results. Preliminary results showed that the calibration curve for the selected conditions is linear to approximately 5 X M H2OP Evaluation of the proposed method was accomplished by determination of HzOz in eight rainwater samples. Eight working standards from 3.56 X to 1.80 x IOv7 M were used for calibration. The resulting
2.2 -80 4.OC
-2.5 2.2'
-50 -31
-2.1
-83
-1.7 -4.3c
-3.8
-11
-3.8
1.3
-93
-1.8
-1.3 -57
-2.1d -2.5' -1.3f
"3.6 X lo4 M Mn(I1) except as noted. *3.6 X lo4 M Fe(II1) M Mn(I1) or Fe(II1). d1.8 X M except as noted. c1.8 X Co(II), Cu(II), Ni(II), Zn(I1). e1.8 X M Mn(II), Fe(III), Co(II), Cu(II), Ni(II),Zn(I1). '3.6 X 10" M Mn(II),Fe(III),Co(II), Cu(II),
Ni(II), Zn(I1). calibration curve was obtained by injecting standards, and samples in a sequence which ensured at least duplicate measurements can be described as follows: Absorbance = (6.50 f 0.04) X lo3 [H202] (-0.001 f 0.001) with a standard error of estimate 0.002 for n = 27. The mean absorbance and standard deviation observed for 4.47 x lov6 M and 8.9 X lo-' M standards during calibration were 0.0272 f 0.0009 ( n = 6) and 0.0053 f 0.0003 ( n = 5), respectively. Determination of H202'in the rain samples is summarized in Table I. Separate portions of samples 1,5, and 7 were spiked with 3.44,0.68, and 3.44 pM HzOz,respectively. Recoveries were 3.32,0.56, and 3.20 pM by the FIA method and 1.79,0.82, and 2.00 pM by the chemiluminescence (CL) procedure, respectively. These spike recoveries and results summarized in Table I illustrate the erratic and generally lower results obtained by the CL procedure. Four samples were subjected to additional study after addition of Mn(I1) and Fe(II1) which have been shown to cause interference with the CL method (2). Results based on the CL method are decreased when Mn(I1) or Fe(II1) are added while results from the FIA method remain essentially unchanged; see Table I. The addition of up to 3 X M of Zn(II), Ni(II), Co(II), and Cu(1I) individually or combined with Mn(I1) and Fe(II1) did not alter FIA results. The method has been demonstrated to be rapid and precise for concentrations of H202up to 4 x M. Accuracy compared to the luminol CL procedure is improved because Mn(I1) and Fe(II1) interference is not intrinsic to the described method.
+
LITERATURE CITED (1) Kok, G.L.; Holler, T. P.; Lopez, M. 9.;Nachtrieb. H. A.; Yuan, M. Environ. Sci. Techno/. 1978, 72, 1072. (2) Ibusu'ka, T. Atmos. Environ. 1983, 77, 393. (3) Ruzlcka, J.; Hansen, E. H. Anal. Chim. Acta 1980, 174, 19. (4) Olsson, B. Anal. Chim. Acta 1982, 136, 113. (5) Tamaoku, K.; Murau, Y.; Aklura, K.; Ohkura, Y. Anal. Chim. Acta 1982, 736, 121. (8) Day, R. A,, Jr.; Underwood, A. L. "Quantitative Analysis Laboratory Manual", 4th ed.; Prentice-Hall: Englewood Cliffs, NJ, 1980; pp 98-99. (7) Nakahara, S.;Yamada, M.; Suzuki, S. Anal. Chlm. Acta 1982, 747,
255.
RECEIVED for review July 9, 1984. Accepted September 10, 1984. This work was supported in part by the University of Central Florida Division of Sponsored Research.
