594
Anal. Chem. 1986, 58,594-597
Registry No. Sephadex G-25,9041-35-4;phosphate, 1426544-2; water, 7732-18-5.
(IO) Matsuhlsa, K.; Ohzeki, K.; Kambara, T. Bull. Chem. SOC.Jpn. 1982,
LITERATURE CITED
(11) Yoshlrnura, K,; Motomura, M.; Tarutani, T.; Shlrnono T. Anal. Chem. 1984, 56, 2345-2349. (12) Yoshimura, K.; Waki, H. Talanta, In press.
(1) Kircher, C. C.; Crouch, S. R. Anal. Chem. 1982, 5 4 , 879-864. (2) Heslop, R. E.; Person, E. F. Anal. Chlm. Acta 1987, 39, 209-221. (3) Harry, L.; Rowe, J. J.; Grirnaldi, F. S. Anal. Chem. 1955, 27, 258-262. (4) Yoshirnura. K.; Waki, H.; Ohashi, S. Talanta 1978, 2 3 , 449-454. (5) Yoshirnura, K.; Nigo, S.; Tarutani, T. Talanta 1982, 2 9 , 173-176. (6) Waki, H.; Korkisch, J. Talanta 1983, 30,95-100. (7) Yoshimura, K.; Waki, H. Talanta 1985, 32,345-352. (6) Tanaka, T.; Hilro, K.; Kawahara, A. BunseklKagaku 1979, 28,43-47. (9) Matsuhlsa, K.; Ohzekl, K.; Karnbara, T. Bull. Chem. SOC.Jpn. 1981, 2675-2677.
3335-3336.
(13) Horwltz, W., Ed. “Official Methods of Analysis of the Association of Official Analytical Chemists”, 12th ed.; AOAC: Washington, DC, 1975; p 622. (14) Going, J. E.; Eisenreich, S. J. Anal. Chim. Acta 1973, 70,95-106. (15) Waki, H. “Ion Exchange Technology”; Naden, D.,Streat, M., Eds.; Ellis Horwood: Chichester, 1984; pp 595-602. (16) Yoshimura, K.; Ohashi, S. Talanta 1978, 25, 103-107.
RECEIVED for review April 3, 1985. Resubmitted October 4, 1985. Accepted October 4, 1985.
Automated Fluorometric Method for Hydrogen Peroxide in Air Allan L. Lamus,* Gregory L. Kok, J o h n A. Lind, Sonia N. Gitlin, B r i a n G. Heikes, a n d Richard
E. S h e t t e r
National Center for Atmospheric Research, P.O. Box 3000, Boulder, Colorado 80307
A fluorometrlc method for measurlrlg H,O, vapor in air utlllzes peroxidase enzyme to catalyze the reactlon In which hydroperoxides cause dlmerlratlon of (p-hydroxypheny1)acetic acld. I n a second channel, H,O, is seiectlvely decomposed by catalase so that the fluorescence slgnal Is due only to organic hydroperoxldes. The difference between the two slgnals Is a measure of H20, vapor. The H 2 0 2 vapor Is collected by means of a glass coil through which air and water flow concurrently. The coefflcent of variatlon Is 0.5 % at 2.5 parts per blllion by volume. The standard deviation of the base line is 10 parts per trllilon by volume (pptv) under iaboratory condltlons. Thls standard deviation has varied between 3 and 33 pptv during ground-based field missions, and was 70 pptv on alrcrafi flights. Thirty seconds is requlred for the slgnal to change from 10 to 90% of Its maxlmum value.
a peak emission wavelength of 400 nm (11). The peroxide concentration is directly proportional to the fluorescence intensity. Peroxidase also catalyzes the reaction of organic hydroperoxides to form the fluorescent dimer. In order to distinguish HzOzfrom organic hydroperoxides,the technique utilizes a dual-channel flow system with a dual-cell fluorometer. The reaction described above yields a measure of both the hydrogen peroxide and organic hydroperoxides in the first channel. In the second channel, the enzyme catalase is added to selectively destroy hydrogen peroxide before the peroxidase-catalyzed reaction occurs. The second channel therefore provides an analytical blank for the determination of HzOP The two signals are produced simultaneously by the system, which is automated by means of a Technicon AutoAnalyzer peristaltic pump.
Hydrogen peroxide is believed to dominate oxidation processes that convert SOz to HzS04in clouds a t pH values less than 4.5. The predominant source of HzOzdissolved in cloud water is H 2 0 zvapor formed photochemically in the atmosphere (1-5). Concentrations of H20zvapor observed by the described technique have ranged from 30 parts per trillion by volume (pptv) to 4 parts per billion by volume (ppbv) in the eastern U.S.A. Attempts to collect HzOz from air by scrubbing with water-filled impingers have been hindered by artifact formation of HzOz in the solution (6-9). Earlier methods utilized detection of the dissolved HzOzby a luminol technique (6). Interferences observed in the measurement of HzOz in cloud water by the lurninol method may pose a problem in the measurement of the vapor (10). This paper describes an alternative way to measure HzOZ vapor in real time. A stripping coil is used in combination with a fluorometric analysis of HZOp The method as applied to aqueous samples has been described in detail (10). The HzOZvapor technique incorporates changes that accommodate a lower and narrower concentration range of HzOz in the solution used for stripping HzOz from air. The technique is based on the selective catalysis of HzOz decomposition with (p-hydroxypheny1)acetic acid (POPHA). The reaction products are water and the dimeric product, 6,6’-dihydroxy-3,3’-biphenyldiacetic acid. The dimeric product fluoresces with a peak excitation wavelength of 320 nm and
EXPERIMENTAL SECTION The H202 is stripped from the atmosphere by means of concurrently pulling the air sample and scrubbing solution through a Technicon AutoAnalyzer coil (6, 12). The scrubbing solution M potassium acid phthalate in water adjusted to pH is 5 X 6.0 with NaOH. The tube length of the 10-turn, 2-mm4.d. coil is approximately 50 cm. Air sampled at 2 L/min has a residence time of 0.05 s. Scrubbing solution is pumped into the inlet of the coil at a rate of 0.42 mL/min, giving an air-to-water ratio of 4800 (Figure 1). The scrubbing solution, which is impelled by the air as it flows through the horizontal coil, forms a thin film on the glass, providing a large surface for gas exchange. The system is provided with a stripping coil for each channel. For maximum accuracy, the pump tubes introducing water into the coils should be calibrated for delivery rate. Air and scrubbing water are pumped from the collection coil into a vertical separator tube. The water plus about 0.3 mL/min of the scrubbed air is pumped by the AutoAnalyzer from the bottom of the separator. The sum of the flow rates of the pump tubes delivering the scrubbing water and the reagents into the system is approximately 0.3 mL/min less than the sum of flow rates of the pump tubes leading from the fluorometer cell and the debubbler exiting the system (Figure 2). This difference causes about 0.3 mL/min of the scrubbing air t o be pumped through the system. The air segments the reaction stream with bubbles helping to maintain sharp concentrationgradients along the stream. The sample conditioning reagent is added (0.16 mL/min) to each stream of scrubbing solution. The conditioning reagent M formaldehyde and 0.02 M phthalate (Table I) contains 5 X buffer adjusted to pH 6.0. The former, by forming hydroxy-
0003-2700/86/0358-0594$0 1.5010
@ 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. i o Pump
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Table I. Reagent Concentrations Used Stripping Solution
I/,'
O.D.
5 x low3M KHPhthalate," adjusted to pH 6.0 with NaOH Sample Conditioning Reagent 0.02 M KHPhthalate, adjusted to pH 6.0 with NaOH; 8.4 X M CH20' tetrasodium EDTA;b 5 X
5cm,lOmm I D
Sample Conditioning Reagent with Catalase 0.02 M KHPhthalate, adjusted to pH 6.0 with NaOH; 8.4 X M CHzO M tetrasodium EDTA; catalase protein;d,e5 X 10TurncoiI f Stripping Solution
Solulio
Fluorescence Reagent M 0.085 M KHPhthalate, adjusted to pH 6.0; 3.0 X @-hydroxypheny1)aceticacid;f 8 purpurogalin units peroxidas@/mL of reagent
Flgure 1. Pyrex glass stripping coil and separator. Typical flow rates
are
1.0
slm air and 0.42 mL Stripping solution. SbMPLE bIR
*
TECHNICONPUMP
0.1 M NaOH"
PUMP
STRIPPER
'
Potassium hydrogen phthalate, Fischer Scientific Co., P-243; adjust pH by using NaOH. *Sigma Chemical Co., ED 4SS. J. T. Baker, 37% formaldehyde, reagent grade. Sigma Chemical Co., Stock C-100. e See text for adjusting catalase concentration. f Fairfield Chemical Co., Blythewood, SC. BSigma Chemical Co. Stock P8375. TvDe VI. J. T. Baker Co.
SEPARbTOR STRIPPING SOLUTION CONOlTIONING REAGENT FLUORIMETRIC REbGENT NoOH
0 16
I
0 I6
I
O 16
I WbSTE
Base
DEBUBBLER
WASTE L--->
CELL EFFLUENT DEBUBBLER
Flgure 2. Diagram of the AutoAnalyter pump manifold.
methanesulfonic acid, completely eliminates an interference caused by SOz in the reaction forming the fluorescent dimer (10). For the reagent blank stream, the conditioning reagent is identical except for the addition of catalase, which selectively decomposes HzOz A five-turn mixing coil, 2 mm i.d., follows the addition of each conditioning reagent (Figure 2). Each stream then receives the fluorescence reagent (Table I) at 0.16 mL/min. Finally, 0.1 N NaOH is added at 0.16 mL/min to raise the pH above 10 a t which the fluorescence signal of the dimer is maintained a t a maximum level. A two-turn mixing coil is inserted after the addition of each of these reagents. Each stream enters a fluorometer cell. The excitation and emission wavelengths of the POPHA dimer are 320 and 400 nm, respectively. Details of the dual beam fluorometer have been described (10). The purity of the POPHA used has a large influence on the noise and background signal of the analytical technique. POPHA from several different manufacturers was examined, and the POPHA supplied by Fairfield Chemical is acceptable with no added purification. In analytical work with HzOzit is important that all solutions by prepared from water that is free of bacteria as well as ionic impurities. In these studies, solutions were prepared from water purified by a cartridge deionization and organic removal system (Millipore Corp.). The resistance of the final water was greater than 1.8 X lo7 0 cm-'. Bacteria that rapidly decompose H20zmay be present in cartridge deionization systems. It is important that a fiiter to remove bacteria be present on the outlet of the cartridge system and that the system be cleaned frequently. Aqueous HzOzstandards were prepared by serial dilution of a stock HzOz standard. The HzOz concentration of the stock standard is determined by titration with KMnO,. The stock H20, standard (1%) is prepared by dilution of commercially available 30% HZOz The 1% standard is preserved by the addition of 5 X 10" M sodium stannate. Stock HzOzsolutions prepared in this manner are found to decay at a rate of about 1%/month. Working standards are prepared daily. Glassware to be used for the first time in preparing H20zstandards should be washed with soap, rinsed, and allowed to soak in deionized water for several days with frequent water changes. TOcalibrate the catalase channel, all traces of catalase must be removed from the system. This is accomplished by replacing
the catalase conditioning reagent with 0.1 N HC1 and running the flow system for a few minutes. The strong acid will denature the catalase instantly. After the removal of the catalase, the catalase-free conditioning reagent is set in place of the catalase conditioning reagent and the channel calibrated with H202in the regular manner. Though catalase does not react with ethyl hydroperoxide, n-propyl hydroperoxide, tert-butyl hydroperoxide, or peroxoacetic acid, under the conditions of the test, there is a slight reaction with methyl hydroperoxide. To minimize this problem, sufficient catalase is added to the conditioning reagent to remove all but 3% of the hydrogen peroxide. The catalase working solution is 43 ppm catalase protein (1720 sigma units L-l). It is prepared by a 1:lOOO dilution of the catalase reagent in 0.02 M phthalate buffer (pH 6.0). The commercial catalase reagent (Table I) is a suspension that should be well-shaken before dilution. The working solution should be allowed to stand for at least 4 h before use. The catalase conditioning reagent is formed by further diluting 7 mL of the catalase working solution into 200 mL of 0.02 M phthalate buffer (pH 6.0). To adjust the catalase concentration, catalase is purged from the tubing as described above. The stripping solution through the coil is then temporarily replaced with a 2.47 X lo-' M aqueous H202standard in 0.005 M phthalate buffer (pH 6.0). If needed, additional catalase working solution is added to the conditioning reagent until the HzOz signal is reduced to 3% of its value without catalase. A test manifold was constructed to investigate the effects of interferences and of varying experimental conditions. This system consisted of a PFA Teflon manifold, a sample pump, an AADCO Model 737 pure air generator, a pressure transducer, and metering valves. After comparisons with stainless steel, borosilicate glass, TFE, and FEP Teflon, PFA Teflon was determined to be the best material in terms of least absorption of HzOzand the shortest equilibration time. The manifold utilizes 1.27-cm-0.d. PFA Teflon and injection-molded PFA fittings. Sampling of air through a 7.62 m length of 1.27-cm-0.d. PFA tubing a t 20 standard liters per minute (slpm) did not introduce a noticeable loss of HzO?. Standards of HzOzvapor were generated by equilibrating air with aqueous HzOzstandards (13). Equilibrium concentrations as a function of temperature are described by the Henry's law constant: KH = exp[(6621/n - 11.001 M atm-l (13). The generator apparatus (Figure 3) consists of a tube lined with glass-fiber standard runs down the filter while filter paper. A liquid H202 air supplied by an AADCO Model 737 pure air generator runs in a counter current flow. The tube is thermostated at 18 "C. The concentration of Hz02vapor in the standard depends on
596
ANALYTICAL CHEMISTRY, VOL. 58,
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PEROXIDE SOLUTION 0005" SPACER
..
I
I
125-
I W
FROM WATER B A T H 4 1
Y 150-
loo-
I Y
7 5 r
.=I W
a
50-
COOLING
25
JACKET\
t/
OV 0
i I
I
I
I
2
3
FLOW RATE (Standard
I
I
BUeBLERs CONSTANT TEMPERATURE WATER BATH
AIR IN
L
T
O WASTE
Flgure 3. Vapor-phase standard generator. Typical flow rates are mL/min aqueous standard and C 1 L/min zero air.
