Anal. Chem. 1985, 57, 917-922
equally similar to each of the desired anal* spectra may allow a further improvement in this method. The individual sensor loadings into the perpendicular projection axis, equivalently the final rotation vector, indicates the sensors which are the most highly correlated with those features of the mixture not fitted by the calibration spectra. If the analysis is overdetermined with respect to the number of sensors employed, then elimination of those sensors which load most strongly into the fiial rotation vector may result in decreasing the overall effect of the background components on the mixture response. This suggests an additional criteria which might be useful for optimizing the sensor selection in a multicomponent analysis. While both of these two quantitation approaches provided improved results, significant errors may still occur. The problem of background detection increases as more known analytes are present due to the increased likelihood that the background can be modeled as a linear combination of the analvtes. These difficulties i m d v that identification of all samile components, both desired"analytes and interferents, affecting the measured response is still a worthwhile goal for obtaining the most accurate analytical results.
017
LITERATURE CITED Warner, I. M.; Davldson, E. R.; Christian, G. D. Anal. Chem. 1977, 49, 2155-2159. Leggett, D. J. Anal. Chem. 1977, 49, 276-281. Gayle, J. B.; Bennett, H. D. Anal. Chem. 1978, 50,2085-2089. Haaland, D. M.; Easterling, R. G. Appl. Specfrosc. 1982, 36, 665-673. Lawton, W. H.; Sylvestre, E. A. Technometrics 1971, 13, 617-633. Martens, H. Anal. Chlm. Acta 1979, 112, 423-442. SpjDtvoll, E.; Martens, H.; Voiden, R. Technomefrics 1982, 2 4 , 173-180. Kaiser, H. Pure Appl. Chem. 1973, 34, 35-61. Saxberg, B. E. H.; Kowalski, B. R. Anal. Chem. 1979, 5 1 , 1031- 1038. Nattrelia, M. G. "Experimental Statistics"; Government Printing Office: washington, DC, 1963; National Bureau of Standards Handbook 91; Chapter 5. Sharaf, M. A.; Kowalski, B. R. Anal. Chem. 1981, 53, 518-522. Sharaf, M. A.; Kowaiski, B. R. Anal. Chem. 1982. 5 4 , 1291-1296. Zschelle, F. P.; Murray, H. C.; Baker, G. A,; Peddicord, R. G. Anal. Cham. 1962, 34, 1776-1780. Kalivas, J. H. Anal. Chem. 1983, 55, 565-567.
RECEIVED for review June 18,1984. Resubmitted November 27, 1984. Accepted November 27, 1984, This work was supported in part by the Office of Naval Research.
Automated Fluorometric Method for Hydrogen Peroxide in Atmospheric Precipitation Allan L. Lamus,* Gregory L. Kok,Sonia N. Gitlin, and John A. Lind National Center for Atmospheric Research, P.O. Box 3000, Boulder, Colorado 80307
Scott E. McLaren Atmospheric Science Research Center, 1400 Washington Avenue, Albany, New York 12222
An automated analytlcal technlque for the determlnatlon of hydrogen peroxide (H202) In the llquld phase has been developed. The chemlstry of thls technlque Is based on the reactlon of H,02 wlth horseradish peroxldase and p hydroxyphenylacetlc acid (POPHA). The resultlng reactlon forms the fluorescent dlmer of POPHA. By use of conventlonal fluorescence detection technlques a detection llmlt of 1.2 X lo-' M (0.4 ppbm) H,O, Is obtalned for a 1.5-mL aqueous sample. The coeffklent of varlatlon Is 0.66% at 1.6 X lo-' M (53 ppbm). The analytical chemical reactlon responds stolchlometrlcally to both H20, and organlc hydroperoxides. To dlscrlmlnate H202from organlc hydroperoxldes, a novel dual-channel chemlcal flow system has been devlsed to separately determlne total hydroperoxides and organlc hydroperoxides. The concentration of H,O, Is determined by the dlfference between these two measurements. Th!, system has been tested extensively for potentlal Interferences commonly found In environmental aqueous samples, and none has been observed.
analytical method for Hz02(5,6) provided the first approach for determining H202at the 10 nM level in precipitation. The luminol technique has been modified by adapting hemin as a catalyst (7). Recently a scopoletin based enzyme technique has been used for the determination of H202in precipitation samples (8). Interferences were observed in the luminol technique applied to atmospheric precipitation samples collected during autumn in Boulder, CO. The interference was detected by the persistence of a positive signal for H202,even though the sample had been treated with the enzyme catalase. The selective decomposition of H202by catalase has been used earlier to identify H 2 0 2in atmospheric samples (9) catalase
2H202 2H20 + 02 (1) Peroxidase is another enzyme characterized by selectivity toward hydroperoxides. In the presence of a hydrogen donor molecule such as p-hydroxyphenylacetic acid (POPHA) the enzyme peroxidase catalyzes the reduction of H 2 0 2via the following reaction (10): CHPCOOH
-hfi w
CHpCOOH CHpCOOH
PEROXIDASE
Hydrogen peroxide rapidly oxidizes bisulfite ion in water a t pH values below 4.5, and therefore is currently believed to contribute significantly to the generation of sulfuric acid in atmospheric precipitation (1, 2). Studies on H202in precipitation samples have been limited by the availability of sensitive analytical techniques. Early studies on H 2 0 2in precipitation were conducted using iodometric techniques ( 3 , 4 ) . The luminol chemiluminescence 0003-2700/85/0357-09 17$01.50/0
2
v OH
+ He02
OH
(2)
OH
The dimeric product fluoresces with a peak excitation wavelength of 320 nm and peak emission wavelength of 400 nm (IO). The stoichiometry of reaction 2 indicates that for every hydroperoxide (-0-0-H) bond broken, one dimer is formed. The fluorescence of this dimer is therefore directly 0 1985 American Chemical Society
918
ANALYTICAL CHEMISTRY, VOL. 57, NO. 4, APRIL 1985
SAMPLE S A M P L E COND. R E A G E N T
0.16
FLUORIMETRIC REAGENT
0.16
NaOH Fluorescence AIR
0.32 I
DELAY COIL
MIXING COILS
r--l
Fluorescence Cell H 20
WASTE
0
WASTE
0
so so
I
I
WASTE SYRINGE
Flgure 1. Technicon AutoAnalyzer pump niariifold and flow system.
proportional to the peroxide concentration. An automated analytical technique based on the selectivity of the two enzymes, catalase and peroxidase, has been developed in order to provide a highly specific method for atmospheric samples. In addition, comparative analyses of HzOz in cloud water were conducted between the luminol method and the present technique at Whiteface Mountain, NY. The technique utilizes a dual channel flow system and dual cell fluorometer which provides simultaneously the fluorescent signal resulting from the peroxidase reaction (eq 2) and an analytical blank derived from the catalase reaction (eq 1). The chemical reagents are identical in both channels, except for the addition of catalase to the second channel which destroys before the addition of POPHA. the H202 The catalase analytical blank is provided for two reasons: first, the reaction of some organic hydroperoxides and POPHA will also be catalyzed by peroxidase; second, fluorescent organic substances could conceivably occur in polluted rain which might interfere with the fluorescence signal of the POPHA dimer. I t should be pointed out that only insignificant catalase signal) have been blank signals (e.g., less than 1%of the H202 observed in precipitation samples collected a t Boulder and Denver, CO, or in cloud water samples collected a t Whiteface Mountain, NY. The Henry's law coefficients of both methylhydroperoxide and peroxoacetic acid are so much lower than that of HzOzthat significant scavenging of these organic substances from air by cloud droplets or rain is not likely (11).
EXPERIMENTAL SECTION A detailed description of the reagents is given in Table I. The flow system is operated with a Technicon AutoAnalyzer peristaltic pump. The pump manifold is comprised of standard AutoAnalyzer pump tubes and components (Figure 1). Since reagent addition and timing are critical, the use of Technicon flow-certified pump tubes is advisable. The sample (1.5 mL) is loaded, using a glass syringe with a Teflon plunger, into a sample injection valve and split equally between the two channels. The sample channel is initially segmented with air bubbles in order to maintain sharp concentration gradients along the stream. The sample conditioning reagent (Table I) is then added. Potassium hydrogen phthalate buffer adjusted to pH 5.5 maintains the sample in the appropriate pH range for both the catalase and peroxidase reactions. The buffered reagent also contains EDTA to prevent possible interferences by metal ions.
Table I. Reagent Concentrations Used
Sample Conditioning Reagent 0.35 M KHPhthalateO, adjusted to pH 5.5 8.4 X M tetrasodium EDTAb Sample Conditioning Reagent with Catalase 0.35 M KHPhthalate, adjusted to pH 5.5 8.4 X M tetrasodium EDTA 490 u units of catalase/mLc of reagent Fluorescence Reagent 0.35 M KHPhthalate, adjusted to pH 5.5 8.0 X M p-hydroxyphenylaceticacidd 8 purpurogallin units of peroxidase'/mL reagent Base 0.4 M NaOHf Potassium hydrogen phthalate, Fischer Scientific Co. P-243. Adjust pH using 10 N NaOH. *Sigma Chemical Co., ED 4SS. CSigmaChemical Co., Stock C-100. dKodak (Eastman Kodak Co.), Lot No. C8B. "Sigma Chemical Co., Stock P8375, Type VI. 'J.T. Baker Co. The fluorescence reagent (Table I) next joins the sample stream. The POPHA must be purified before use by recrystallizing from aqueous solution. Activated charcoal is used in the recrystallization to remove impurities. All other reagents can be used as received. The fluorescence reagent can be kept for several days and should be discarded when either the base line becomes unsteady or significant degradation of the linearity of response is observed. NaOH (Table I) is then added to the sample stream. The fluorescence quantum yield of the POPHA dimer diminishes below pH 9.0 but remains at a maximum above pH 10.0. The addition of NaOH maintains the reaction stream at pH 10.0 or higher permitting analysis of samples as acidic as pH 2.0 without measurement artifact. The reaction stream passes through a five-turn, 2 mm i.d. glass mixing coil after the addition of each reagent. The second sample channel is identical with the first, except that the sample conditioning reagent contains catalase in addition to the other constituents. The catalase used was obtained from Sigma Chemical Co. This material, obtained from bovine liver, is doubly crystallized and suspended in water containing 0.1% thymol with an indicated activity of 35 300 Sigma units per mg of protein. One Sigma unit of catalase activity will decompose 1.0 X lo4 mol of HzOz/min at pH 7.0 at 25 O C , while the concentration of HzOz falls from 10.3 X to 9.2 X IU " mol/mL of reaction mixture.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 4, APRIL 1985
919
PRIMARY INTERFERENCE FILTER 320 n m FLUOR FLOW
FLUORESCENCE
INTERFERENCE
SOURCE REFERENCE
SECONDARY INTERFERENCE
SECONDARY INTERFERENCE FILTER 400nm
ii 1PHOTOMULTIPLIER
TUBE
PHOTOMULTIPLIER TUBE
\
Flgure 2. Optical diagram for the dual beam fluorometer.
Hz02standards were prepared by serial dilution of a stock HzOz standard. The H202concentration of the stock standard is determined by titration with KMn04. A l % Hz02 standard is prepared by dilution of commercially available 30% H20z. The 1% standard is preserved by the addition of 5 X M sodium stannate. Stock H202solutions prepared in this manner are found to decay a t a rate of about 1%per 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. To calibrate the catalase channel all traces of catalase must be removed from the system. This is accomplished by replacing the catalase reagent with 0.1 N HCl and running the flow system for a few minutes. The strong acid will denature the catalase instantly. After the removal of the catalase, water is set in place of the catalase reagent and the channel calibrated with H202in the regular manner. In analytical work with H202it is important that all solutions be 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 lo7Q. Bacteria may be present in cartridge deionization systems which rapidly decompose H202. It is important that a filter to remove bacteria be present on the outlet of the cartridge system and that the system be cleaned frequently. Methyl hydroperoxide, ethyl hydroperoxide, and n-propyl hydroperoxide used to test the method were synthesized by the procedure of Riche and Hitz (14). These peroxides were purified through repeahd extractions and washings with water. Technical grade tert-butyl hydroperoxide was obtained from Mallinkrodt. Peroxoacetic acid, 40%, was purchased from FMC Corp, Buffalo, NY. Methyl hydroperoxide and peroxoacetic acid were individually assayed by using iodometric techniques (15). T o use the instrument for the quantitative determination of methyl hydroperoxide and peroxoacetic acid up to concentrations
of about 3 x M, it is sufficient to calibrate with HzOz For higher concentrations, or for organic hydroperoxides of higher molecular weight, calibrations with the specific compounds are necessary. Except in the case of peroxoacetic acid the rate of the peroxidase-catalyzed reaction is slower for these compounds and a t higher concentrations under the described conditions of the automated test incomplete reaction may lead to nonlinearities. An optical diagram of the dual-beam fluorometer is shown in Figure 2. The instrument is comprised of two fluorometer units with a common excitation source. Both beams are identical in terms of the optical and electronic components. The fluorometer is designed using an H85A3/UV mercury arc lamp as the excitation source and Hamamatsu R268 photomultiplier tube for fluorescence detection. Since the optical system is designed to be used specifically with the peroxidase/POPHA reagent system, interference filters rather than monochromators are used to isolate the excitation and emissions wavelengths, 320 and 400 nm, respectively. The optical system is rigidly mounted on an aluminum base plate. The instrument is designed for use on an aircraft in measuring cloud water composition. The unit can be mounted into a standard electronic rack panel. The weight of the instrument is 22 lbs, and its dimensions are 18 X 10 X 15 in. It requires 120 W a t 115 V. For the complete fluorometer unit two chassis are required: one contains the fluorometer and the reagent flow system while the second contains the signal processing electronics and the readout unit. The electronics system, shown in Figure 3, is comprised of two identical high voltage/electrometer units for detection and amplification of the fluorescence signals, and a common system monitor to track the high voltage power supplies and the excitation lamp output. The sensitivity of the two optical channels is adjusted by changing the high voltage'applied to the photomultiplier tubes. In this manner the signal output from each detector can be adjusted to be equivalent for a given peroxide concentration. The high voltage applied can be read out at the system monitor.
920
ANALYTICAL CHEMISTRY, VOL.
PHOTOMULTIPLIER A
1
EXCITATION L A V F MONITOR
ANALOG ELECTROMETER A
1
1
L3W PASS
'
\SYSTEM MONITOR
4NALOG 1 PMOTOMU-TIP_iER B
57, NO. 4, APRIL 1985
ELECTROMETER
LOW PASS
SIGNAL OUT B
Figure 3. Signal processing system for the dual beam fluorometer.
The photocurrent representing the fluorescence signal is amplified with an analog electrometer. Current suppression from lo4 to lo4 A is provided if suppression of high background signals is necessary. To reduce the noise in the signal output, a low pass filter, 0.1 Hz frequency cutoff, can be switched into the circuit followingthe electrometer. At low signal levels or high suppression currents this filter will reduce the high frequency noise in the output. At higher signal levels the filter can be switched out for improved response times. The signal output is 0-1 V full scale. Further details on fabrication of the fluorometer are available from the authors.
ditions under which the H 2 0 2is completely removed by the catalase while organic hydroperoxides are left unreacted to provide an accurate analytical blank. Methyl hydroperoxide, ethyl hydroperoxide, n-propyl hydroperoxide, tert-butyl hydroperoxide, and peroxoacetic acid were investigated. Only methyl hydroperoxide exhibited an appreciable reaction with catalase under the conditions of the automated test. If MeOOH is destroyed by too much catalase, the background signal is too low, and the difference between the signals of the two channels would indicate a spuriously high H202value. On the other hand, incomplete destruction of the H202 by too little catalase would yield too high a background signal, and a final HzOzvalue which is spuriously low. To investigate the effect of these two compensating errors, the rates of conversion of MeOOH and HzOzby catalse under the recommended instrumental conditions were studied. At 22 "C and a constant 40-s reaction time, the rates of loss of MeOOH and H20zwere studied as a function of catalase Concentration. The following expressions describe the rates occurring specifically under the conditions of the automated instrument: In (HzOdfinal = [-(1.7 f 0.1) X 10-6](catalase)(At) (H2O~)initial
[-(0.072 f 0.008) X 10-6](catalase)(At)
RESULTS AND DISCUSSION By use of the reagent concentrations given above, the detection limit is 0.4 ppbm. At 1.56 X 10" M HzOzthe standard M, equivalent to a coefficient of deviation is 1.02 X variation of 0.7%. Plots of peak height vs. concentration are linear with concentration up to about 1.8 X M or 0.6 ppm HzOz. Most values we have measured a t Boulder, CO, or Whiteface Mountain, NY, are below this concentration. Above this value the curve becomes slightly nonlinear. We have adjusted the concentration of reagents to accommodate H202concentrations from 1.1 X M (0.4 ppbm) M (5000 ppbm) in order to eliminate the need to 1.5 X to dilute samples during field measurements. At concentrations above 0.6 ppm, the calibration points have been readily fitted by a quadratic regression equation with a correlation coefficient of 0.9998. A considerable effort was made to discover the cause of the nonlinearity a t higher concentrations. A likely cause in the case of fluorometry is the attenuation of exciting radiation in the path of the beam as a result of absorption by higher concentrations of absorbing molecules. However, dilution of the solutions of the dimer of POPHA by a factor of 10 did not alleviate the nonlinearity. Another possibility is a decreasing yield of dimer from the higher concentrations of HzOP However, no improvement was observed after increasing the concentrations of POPHA or peroxidase, changing the pH, or increasing reaction time. The possibility that the buffer, present in relatively high concentration, interferes chemically with the reaction was disproved since using Tris, glycine, ammonia, or borax buffers in lieu of potassium biphthalate gives a similar effect. Reaction involving enzymes frequently become nonlinear after the initial stages. However, reducing the reaction time in the automated test by a factor of 4 did not ameliorate the problem. Given the fact that the analytical precision did not decrease at the higher H 2 0 zconcentrations in spite of the slight nonlinearity, no further efforts were made. The peroxidase channel will yield a signal for organic hydroperoxides as well as for Hz02. Dialkyl peroxides do not give a signal. Since catalase does react slowly with some organic hydroperoxides, it is necessary to establish the con-
where (catalase) is activity of catalase as Sigma units per liter and At is time as seconds. Catalase reacts 24 times faster with HzOzthan with MeOOH under the conditions of the automated methods. To minimize errors introduced by MeOOH, we use a concentration of 2 ppm catalase protein in the reaction mixture. The catalase in the reagent reservoir is 8 times more concentrated to compensate for the dilution factor, indicated in Figure 1,to a final activity of 70600 Sigma units per liter. The application of the above empirical rate expressions yield the following errors in HzOzdetermination for the worst case in which the organic peroxide is entirely MeOOH and is present a t the indicated percentages of total hydroperoxide: %MeOOH % H,O, error
0%
5%
-0.82%
+ 0.13% + 1.2%
10%
20% t 3.5%
These small errors represent maximum errors to be expected from this source, since in actual practice the background signal in atmospheric samples will probably not represent MeOOH exclusively. MeOOH is the only organic peroxide likely to be present in atmospheric samples with sufficient reactivity with catalase to cause a problem. When operated at higher pH values, e.g., using Tris buffer adjusted to pH 8.6, the test is affected by the adduct of formaldehyde and bisuilfite ion (hydroxymethanesulfonicacid). Normally, bisulfite ion will not coexist with H 2 0 2in precipitation samples. However, the adduct of formaldehyde and bisulfite ion can coexist with H,02 at pH 5 (12). The hydrolysis rate increases with pH (13) and when the adduct encounters the buffer at pH 8.6 it releases sufficient bisulfite to interfere with the formation of the dimer. The adduct at 5 X 10" M reduces the signal of HzOz(5.6 X M) by 6 % . This interference was eliminated by first adding formaldehyde (1 X M) to the sample stream through a 0.16 mL min-' pump manifold tube. With formaldehyde present, the adduct a t 1.5 M X causes no interference in the measurement M H20,. Running of H z 0 2at either 1.5 X 10" or 1.5 X the test at pH 5.5, as described in the Experimental Section, eliminates the interference by hydroxymethanesulfonic acid and therefore the need to introduce formaldehyde reagent.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 4, APRIL 1985 400 I l
Table 11. Sample Deterioration Tests date
pH
[H,021, pM
4/7/82 5.3 2.90 5/4/82 5.9 25.7 2nd Determination 5/11/82 5.5 3.20 5/12/82 5.4 1/27/82 5.5 24.1 2nd Determination 7/28/82 5.5 3.1 7129182 12.6 8/4/82 28.8 6/9/83 3.1 2nd Determination 6/14/83 3.5 2nd Determination average a
350
decay/hours % loss per h -80%/48 -26%/5 -100%/5 - 52%/5 -30%/24 -80%/16 -100%/24 -21%/16 -22%/16 -100%/16 -21%/0.75 -34%/1.2 -7%/1.8 - 10%/2.1
1.7 5.2 20 10.4 1.3
l
I
I
i
l
l
'
~
-
921
/
l
I
0
I
E
9
a I
0" N
I
5.0
4.2 1.7 1.4
6.3 28.0 28.0 3.9 4.8 5.6 i 8.1
Not including replicate determinations.
There is no significant change in sensitivity a t the lower pH. Both negative and positive interferences were tested by adding known quantities of substances to standard solutions of HzOz. We have not found interfrences by commonly occurring atmospheric trace constituents. Ferrous ion and hydroquinone tend to eliminate the HzOzsignal by reaction with HzOF Ammonium, potassium, sodium, ferric, and manganous ions had no effect. Iodide, bromide, chloride, phosphate, nitrate, and benzoate ions had no effect. Formaldehyde, methanol, glyoxal, acetone, methylamine, dimethylamine, methyl ethyl ketone, toluene, benzene and peroxyacetylnitrate had no effect. Nitrite in neutral solution shows neither a positive nor negative interference. However, below pH 3.0, nitrite ion chemically reduces HzOz(17). Bisulfite ion below pH 4.6 also rapidly reacts with HzOz in the sample. By far the greatest source of error is loss of HzOzwith time in collected samples. We have found that glass and Teflon containers, though superior to other types, still do not prevent significant loss of HzOz. The addition of common preservatives for HzOz such as sodium stannate and acetanilide to samples of precipitation does not prevent appreciable loss. Refrigeration of samples does appear to have a beneficial effect, but by no means eliminates the problem. Sample deterioration clearly is a major problem. Data indicating the problem for samples collected at Boulder and then refrigerated are given in Table 11. Since the duration of precipitation events may be several hours or more, a technique for fixing HzOz concentrations is badly needed. Similarly, the collection of cloud water samples by aircraft with subsequent analysis a t laboratories several hours later can introduce a large loss of HzOz. Since the dimer of p hydroxyphenylacetic acid formed in this test is stable for several days, we have been exploring the possibility of forming the dimer by immediately reacting the HzOz with reagent stored in the sample collection vessel. At the present time, this approach appears successful and will be discussed in a later paper. In standard addition studies it is necessary to work rapidly and to measure the HzOzconcentration in the original sample immediately before each addition. In ambient precipitation samples HzOz can decay rapidly. If the data are not corrected for this HzOz decomposition, a false bias in the standard addition data will appear. Matrix effects have not been observed in samples at Boulder or Denver, CO. However, it is impossible to evaluate the peroxidase HzOzanalytical technique for all of the differing sample matrix conditions which
v -
0
20
40
60
80
100
I20
140
I60
I80
ENZYME H 2 0 2 Ippbm)
Figure 4. Intercomparison between the luminol method and the peroxidase enzyme technique on cloud water samples collected at Whiteface Mountain, NY. Upper line represents least-squares regression fit: lower line represents perfect agreement between tests. The ordinate equals luminol results (ppbm) and the abscissa equals enzyme results (ppbm). Concentration range is zero to the median H,O, value.
may be encountered. It is recommended that the standard addition be conducted on a portion of the precipitation samples collected in each sampling program. As part of testing this technique, measurements of HzOz in 284 cloud water samples at the peak of Whiteface Mountain were made simultaneously using the luminol and enzyme systems. The data indicated that the percentage difference between the luminol and enzyme methods is a function of peroxide concentration and that at values below 1.5 X M HzOz(0.5 ppm) the discrepancies can be quite large. The median concentration of HzOzby the enzyme method was 5.4 X M (0.18 ppm). A paired sample t test applied to the 284 comparisons indicated that the two techniques are statistically different at a significance level less than 0.001. Including all data pairs (maximum observed HzOz = 3815 ppbm) the regression equation relating the two data sets is luminol (H202)= 1.01 enzyme (H,O,)
+ 71.05
(3)
with a correlation coefficient of 0.98. Clearly the correlation using the entire concentration range was reasonably good. The median value of H202by the enzyme technique was 183 ppbm. By use of only data points below the median value, the regression equation becomes luminol (H,O,) = 1.5 enzyme (H,O,)
+ 48.58
(4)
with a correlation coefficient of 0.86. The 95% confidence limits of the correlation coefficient were 0.81 and 0.90 using the Fisher Z transformation. The lower line in Figure 4 indicates the plot anticipated for perfect agreement between the two methods, and the upper line shows the actual linear regression represented by eq 4. The data show a tendency for the luminol signal to be higher than the enzyme signal. Catalase was added to samples to destroy HzOzprior to testing by the luminol method. However, only a portion of the signal was eliminated. The residual signals were comparable to the differences between the HzOzconcentrations indicated by the two analytical tests, suggesting a positive interference in the luminol method. ACKNOWLEDGMENT Very valuable and appreciated contributions were made by Paul Sperry and Bruce Gandrud. LITERATURE C I T E D (1) Penkett, S. A.; Jones, B. M. R.; Brice, K. A,; Eggleton, A . E. J. Atmos. Envifon. 1979, 73, 123-137.
922
Anal. Chem. 1985, 57,922-926
(2) (3) (4) (5)
Molter, D. Armos. Environ. 1980, 14, 1067-1076. Schone, E. Z . Anal. Chern. 1984, 3 3 , 127. Matsui, H. J . Mefeorol. SOC.Jpn. 1949, 2, 380-381. Kok, G. L.;Darnaii, K. R.: Winer, A. M.; Pitts, J. N., Jr.; Gay, B. W., Jr. Environ. Sci. Technol. 1978, 12, 1077. (6) Kok, G. L. Atmos. Environ. 1980, 14, 656. (7) Yoshizumi, K.; Aokikazuyuki, I. N.; Toshichi, 0.; Toshimi, K.; Shujkamakura; Tajima, M. Atrnos. Environ, 1984, 18,395-401. (8) Zika, R.; Saitzman, E.; Chameides, W. L.; Davis, D. D. J . Geophys. Res. 1982, 87,5015-5017. (9) Heikes, B. G.; Lazrus, A. L.; Kok. G. L.; Kunen, S. M.; Gandrud, B. W.; Gitiin, S. N.; Sperry, P. D. J . Geophys. Res. 1982, 87,3045. ( I O ) Guiibauit, G. G.; Brignac, P.; Juneau, M. Anal. Chem. 1988, 40, 1256. (11) Lind, J.; Kok, G. L., submitted for publication, (12) Richards, L. W.; Anderson, J. A.: Blumenthai, D. L.; McDonaids, J. A,: Kok, G. L.; Lazrus, A. L. Atmos. Environ. 1983, 17, 911. (13) Kok, G. L.; Gitiin, S. N.; Lazrus, A. L., submitted for publication, (14) Rieche, A,; Hitz, F. Ber. Dtsch. Chem. Ges. 1920, 62, 2458. (15) Johnson, R. M.; Siddigu, I . W. “The Determination of Organic Peroxides”; Pergammon Press: London, 1970. (16) Aubar, M.; Taube, H. J . Am. Chem. SOC. 1954, 76,6243-6247. (17) Bhattacharyya, P. K.; Veeraraghavan, R. J . Cbem. Kinetics 1977, 6 0 , 629-640.
RECEIVED for review October 15, 1984. Accepted December 26, 1984. The development of the analytical method was funded by the Electric Power Research Institute under Contract 1630-12. The intercomparison between the peroxidase and luminol methods was funded by the Environmental Protection Agency. Although the research described in this report has been funded in part by the United States Environmental Protection Agency through interagency agreement EPA-AD49F2A182 to the National Science Foundation, it has not been subjected to the agency’s required peer and policy review and therefore does not necessarily reflect the views of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The National Center for Atmospheric Research is sponsored by the National Science Foundation.
Determination of Acidity Constants by Solvent ExtractionlFlow Injection Analysis Using a Dual-Membrane Phase Separator Lynette Fossey and Frederick F. Cantwell* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
The absorbances of both the organic and aqueous phases In a solvent extraction/flow Injection analysis system are slmultaneously monitored. Acidity constants are determlned from straight llne plots relating the ratlo of peak areas In the aqueous and organlc phases, A,/A,, to the hydrogen ion actlvlty of the aqueous phase. Theoretical equatlons descrlblng this relatlonshlp for both HA and BH’ charge type acids are derived and verified experimentally uslng 3,5-dimethylphenol (pK, = 10.09 f 0.01) and p-toluldlnlum Ion (pK, = 5.28 f 0.01). The dlstrlbutlon coefficlent of the neutral conjugate species is also obtained durlng the experiment. Some distinct practlcal advantages to using the dual-membrane device over the slngle-membrane devlce are dlscussed.
The use of solvent extraction for determining acid-base dissociation constants (1-7) is particularly attractive for compounds that have low solubility in water and whose two conjugate species have the same absorption spectrum. For such compounds, the low solubility precludes accurate pK, determinations by potentiometric titration in water, and the similarity of spectra precludes the use of the spectrophotometric technique. However, the labor and time involved diminish the attractiveness of batchwise solvent extraction for this purpose. Continuous extraction systems employing rapid phase separation make it possible to perform solvent extraction measurements much more rapidly and conveniently and therefore make pKa determination by solvent extraction more attractive (8). Solvent extraction/flow injection analysis (FIA) employing either one or two porous membranes as phase separators has been shown to give precise, accurate, and very rapid analytical determinations of drugs in pharmaceutical
tablets (9, 10). In the present paper we use a solvent extraction/FIA technique employing two membrane phase separators which allows simultaneous spectrophotometric monitoring of concentration in both the aqueous and organic phases. Equations are derived which relate peak areas in the organic and aqueous phases to hydrogen ion activities in the aqueous phase and which permit the determination of acidity constants of both HA and BH+ charge type acids. Validity of the equations is experimentally demonstrated using 3,5dimethylphenol and p-toluidinium ion as test acids.
THEORY The relationship between sample peak area in the organic phase A,,, and the hydrogen ion concentration in a solvent extraction/FIA system has been derived for a BH’ type acid in earlier papers from this laboratory (9,10). In the present paper, we are concerned with determining a “mixed” acidity constant which incorporates the concentrations of the protonated and deprotonated sample species and the activity of the hydrogen ion. The equation relating peak areas in the organic phase with hydrogen ion activity in the aqueous phase can be written as
Here b is the path length of the spectrophotometer flow cell, f is a response factor which relates the absorbance from the detector to a count rate on the integrator, n is the moles of sample injected, tB,, is the molar absorptivity of the sample in the organic phase, KB is the distribution coefficient of the conjugate base B, K, is the acidity constant of BH+, Faand F, are flow rates of the aqueous and organic phases, respectively, and aH is the hydrogen ion activity in the aqueous phase. This equation is similar to eq 4 in ref 10 if the “system constant” in that paper is defined as K = fbtB.,.
0003-2700/85/0357-0922$01.50/00 1985 American Chemical Society
