1192
Anal. Chem. 1986, 58, 1192-1194
ACKNOWLEDGMENT We gratefully acknowledge the technical assistance of Judy Sophianopoulos and Matthew T a r r for acquisition and preparation of the oil refinery sample discussed in this paper. Registry No. Nap, 91-20-3; Acy, 208-96-8;Ace, 83-32-9;Flo, 86-73-7;Phe, 85-01-8;Ant, 120-12-7; Flu, 206-44-0; Pyr, 129-00-0; 1,2-BA,56-55-3;Chr, 218-01-9; BbF, 205-99-2; BkF, 207-08-9 B e , 50-32-8; DPA, 53-70-3; IPy, 193-39-5;BPe, 191-24-2;Per, 198-55-0.
LITERATURE CITED Merller, M.; Alfhein, J. Atmos. Environ. 1980, 14, 83. Salamone, M. F.; Heddle, J. A,; Katz, M. Environ. I n t . 1979, 2 , 37. Josephson, J. Environ. Sci. Techno/. 1981, 15,20. "Chemical Analysis and Biological Fate: Polynuclear Aromatic Compounds"; Cooke. M., Dennis, A. J., Eds.; Battelle: Columbus, OH, 1980. (5) "Polycyclic Hydrocarbons and Cancer"; Gelboin, H. V., Ts'o, P. 0. P., Eds.; Academic Press: New York, 1978. (6) Lee, M. L.; Novotny, M.; battle, K. D. "Analytical Chemistry of Polycyclic Aromatic Hydrocarbons"; Academic Press: New York, 1981. (1) (2) (3) (4)
(7) "Polynuclear Aromatic Hydrocarbons"; Jones, P. W., Leber, P., Eds.; Ann Arbor Sclence: Ann Arbor, MI, 1979. (8) "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjorseth, A., Dennis, A. J., Eds.; Battelle: Columbus, OH, 1980. (9) Natusch, D. F. S . ; Tomkins, B. A. Anal. Chem. 1978, 50, 1429. (10) Wise, S. A.; Cheder, S . N.; Hertz, H. S . ; Hilpert, L. R.; May, W. E. Anal. Chem. 1977, 4 9 , 2308. (11) Sonnefeld, W. J.; Zoller, W. H.; May, W. E ; Wise, S . A. Anal. Chem. 1882, 5 4 , 723 (12) Chmieiowiec, J.; George, A. Anal. Chem. 1980, 52, 1154. (13) Bjerrseth, A. Anal. Chim. Acta 1977, 9 4 , 21. (14) Swanson, D. H.; Walling, J. F. Chromatogr. Newsi. 1981, 9 , 25. (15) Swartz, G. P.; Daisey, J. M.; Lioy, P. J. Am. Ind. Hyg. Assoc. J . 1981, 42, 258. (16) Lee, F. S.-C.; Pierson, W. R.; Ezike, I . "Polynuclear Aromatic Hydrocarbons: Chemlstry and Biological Effects"; Bjerrseth, A,, Dennis, A. J., Eds.; Battelle: Columbus, OH, 1980, 543.
RECEIVED for review July 12, 1985. Resubmitted January 9, 1986. Accepted January 14,1986. This work was supported by the Department of Energy Grant DE-AS05-82ER60100.
Derivatization Technique for the Determination of Peroxides in Precipitation Gregory L. Kok,* Kathleen Thompson,' and Allan L. Lazrus
National Center f o r Atmospheric Research, P.O. Box 3000, Boulder, Colorado 80307 Scott E. McLaren
Atmospheric Science Research Center, State University of N e w York at Albany, Albany, N e w York 12222
A derivatization technique has been developed for the combined determination of hydrogen peroxide and some organic hydroperoxidesin precipitation samples. A fluorescent dimer is formed via the reaction of peroxides with p-hydroxyphenylacetic acid and horseradish peroxidase. The resulting dimer Is stable for a minimum of 5 days, eliminatlng dlfficulties caused by the decomposition of peroxides in stored samples. The detection limit is 3 X lo-* M, based on 3 times the standard deviation of the blank.
The accurate determination of hydrogen peroxide (H,O,) in cloud and precipitation samples is important for determining the amount of H 2 0 2 available for the oxidation of bisulfite ion (1,2). Measurements of H20zin cloud water a t Whiteface Mountain, NY, show a strong seasonal variation, with winter concentrations on the order of M and summer concentrations 2 orders of magnitude higher ( 3 ) . However, a major difficulty in obtaining reliable analytical data is the rapid decomposition of H,Oz in collected samples. Decomposition rates of up to 5% per hour have been measured ( 4 ) . Under these conditions it is important t h a t HzOzbe determined immediately or derivatized for accurate analytical results. In this paper a technique is presented to rapidly derivatize peroxides to the stable p-hydroxyphenylacetic acid dimer, which can be quantified by fluorescence several days after prepararation. 'Present address: 1529 Seymour Ave., North Chicago, IL 60064. 0003-2700/86/0358-1192$01.50/0
Table
I. P e r o x i d e R e a g e n t C o m p o s i t i o n o
components
concn, M
tris(hydroxymethyl)aminomethaneb(Tris)
0.5 Na2EDTAc 0.005 formaldehyde (HCHO)d 0.26 p-hydroxyphenylacetic acid' 0.15 horseradish peroxidase' g aAdjust the final pH to 9.0 using concentrated HC1. bFisher Scientific. Mallinckrodt Chemical. Baker Chemical, 37% stock solution. e Fairfield Chemical, Co., Blythewood, SC. {Sigma Chemical Co., P-8250, Type 11. 9150 units/100 mL solution.
THEORY T h e analytical procedure for peroxides is based on the peroxidase enzyme fluorescence technique developed by Lazrus et al. (4). This technique uses the formation of a fluorescent dimer from the reaction of hydroperoxides, p hydroxyphenylacetic acid, and peroxidase for quantitation. The reaction chemistry is as follows: CHzCOOH CHzCOOH
CH2COOH
PEROXIDASE
OH
OH
OH
Extensive studies have shown the technique to be reliable and free from interferences typically present in precipitation samples. T h e original implementation of the peroxidase analytical technique utilizes a dual-channel flow system 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
1193
(mllmin)
Table 11. Time Stability and Interference Test Solution Composition cation
concn, pm
anion
concn, pm
Cd(I1) Cr(II1) Co(I1) Ni(I1) Mn(I1) V(V) Pb(I1) Fe(II1) Cu(I1) Zn(I1) MI) NH4+ H+
0.05 0.05 0.05 0.05 0.05 0.05 0.5 0.5 0.5 0.5 5 20 90
S042NOSc1HCOO-
30 30 22 5 I
WASTE
Figure 1. Flow system for fluorometric determination of the dimer. The valve is used for manual sample injection only.
tively insensitve to the reagent-to-sample ratio. At peroxide M and above, care must be taken to keep concentrations of the reagent-to-sample ratio reasonably constant. In these studies the stock reagent solution and all samples were stored a t 4 "C in srew-cap polyethylene culture vials. This minimizes time-dependent formation of the dimer from the p hydroxyphenylacetic acid. If the reagent or the derivatized samples are subjected to elevated temperatures, the p-hydroxyphenylacetic acid will spontaneously convert to the dimer. At 20-25 "C the change in the response measured from a blank is less than 1 X M/day as HzOz. For blanks stored a t 35 "C this change increases to over 1 X lo4 M/day. The reagent should be prepared fresh every 4 days since the peroxidase activity decreases and reproducibility of the measurement decays beyond that period. The reaction with HzOzto form the dimer is complete in less than 60 s. The reaction of p-hydroxyphenylacetic acid and peroxidase with peroxyacetic acid is faster than with HzOzand slower by a factor of 2 or 3 for methyl hydroperoxide and other short, straight-chain organic hydroperoxides (5, 6). The dimer is quantitated by fluorescence detection using an excitation wavelength of 320 nm and measuring the emission intensity a t 400 nm. Since the analytical procedure does not require any critical timing procedures, the analysis can be performed either by manual means in a batch mode or by using a flow system. If the analysis is performed manually, sufficient aqueous NaOH must be added to the reaction mixture to raise the pH above 10. This pH assures maximum fluorescence yield from the dimer. If a large number of samples are to be analyzed, it is useful to implement the flow system, shown in Figure 1. This provides for automatic addition of NaOH and will give better reproducibility than manual fluorescence analysis. In this system HzOis the sample carrier and a rotary or slide injection valve with a 1-mL loop is used for sample addition. The flow system can be adapted to operate with an automatic sampler by replacing the HzO reservoir with the automatic sampler and removing the injection valve. Any type of unpressurized automatic sampler can be used. In these studies all analyses were carried out using the flow system with a Technicon Instrument Autosampler IV having a 1:2 sample-to-wash ratio. Standards for HzOzare prepared by serial dilution of a stock H20zsolution ( 4 ) . Water obtained from most cartridge deioniM or more HzOz. zhtion systems or distillation units contains In preparation of standards, this "intrinsic" HzOzwill displace the intercept of the calibration curve. At peroxide concentrations of lo-' M and above this will not present a problem. Below peroxide concentrations of 10" M this "intrinsic" peroxide gives
"All chemicals are reagent grade. ~~
chemistry for determination of both the total peroxide content and total peroxides less hydrogen peroxide (4). The difference between the two channels gives t h e net HzOzconcentration. In this study, t h e fluorescent dimer is formed in a batch reaction at the point of sample collection. Both HzOz and organic hydroperoxides are converted t o the stable dimer, so that analysis can be carried out at a later time and a t a location separate from the sample collection point.
EXPERIMENTAL SECTION The components of the fluorometric reagent and concentrations used are given in Table I. The Tris acts as a buffer to maintain the pH of the final solution between 8 and 9. Under the reaction conditions employed in this analytical technique, this pH range gives the best stability of reagent and reaction products. Potential interferences are masked by the use of EDTA to complex the transition metals and HCHO to reduce interference from hydroxymethanesulfonate. The p-hydroxyphenylacetic acid and peroxidase comprise the active components in the reagent. The reagent has been designed to derivatize peroxides up to concenM for a 1 5 0 volume dilution of reagent to trations of 1 X sample. If a higher concentration of peroxide is anticipated, the dilution must be reduced to provide sufficient p-hydroxyphenylacetic acid for dimer formation. If very low concentrations (
