(21) H. L. Kahn and S . Slavin, At. Absorption Newsl., I O , 125 (1971). (22) J. Janouskova, Z. Sulcek. and V. Sychra. Chem. LiStY. 68, 969 (1974): Chem. Abstr., 82, 021594E. (23) G. P. Sighinolf, At. Absorption Newsl., 11, 96 (1972). (24) C. R. Parker, J. Rowe, and D. P. Sandoz, Amer. Lab., 6, 53 (August 1973). (25) K. G. Brodie and J. P. Matousek, Anal. Chem., 43, 1557 (1971). (26) D. C. Manning and F. J. Fernandez, At. Absorption Newsl., 9, 65 (1970). (27) F. J. Fernandez, At. AbsorptionNewsl., 11, 123 (1972). (28) J. H. Runnels, Ruth Merryfield, and H. B. Fisher, Anal. Chem., 47, 1258 (1975). (29) H. Massman and S.Gucer, Spectrochim. Acta, Part 8. 29, 283 (1974). (30) C. W. Fuller, Anal. Chim. Acta, 62, 442 (1972). (31) C. W. Fuller, Analyst, 99, 739 (1975). (32) G. L. Everett, T. S. West, and R. B. Williams, Anal. Chim. Acta, 70, 294 (1974). (33) "Rare Metals Handbook". 2nd ed., C. A. Hampel, Ed., Reinhold, New York, N.Y., 1961, p 40. (34) S. H. Kagler. Erdoel Kohle, Erdgas, Petrochem. Brennst.-Chern., 27, 514 (1974).
(35) H. Nickel, Spectrochim. Acta, Part 8,23, 323 (1967). (36) "Comprehensive Inorganic Chemistry", J. C. Bailar, Ed., Pergamon Press, London, 1973, p 564. (37) M. S. Black and R. E. Sievars, Anal. Chem., 45, 1773 (1973). (38) T. M. Florence. Y. J. Farrar, L. S. Dale, and G. E. Batleg. Anal. Chem., 46, 1874 (1974). (39) D. J. Von Lehden, R. H. Jungers, and R. E. Lee, Jr., Anal. Chem., 46 239 (1974).
for review May 9, 1975. Accepted August 49 1975. Contribution of the Trace Metals Project Participat-
ing Laboratories: Atlantic Richfield Company, Harvey, Ill.; Chevron Research Company, Richmond, Calif.; Exxon Research and Engineering Company, Linden, N.J.; Mobil Research and Corporation? N*J*; and Phillips Petroleum Company, Bartlesville, Okla.
Chemiluminescent Flow Method for Determination of Formaldehyde Danuta Slawinska' and Janusz Slawinski2 Department of Chemistry and Department of Biochemistry, University of Georgia, Athens, Ga. 30602
Formaldehyde and gallic acid oxidized with aqueous alkaline hydrogen peroxide produce relatively strong chemiiuminescence in the spectral range 560-850 nm (the TrautzSchorigln reaction). The kinetics of this system have been measured as well as the chemiluminescence spectra and absorption and fluorescence spectra of intermediates and products. The effects of order of reagent addition and variations in oxygen pressure on chemiluminescence have also been determlned. Heat generation and variations in dlssolved oxygen concentration have been measured as a function of tlme and order of reagent addition. The results are discussed in terms of the reaction mechanism with particular emphasis on the possible role of singlet molecular oxygen. The effects of reagent concentrations, pH, temperature, rate flow, and interfering compounds on the maximum of chemiiumlnescence intensity were measured. Chemiluminescence Intensity is linearly proportional to formaldehyde concentration from lo-' to 10-2M. Using the optimized system, a simple and rapid flow method for formaldehyde determination in water was developed with a mean error less than 1 % and a detection limit of 1 pgA.
Chemiluminescent and bioluminescent methods offer certain advantages for chemical analysis (1-7). Hercules, Seitz, and coworkers, combining enzyme-induced reactions with the sensitive luminol chemiluminescence (CL)-system and performing analyses in a flow system have developed new CL techniques (1, 4, 8-10), These techniques are very successful because of CL's unique advantages of sensitivity, specificity, and simplicity. The oxidation of luminol in basic solution is one of the most efficient, best known CL reactions and is widely used for analytical purposes ( I , 4, 11-1 3 ) . Several other reactions appear to have analytical potential (7, 14-1 7 ) and further developmental work might proPresent address, Institute of Physics, Technical University, Szczecin, Poland. Present address, Department of Physics, Agricultural Academy, 71-424 Szczecin, Poland.
vide the basis for analytical applications not possible with luminol. The Trautz-Schorigin reaction (TSR) emits a strong orange luminescence lasting several minutes. This is a vigorous reaction between concentrated alkaline H202 and CH20 to which a polyhydric phenol such as pyrogallol is added (18). There are some spectrometric (19, 20) and chemical (21) data suggesting a singlet oxygen formation. However, details of the mechanism and the exact nature of the emitters are not clear. The classical TSR is not convenient for analytical purposes. The modified TSR, involving diluted reagents and gallic acid which is more stable than pyrogallol, has been applied for the determination of CHzO (22),proteins ( 2 3 ) ,and tannins ( 2 4 ) .Recently, CL was reported from the base catalyzed decomposition of the CH20-Hz02 system, which is thought to be a model for lipid peroxidation in biological membranes ( 2 5 ) .Therefore, a detailed understanding of the TSR is also important for biological chemistry. Determination of CH2O in natural waters is of considerable interest. CH20 and related compounds enter waters through petroleum, food, biochemical, plastic, and other industrial wastes. Moreover, CH20 is formed in photochemical reactions in polluted urban atmospheres (26). There are a number of methods available in the literature to determine CH20 (27-30). However, only a few of them have satisfactory sensitivity and have been tested to determine CHzO in natural waters (28,29). This work deals with the mechanism of TSR chemiluminescence and its analytical application. It further describes the development of the previous method (22) for CH2O determination.
EXPERIMENTAL Apparatus. Chemiluminescence kinetics, the effect of reagent concentrations and inhibitors on CL intensity, were measured using an RCA 6655 photomultiplier with an S-11 cathode operating with a Keithley 414 microammeter and a Keithley recorder. Thermometric measurements of thermal effects were performed on a YSI Model 42SC Telethermometer. Oxygen concentration in the diluted solution was determined using an oxygen electrode operating with a Keithley Digital Elec-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2101
SAMP
TO W A S X
1 G4UIC 4CiO
H2 02 '--NITROGEN
Figure 1. Diagram of the flow system for the chemiluminescent determination of formaldehyde
trometer. The system was calibrated with high purity 0 2 and N2 and a constant temperature of 25 & 0.1 "C was maintained. Simultaneously, CL from the same solution was measured with a 9658R EM1 ph0,tomultiplier. Fluorescence spectra of gallic acid oxidation products were recorded on an Amino-Keirs spectrophosphorimeter. Absorption spectra of gallic acid oxidation intermediates and products were recorded on a Zeiss spectrophotometer. Chemiluminescence spectra of the TSR were measured with a fluorometer constructed from Aminco-Building Blocks. The monochromator was an Aminco Grating Monochromator. Two detectors were used alternately: 1) a Hamamatsu R818 high cathode sensitive p,hotomultiplier with spectral sensitivity in 185-850 nm and 2) an EM1 9558QA photomultiplier with an S-20 cathode. The photomultipliers were powered at 1100-1250 V by a Kepco regulated supply, Model ABC 2500M. The amplifier used was a HewlettPackard x-y Model 7005B. The spectral distribution of CL was measured under flow conditions. The reactants, H202 plus gallic acid and CH20 plus NaOH, were placed into two 50-ml syringes which were mounted on a Harvard Apparatus Model 600-2-200 syringe pump. The outlets of the syringes were connected with Teflon tubing to a special Y-shaped glass tubing with platinum gauze. T h e gauze produced a turbulent flow which greatly enhanced the mixing. While the solution was flowing, an emission spectrum was recorded. Since flow artifacts were frequently observed, the true spectrum was determined from a composite of several runs. The slit widths were 0.5 and 1mm which correspond to the 4- and 8-nm band pass, respectively. The fluorimeter was calibrated using a low-pressure Hg lamp. Spectra were corrected for the spectral response of the photomultipliers. Additionally, an RCA C7007A photomultiplier with an infrared sensitive cathode, cooled to -40 "C and equipped with interference filters having T,,, a t 1270, 1060, and 765 nm was used to check for possible emission from the lAS and lZ+,0 2 at these wavelengths. For the analytical assay of CH20, the reaction was carried out in a flow system observing steady-state CL intensity. The CL produced by the oxidation of gallic acid and CH20 with HzOz plus NaOH was followed in a continuous flow system using the apparatus shown in Figure 1. The system used three 50-ml plastic syringes, containing gallic acid with H202, NaOH, and an aqueous background. The syringes were driven by a Harvard 600-2-200 syringe pump, as described above. Nitrogen gas was bubbled through the cell to assure uniform mixing. Chemiluminescence intensity was detected by an R818 Hamamatsu photomultiplier tube attached to the face of the cell. The signal was amplified by an Keithley Solid-state 610C Electrometer. The response was recorded on a Hewlett-Packard Model 7127A Strip Chart Recorder. Chemicals. Gallic acid and CH20 (analytical grade from Fisher Scientific Company) were used as received. Stock solutions of 10-2M CHlO were prepared daily from concentrated (37.50%) formaline. The 5 X 10-2M gallic acid, 1.8 X 10-'M H202, and 6 X 10-'M NaOH solutions were prepared daily by dissolving an appropriate amount of reagents in water. Other aldehydes were analytical grade from Eastman Organic Chemicals. Paraformaldehyde, K2C03, and NaOH were from Baker Chemical Company, KO2 from Alfa Inorganic (Ind.), alltrans-p-carotene, bilirubin, and biliverdin from Sigma (St. Louis). Special solvents such as DMSO and 99.5% DzO were from Matheson Coleman and Bell (dried over a zeolite) and from Columbia Organic Chemicals. Solutions were prepared using water from a Continental Water Conditioning Company deionization system.
v
400
500
600
700
WAVELENGTH, n m
Figure 2. Absorbance, fluorescence, and chemiluminescence spectra of gallic acid oxidation products (1, 2) Absorption spectra of the solution with initial concentrations 200 fiM gallic acid + lOmM H202 and lOmM K&03 after 1 min (1) and 12 hr (2) of reaction. (3)Fluorescence spectrum of 2.5mM gallic acid + lOmMKOH oxidized during 2 min with dissolved 300pM 02.A,, = 365nm. (4) Chemiluminescence spectrum (31).Conditions as in 3. Both spectra were measured at 24 O C and corrected for the spectral response of a photomultiplier
nism, 2) quantitative aspects of the modified Trautz-Schorigin reaction and its optimization for analytical purposes, and 3) the application of the optimized system to CHzO analysis.
PHYSICO-CHEMICAL CHARACTERISTICS AND MECHANISM OF CL I N THE TRAUTZSCHORIGIN REACTION The Trautz-Schorigin reaction involving four substrates and water as the solvent is a complex system. Therefore, CL of "partial systems", Le., polyphenols and aldehydes oxidized separately with H202 or 0 2 was first studied. This simplification also gives information on the analytical aspects, namely: 1) what is the CL background of the gallic acid-H202(02)-OH- system in the absence of CH20, and 2) what is the selectivity of the TSR for CHzO relative to other aldehydes. CL for the Gallic Acid-Oz( HzOz)-OH- System. Gallic acid (I), R = COOH, and other polyphenols when oxidized with molecular 0 2 or H202 in aqueous 10-4-10-2M solution a t pH 8-12, give weak CL. Emission spectra consist of two bands-one blue and one red, with maxima a t 475-505 nm and 635 nm (31-35). The CL spectrum of (I)-Oz-KOH is shown in Figure 2. The oxidation of I proceeds gradually in several stages involving colored intermediates I1 and 111. Final oxidation products contain IV-VI1 and a brown, low molecular weight, water-soluble polymer (P) of unknown structure. Absorption spectra of the solution during and after the oxidation are given in Figure 2. The final oxidation products of the polyphenols fluoresce in the 420-700 nm region with maxima at 470-560 nm (31-33). The fluorescence of gallic acid oxidation products in an aqueous solution after 2 minutes with the maximum at 565 nm is par-
RESULTS AND DISCUSSION The development of the chemiluminescent method for CHZO determination was divided into three stages, namely: 1) basic investigation of the chemiluminescence mecha2102
,
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
R
k
I
II
IV
Ill
V
VI
12
Figure 4. Thermal effects and chemiluminescence in the TrautzSchorigin reaction
TIME
(MIN)
Figure 3. Kinetic curves of chemiluminescence
+
The system: lOmM gallic ecid .t 1M H202 1M KzC03, containing different aldehydes: (1) CHzO, (2) gallaldehyde, (3) glyoxal, (4) methylglyoxal, (5)crotonaldehyde. (6) propionaldehyde. (7)acetaldehyde, (8)dodecanal, (9) hexanal in concentration 1M, (10) chemiluminescence resulting from the addition of CH20 into the system 9. Curves 1, 2, and 10 have the light intensity scale diminished three times
ticularly strong (Figure 2, curve 3). In DMSO solution, the fluorescence band is narrower than that in aqueous s o h tion (Fluorescence Width Half Maximum = 1100 cm-l VS. 1540 cm-l in water) and has its maximum at 500 nm. The oxidation of gallic acid (I) (R = COOH) and other polyphenols in the presence of 10-3-lM H202 results in: a) an increase of CL intensity by a factor of 10-100, b) a decrease of the absorbance of oxidized solution (bleaching) in comparison with those without H202, and c) a decrease in the pH of solution during the oxidation by 0.2-0.3 pH unit. Chemiluminescence and fluorescence spectra are either the same as with 0 2 , or blue-shifted, and they are dependent on the time of reaction ( 3 1 ) .These findings are assigned to the oxidative degradation of I11 and IV intermediates as well as of polyiners (33, 35, 3 6 ) . The data may be summarized in the following scheme: I
+
O,,OOH-.O.-
OH-
+ fast
ii,-/o;* Ii
------ * If1 slou
red-pink
bluegreen
\
+
ery slow
L
I
V v-VI1
fast
IOLx.
+ co, + IO2*
A CL mechanism involving emission from singlet oxygen and intermolecular energy transfer has been proposed (31, 32):
+
(O-('A,))? 200?(38,-) hr O2('A,)0fZ,-) -* 20J3Z,-) + hu o?(lAg)o?(l&+) + Po m2(1&-) + IP* +
P*
-
P
+
hr
X = 643nm X = 478nm
X =3i5-j05 nm
Traces of heavy metal ions, colloids, and oxidases strongly enhance both the oxidation rate and CL intensity. Since CL of the I-H202(02)-OH- system sets the limit of CH20 detectability, i.e., a background, particular precaution is necessary to purify reagents and water.
(A, 6. and C ) different combinations of substrates and the sequence of their mixing, (D)a differential transformation of the curve C , T = fit). Concentrations: (1) 3 M H202, (2) 40mM gallic acid, (3) 2.6M CHZO and 0.9M K2C03. 0 0 0 temperature [ " C ] , curves T = Rt), -- chemiluminescence intensity /cL = Rt), A A A the rate of temperature changes, curves AT/At = R t )
- - - -
- - - -
CL of Aldehydes-H202(02)-0H- Systems. The oxidation of CH20 + H202 NaOH in aqueous solution is accompanied by very weak CL. Because CL intensity is very low, only part of the spectrum has been measured, ranging from 450 to 650 nm ( 3 7 ) . Recently, CL from the CH20 H202-KCN system has been studied ( 2 5 ) . CHzO H202 form a-hydroxy-methyl peroxide which decomposes rapidly in the base-catalyzed reaction when KCN is added. No CL spectrum was reported. Thermal decomposition of CH300CH3 plus 0 2 in the gas phase a t 120-180 "C results in a red, weak CL ( 3 8 ) . The blue part of the spectrum is observed a t 430 nm and spreads over 550 nm into the red. The total CL spectrum could not be measured however. The question arises if other aldehydes can react with I H202 OH- and interfere with CH20 determination. The effect of some aldehydes on the Trautz-Schorigin reaction is shown in Figure 3. Glyoxal (CH0)2 and methylglyoxal (pyruvate aldehyde) gave CL intensity comparable with that of CH20. When injected into the reaction mixture rapidly, glyoxal gives an initial CL intensity even higher than CH20, but the emission has the character of a flash. In order to avoid a very violent reaction, (CH0)2 has to be added gradually. Every drop of (CH0)z added generates strong flashes, shown in Figure 3, curve 3. In the static method of CL intensity measurement, it is possible to distinguish between the instant spike generated by (CH0)2 or CH3CO-CHO and the second main maximum, which in the case of CH20 appears later (Figures 3 and 4). However, in the case of the flow method, both aldehydes will interfere with CH2O determination. Other aldehydes give CL intensity a t least one order of magnitude lower than CH20. When CHpO is added to the solution of I, H202, K2CO3, and aldehyde after several minutes, the CL typical of the TSR appears as indicated by the arrow in curve 10 in Figure 3. Therefore these aldehydes are expected not to interfere with CH20 determination. Effect of Solvents and Inhibitors. The maximum solubility of I in water is 2 x 10-2M a t room temperature. Using a methanolic solution of I, a CL intensity higher by a factor of 3 is obtained. Since l 0 2 * was postulated as an emitter in the TSR (19-21, 3 7 ) , two groups of experiments were performed in
+
+
+
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
+
+
2103
Table I. Effect of Solvents and Inhibitors on Chemiluminescence Substrates and concentrations
Solvent
3mM gallic acid + 0.831 H,O, + 0.45.1.1 KZCO, + 0.8651 CH,O
water
158.O
3mJ1 gallic acid + 0.8N H , 0 2 + 0.86J1 CH,O T 0.4561 KZCO, lOmJI gallic acid + 2.5J1 paraformaldehyde + 0.131 t- BuOK The same
96%
380.5
10mA1fgallic acid + 1OmJf paraformaldehyde i10mA1
DZO DMSO :
Et-OH = 9: l(v'v) DMSO: H,O 1: 1 (v/v)
Etfect of Inhibitors Concentration
I max, re1
saturated H,BO, 0.1M hydroquinone 5md1 cysteine 5mM ascorbic acid 5mM a-naphthol Bilirubm, blliverdin 0.3 m J1 biliverdin 0.8mN bilirubin
Quenching, %
88 and 60" 81.7 10.0 52 .O 10.0 0 62.5
50.0
13.2
10% water
47.0
3.2
0.8m.11 NiC1, 6 ~ J P-carotene l 0.2mA1 biliverdin 5 . 5 ~ 1water 6p-11$-carotene
75.0 74.5 41 .O 67.3 40.0
dry DMSO
KO? Refers to bath chemiluminescence maxima in kinetic curves, respectively, (see, e g , Figure 4C), I,,,, intensity
order to check these' suggestions: 1) the influence of solvents in which 02('Ag) has a longer life time than in water was studied, and 2) the effect of inhibitors, particularly of lAg02-quenchers was tested. The results are summarized in Table I. Adding H3B03 (saturated) before the TSR was started quenches the first stage of CL (spike) by 88% and the second one by 60%. H3B03 added at the minimum on ZL = f ( t ) curves, i.e., between the first and the second CL stages, quenches CL intensity by 71%. It is known that H B B O forms ~ esters with OH groups of polyphenols. The data indicate that CL kinetics are controlled by the rate of I oxidation. This result is confirmed by CL quenching with free-radical scavengers. At higher scavenger concentrations quenching is more effective. Thus, free radicals of semiquinone types I * I1 and 111 are involved in the reactions generating CL. Qaenchers of l A g 0 2 (39, 4 0 ) markedly diminish CL intensity only in nonaqueous solutions. CL intensity in 96% D2O is higher by the factor 2.4 than in H2O. This value is lower than that expected for lAg(O2) with its lifetime 20 wsec in DzO. The discrepancy may be due to the decrease in pK, values of I and H202 in D2O (41). Some quenchers, e.g., bilirubin, give CL in the absence of I. Therefore, the observed quenching effect can be the result of the superposition of CL from quencher oxidation and O2('Ag) quenching. Effect of Temperature. In the temperature range 20-40 "C, the function I,,, = f (l/T) is a straight line with the activation energy E , = 39.5 f 2.4 kJ mol-'. This value is very close to the E , of CL in the oxidation of pyrogallol and the oxidative ring-opening of purpurogalline ( 3 5 ) . The Kinetics of Thermal Effects. Thermometric measurements of the reacting solution temperature T = f ( t ) were performed simultaneously with CL measurements I = f ( t ) for different orders of reagent addition. The results are shown in Figure 4. Although the measurements do not allow calculation of any thermodynamic functions, they give information on the reaction mechanism. The data of Figure 4 and thermochemical calculations implicate four exothermic reactions: 1) The decomposition of the transient a-hydroxyperoxides ( 4 2 ) HO-CH2-OOH and HO-CH200CHz-OH in the base catalyzed reaction: OH-CH200CH2-OH 2 NaOH 2 HCOONa 2 H20 H p A H = 1150 k J mol-l. How-
-
2104
+
+
+
= maximal chemiluminescence
ever, this reaction without I does not generate a strong, red CL (Figure 4A). On the contrary, heat and gas evolution accompany CL quenching. 2) Another mode of a-hydroxy-peroxide decomposition, which involves peroxy-radical dismutation:
CH?O
+
HCOOH
+
HlO
+
02
The enthalpy of this reaction, calculated from the band energies, is - A H = 545 kJ mol-'. The important feature of this reaction is 0 2 formation. Some fraction of the total amount of 0 2 may be formed in the excited singlet states 'Ag and I Z g f . Both reactions 1 and 2 are mutually competitive and their contributions to the over-all reaction can be estimated on the basis of other criteria, e.g. the amount of H2 or 0 2 evolved. (3) 2H?O,
OH-
2H20
+
O2 -A = 2jOk:, mol-'
This reaction produces very little light (Figure 4). Its contribution to the overall reaction is rather small because of strong competition from reaction 1. 4) The oxidation of o-trihydroxy benzenes with H202 at pH 8-11 yields a blue-green radical ion of the intermediate quinone (111). I11 undergoes a further exothermic oxidative ring cleavage to tropolone carbonyl derivatives (V), (VI), (COOH)2, and C02 (35,36).
-AH
=
780-88Okz mal-'
These reactions occur simultaneously with the main emission peak CDE on kinetic curves ZCL = f ( t ) (Figure 4C) and are necessary for CL. The observed thermal effect A T = 0.3-0.4 "C is obviously very low because of low I concentration M). The kinetics of the thermal effect of the complete Trautz-Schorigin system, expressed in the differential form as the rate of temperature change dTldt = f ( t ) , reveals four phases (Figure 4D): 1)an instant ATlAt increase which correlates in time with the CL peak ABC and a redpink, transient 11; 2) a slow increase of ATlAt which corre-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
cl _1
0
, \ 5
TIME
io (min)
15
Flgure 5. Effect of the pressure changes on chemiluminescence
(1) A vacuum line is swilched on and chemiluminescence is recorded upon lower pressure; (1) a vacuum line is switched off and solution equilibrated with air. An additional explanation is in the text
sponds to the ascending part of I C L = f ( t ) in the CD stage; 3) a middle decrease of ATIAt appearing with the second CL maximum D arid the beginning of CL self-quenching. In phases 2 and 3, the reacting solution has a slightly greenyellow color. The last phase, 4) is a slow decrease of A T l A t accompanying a long-lasting “tail” of CL. In this phase, the reaction mixture slowly evolves a gas and is colorless. The temperature increase in the third phase can account for CL temperature-quenching and a diminution of the solubility of gases, e.g. 0 2 . Moreover, the temperature increase accelerates a-hydroperoxide decomposition and, in turn, an evolution of H2 and C02 from K2C03. Effects of the Total Pressure, N2 and 0 2 . If l 0 2 * participates in the Trautz-Schorigin reaction CL either in the gas or liquid phase, then the CL should be pressure-dependent. Figure 5 shows that a rapid decrease of the total pressure causes CL spikes followed by an immediate decrease in CL intensity. When a vacuum line is switched off and the solution equilibrates with air, then a rapid CL decrease is stopped and curves ICL = f ( t ) reveal their normal shape. Repeating this experiment gives qualitatively the same results, but the relative changes of ICL gradually diminish. This simple experiment convincingly indicates that the CL in the TSR is dependent upon a gaseous component being present in the solution. The CL spikes may be due to the formation of gas bubbles under a lower pressure. The escape of bubbles from the solution causes a decrease in the gaseous component concentration and in turn, a CL intensity. The rate of the reaction generating this component critical for CL, is gradually decreasing. Therefore, the observed effect is time-dependent. Gaseous N2 bubbled through the luminescing solution reduces CL intensity by 50%in comparison with I C Lunder pure 0 2 . I C Lin the air-saturated solution is lower by 37%. The use of N2 is advisable for analytical purposes because it depresses CL background more strongly than CL from the Trautz-Schorigin reaction. Dissolved Oxygen Concentration and CL. The concentration of dissolved 0 2 and ICLwere measured simultaneously in the same experiments. Figure 6 shows normalized kinetic curves [&]& = f ( t )and I C L = f ( t ) .I t is evident that the peak of [ 0 2 ] & always accompanies CL. A temporary increase in [02]dls is observed even in solutions constantly flushed with N2. The shape of both kinetic = f ( t ) is similar, but [02]dis curves [ 0 2 ] d i s = f ( t ) and ICL decreases faster than ZCL.The examination of the kinetics and [ 0 2 ] d i s level of different “partial” reaction systems, presented in Figure 6,A-D leads to the conclusion that there are two groups of opposite reactions generating and
Figure 6. Dissolved oxygen concentration and chemiluminescence (A, B, C, and D) different combinations, time, and the sequence of mixing substrates. Conditions: (1) 1M H20, (2) 4mM gaiiic acid, (3) 0.7M CHzO, (4) 0.4M KzC03. 0 0 0 Dissolved oxygen concentration [ M I , Le., curves [02]dla = Rt): chemiluminescence intensity; /CL = qt). Temperature 22 OC f 0.2. Arrows indicate the moment of a substrate injection
- - - --
consuming the dissolved 0 2 . The sources of 0 2 are the following systems, in which [H202 OH-] > [CHzO (I)]:
+
+ + + (4) H202 +
(1) H,O, (2) H,O, (3) H202
OHOHOHOH-
+ + +
+
CH,O I CH20
+
I
In these systems, 0 2 can be generated by two reactions previously mentioned, i.e. (1) 2H202
- -+ O H
2H20
(2) PHO-CH,-OO.
O2 CH2O
+
HCOOH
+
H20
+
0,
in However, as is seen in Figure 6D, the presence of the solution even at a concentration higher than 0.2mM does not produce a strong, red CL. The data of Figure 6,A-D indicate that 0 2 d i s is quickly consumed by 1, I; 2, CH2O; and 3, I CH2O and/or their oxidation products such as 111. However, the addition of I or CH2O to the system of Equation 1 produces only weak CL. Thus, the presence of the dissolved 0 2 is a necessary, but not a sufficient condition for CL. Both 0 2 d i s and oxidation products of I and CH2O are necessary to generate CL in the TrautzSchorigin reaction. Therefore, the most probable assumption is that reaction 2 and oxidation products of I are involved in the CL-limiting step, according to the scheme:
+
H2O CH,O OH
I(reaction 2)
(‘O,---X)* 11, III,P 0 2 dis
(O*-X)
-
-
+ x
”0, ’0,
1
30,
+
hr,
+
X
+
hr,
where X denotes intermediates of I oxidation, consuming 0 2 dis. Details of this scheme will be discussed further. The Evolution of Gaseous Products. In the diluted solution described in Figure 6, the [02]dis is lower than 4 X 10-4M and the gaseous 0 2 does not escape from the solu-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2105
I
6 00
700
800 SUBSTRATE
W A V E L E N G T H , nrn
Figure 7.
Figure 6.
Chemiluminescence spectrum
+
+
Conditions: 2OmMgalllc acid 3 M H202 3MCHzO and 2.5M K2C03. Flow rate, 8.8 ml/min/syringe. Spectrum measured with the Hamamatsu RE18 photomultiplier and corrected for its spectral response; 0 0 0 values of relative chemiluminescence intensity for the spectrum measured and corrected with a EM1 9558 QA photomuttiplier. Vertical segments represent katters of chemlluminescence intensity
--
tion in the form of bubbles. Careful observation of more concentrated reaction mixtures also does not reveal the formation and evolution of 0 2 in the A-D phase of CL. However, in the last phase of the TSR, a violent, gas-evolving reaction takes place. Using Ba(OH)Z, i t was found that C02 evolves from the reaction: 2H202 2CH2O KZCO3 -----t WCOOK 3HzO CO, Moreover, H2 is generated in the previously given reaction. The saturation of the solution with COz diminishes 0 2 solubility. 0 2 dis that was not consumed, undergoes nucleation, forms 0 2 bubbles, and evolves from the solution. This process occurring between the D-E CL stage obviously is not associated with strong CL. Chemiluminescence Spectrum of the Modified Trautz-Schorigin Reaction. The entire CL spectrum has never been measured because of the low sensitivity of light detectors in the red spectral region. Rapid kinetics and a violent course of the reaction create additional difficulties. Bowen and Lloyd (19) have found a narrow emission band a t 630 nm and assigned it to l 0 2 * . The oxidation of CHzO HzOz OH- (37) and thermal decomposition of CH3OOCH3 0 2 (38) are accompanied by a weak, red CL. However, its spectrum which spreads over 550 nm was not measured. The CL spectrum of a modified TSR with tannic acid, evaluated by means of optical filters, has a red emission beyond 570 nm and about a 50 times weaker blue one in the range 440-510 nm with the maximum a t 475 nm (24). These bands were assigned to the radiative deactivation of l 0 2 * and excited anions of carboxylic acids resulting from the oxidative cleavage of polyphenols. The CL spectrum measured in this work is shown in Figure 7 . Both types of photomultipliers give similar results: very broad and structureless bands with slightly marked maxima a t 635-645 nm and 750-760 nm, and a weak shoulder a t 705 nm. The spectrum spreads beyond 800 nm but its exact shape could not be measured. The low spectral sensitivity of both photomultipliers in the range beyond 800 nm caused the correction factor to be very high (>20). Quite recently Lichszteld (43) measured the CL spectra of TSR for several polyphenols. The calibrated system, consisting of a cooled, red-sensitive photomultiplier and a prism monochromator, giving a spectral band width 17 nm a t 700 nm was used. The spectrum for gallic acid spreads from 560-840 nm, with a maximum a t 704 nm, and a small shoulder a t 770 nm. The concentrations used by Lichszteld were half those used here.
+
+
2106
+
+
+
+
CONCENTRATION
[MI
Chemiluminescence intensity vs. concentrations of particu-
lar reagents Conditions: varying concentrations of 5 m M gallic acid, 80mM H202, I m M CHZO, and 0.1 M KOH, temperature 23 'C. The peak height scale for CH20 is given on the right
Additional experiments with an infrared sensitive photomultiplier and interference filters showed the emission a t 1270, 1060, and 765 nm. The relative intensities, corrected for the spectral response of the cathode and filters' transmission are 680:1:1.6, respectively. The observed emission bands could formally correspond to the following transitions of gaseous l 0 2 * :
+
mZ('AJ 202(1Ag)b_0 OZ('Zg+) 02(Ag)Ld OZ('Ag)
---
z02CZg-) 2M3Zg-)
X = ~ n m X = 702 nm 02(3Z,-) X = 762 nm 02(3&)u_,X = 1070 nm Oz(3Z-) X = 1270 nm
Factually, however, the observed emission cannot be assigned to the radiative deactivation of l 0 2 * in a gaseous phase for the following reasons: 1) the spectrum is very broad and does not reveal the structure typical for ( l 0 2 * ) gas, and 2) neither Bowen and Lloyd (19),Bowen (ZO),nor the authors observed the formation of 0 2 bubbles. Thus, the spectral data give inconclusive evidence regarding the nature of the emitters. The most probable assumptions are: 1) l02*-X complex formation, where X is an oxidation product of I. The transfer of energy from lZg+ and lAglAg to the lowest triplet of X ( = 111, P) followed by phosphorescence would be the simplest mechanism. This kind of emitter was postulated by Brabham and Kasha for the CL of the H202-C10--dyes system (44, 45). An emission spectrum of the system besides that of (l02*) gas bands has a new, broad band located in the spectral region of the dye's phosphorescence. This assumption accounts for the experimental data as follows: a) the necessity of I or its oxidation products for CL, b) the presence of dissolved [ 0 2 ] 0.4 mM, c) fast consumption of the dissolved 0 2 by I, CH20, and their oxidation products, and d) quenching effects in nonaqueous systems and CL enhancement in D20. Another result strongly supporting l 0 2 * participation in the observed CL was presented by McKeown and Waters (21). They carried out the TSR in the presence of 9methyl-10-phenylanthracene. No endoperoxide was formed when pyrogallol was oxidized. However, with resorcinol, the reaction, though less vigorous, did give a 5% yield of 9methyl-10-phenylanthracene-endoperoxide. 2) In aqueous solutions, the electric field of the water perturbs the energy levels of lAg and lZ,+ states. If the perturbed levels emit in water then, according to the theory
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
L
k P
io
I1
c
/”
6C
4’ 2
6 10 14 FLOW RATE ( rn I s/m in/ syri n g e )
18
Flgure 9. Chemiluminescence intensity vs. flow rate
-7
Conditions: 5mM gallic acid, 60mM H202, 10kM CH20 and 20mM NaOH. Average values calculated from 4-6 replicates
-6 LOG
-5 -4 -3 -2 [FORMALDEHYDE]
-1
Figure 10. Calibration curve for formaldehyde determination Conditions are as in Figure 9, flow rate 8.6 ml/min/syringe
(46),the emission bands assigned to the solvated 0 2 aq ( l & + ) and [ ( 0 2 ) 2 I a q (lAglAg) should be a t 670 and 787 nm. The bands should be broadened. A hypothetical mechanism of excited species generation, which a t least partially explains experimental findings and the literature (19-22) data, may be proposed as follows:
+
-
CH,O H202 HOCH200H HO-CH2-OOH + HC-CH,-OO H+ HO-CH2-OO- + SQ. HO-CH,+O -I-HQ’ 2HO-CH200. ---t CH20 -I- HCOOH
+
+ +
H,O
IO,+
where SQ- is a semiquinone radical formed from I, e.g., semioxidized form of I and/or 111, and HQ- is an anionic form of polyphenol.
OPTIMIZATION OF THE GALLIC ACID-CH20Hz02-OH- SYSTEM The optimization of the selected system was performed with respect to CL kinetics, intensity and flow parameters. Optimization of the Kinetics. Kinetic curves, I C L = f ( t ) reflect a complex mechanism of light emitting-species formation in consecutive reactions of the type: S , + S * + . . - P * - P f h v
tions. Holding constant concentrations of three reagents in different combinations, profiles of I,,, vs. concentrations of every reagent (CR) were obtained. These results I,, = ~ ( C Rare ) shown in Figure 8. The optimal ratios of concentrations are: [Hz02]/1 = 12, [Hz02]/[CHzO] = 3.5, [H202]/ [NaOH, KOH] = 3 and the initial pH = 11.4. Therefore, the final concentrations in the reaction mixture adopted for this method were 5 mmol of gallic acid (I), 60 mmol of H202, and 20 mmol of NaOH. Optimization of Flow Rate. Relative light emission was found to increase with increasing flow rate as seen in Figure 9. The increasing sensitivity suggests that the chemiluminescent reaction is going to completion within the cell; at higher flow rates, more light is emitted per unit time, resulting in greater peak height. However, an increase in flow rate has a negative effect on reproducibility. The method could be made more sensitive to CH20 concentration but only at the sacrifice of precision. Therefore, accepting a reasonable compromise, the flow rate 8.6 ml/min/syringe was arbitrarily chosen for CH20 determination. Since I C L is [O2]-dependent, the type of gas used for mixing the solution within the cell and its flow rate can both affect CL response. The optimum signal-to-noise ratio occurred a t flow rate 7.5 ml/min Nz. Consequently this flow rate of N2 was used.
Choosing the proper pH, -10.5, and the ratio [H202]/ CHEMILUMINESCENT DETERMINATION OF [CH20] = 1-4, one can observe four stages of CL kinetics FORMALDEHYDE with two maxima, as already presented in Figures 3 and 4. Once the parameters of the CL I-CHZO-HZO~-OH- sysWith an increase of pH and [CH20]/[I] ratio, separation between the B and D maxima decreases and the peaks tem were optimized, the calibration curve was established. overlap. Such a situation is convenient for analytical purStandard solutions prepared freshly by dilution from the poses because higher CL intensity and apparently simpler lOmM CH20 stock solution were placed in the flow line kinetics may be obtained. and steady-state CL I,,, was recorded. The number of replicates was 4-6. The results of the CH2O determination are Optimization of pH. Because the kinetics of the CL reaction are pH-dependent, the influence of several buffers given in Figure 10. The linear range for CH20 analysis is at pH 10.0 and 11.0 on the maximum intensity I,,, of CL, 10-7-10-2M. The limit of detection is 10-7M CH2O in a 2-ml sample a t the signal-to-noise ratio 1:l. Because this using the flow system (Figure 1) was examined. Na3B03and K2HP04-Na3P04 buffers give I,,, 24 and 8 sample is diluted 3 times in the chemiluminescence cell, times lower than that with NaOH or KOH, respectively. the actual detection limit is 2 ng or 1 ppb. The lower limit From the analytical point of view, the ratio I m a x / I ~ ~ of detection is imposed by both the background light emission and the dark current of the photomultiplier. It is pos(where IBG is a stationary ICL without CH20, i.e., background) is important because it often determines the detecsible to decrease the background level using: a) a prelimition limit of the method. Values of the I m a x / I =ratio nary deoxygenation of I ~ were H2O and the NaOH solutions, found to be 5.1, 3.1, and 24.5, respectively, under the optiand b) a lower temperature of the reactants in the CL cell, mal conditions of flow rate, pH and [CHzO] = 10-4M. e.g. 15-20 OC inste.ad of 22-25 OC. Thus, the best conditions for analytical purposes, particuThe sensitivity and the precision of the CH20 determilarly in a flow method, are 0.15M NaOH or KOH. nation may be further increased by cooling the photomultiOptimization of Reagent Concentrations. The sensiplier. One has to realize that red-sensitive photocathodes tivity of the modified Trautz-Schorigin reaction with rehave relatively high dark currents (DC). Moreover, DC is spect to CHzO is markedly affected by reagent concentravery sensitive to temperature changes in the environment.
+
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
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Table 11. Effect of Interfering Compounds on Chemiluminescent Determination of Formaldehyde Relative mean
Concentration
tM1
Compound
CHSOH (CH3)zCO CH3COOH CzHSOH CH3CHO NazS03 NaCl Humic acids Commercial : Serva
A
5 .O 0.27 2mM 0.67 O.lmM 3mM 0.26mM
C,H,OH
0.5
0.5 0.03 0.003 0.1 10pM 0.5mM 0.1 3mM
d
1.58 0.2 0.006 0.46 4 x 10-5 0.006 8 X low4
0.1
+g.o
0.03 0.01 0.02
0.02 0.03 4.7
-30.0 +1.1 -54.4
0.001 0.02 0.09
Conditions are as in Figure 10. The numbers above replicate peaks are molar CHzO concentration
The relative changes in the dark current AN are higher than those of the background (ABG), and the ratio ADC/ABG is equal to 4.5 for [CH20] C 10-6M. It means that for low values of signal-to-background ratio (low concentrations of the contribution of the dark CH20 and low values of I,,, current changes ADC to the total experimental error is higher than that of ABG. Therefore, by keeping the temperature of the photocathode a t a low and a constant level, e.g., -10 OC, it is possible to improve both sensitivity and precision by a factor of about 5. Figure 11 shows the reproducibility when 3 replicates, containing 8 X 10-6M, 3 X 1OW6M,and 6 X 10-7M CH20, were analyzed under optimized conditions. The relative standard deviation of the CL method is about 1%,i.e., similar to that obtained in other instrumental methods for CH20 determination (22, 27-30). The minimum volume of the CH2O solution determined is 2 ml, and the analysis time generally is less than 2 minutes. Effect of O t h e r Compounds on the Determination of CH20. It was reported that some natural waters receiving industrial wastes, and other bodies of water which show the NO.
+16.6 +5.1 -8.8 +11.1 +15.5 -30.0 -2 .o
[MI
+3 .O -14.3
Figure 11. Chemiluminescence response of formaldehyde standard
ANALYTICAL CHEMISTRY, VOL. 47,
of Imax
0.1
run
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%
15.8 1.58 0.012 3.1 4 x 10'4 0.04 0.0016
Fluka Synthetic Pirox OH
Limiting concentration C1
error,
0.01
presence of CH20, also contain other carbonyl compounds (28, 29). Moreover, industrial CH20, e.g., formaline, contains compounds which could interfere with the CL determination of CH20. Therefore the effect of some comof CL of the I-CH20-H202-NaOH pounds upon the I,,, system was investigated. One of the most ubiquitous components of natural waters is a water-soluble fraction of high-molecular, dark polymers-humic acids. They contain phenolic groups and give weak CL when oxidized with H202 or 0 2 in alkaline solution (47, 48). Qn the other hand, humic acids efficiently quench CL generated by peroxyradical recombinations (49). For these reasons, the effect of some commercial and synthetic humic acid preparations on CL in the TrautzSchorigin reaction was tested. In Table 11, limiting concentrations, C1, of interfering compounds, expressed in M and %, are given. C1 is defined as the concentration a t which the change in I,,, of CL does not exceed the mean error of CH2O determination, i.e., 1%. In the second column of Table 11, the concentrations of interfering compounds used in experiments and relative mean errors are tabulated. Because C1 values for some carbonyl compounds are low, it is recommended that it be determined whether these compounds are present in the analyzed sample, and, if they are present, what is their concentration. The quenching effect of humic acids is much stronger than the enhancement one. Therefore, samples containing humic acids in concentrations higher than lo-% have to be diluted or separated. Other phenols present in samples in concentrations lower than lOW3M,or phenol and pyrocatechol at 10-2M, do not interfere with the CH2O determination. When the concentration of the interfering compound in the sample is higher than the value of C1,and moreover, is known and constant, then a new calibration curve, measured in the presence of the interferent may be plotted. If the concentration of CHzO is much higher than its detection limit, the sample may be diluted by a known factor. The dilution will reduce the concentration of the interferent below the C1 value. Effect of Reagents' Age a n d Concentration. It is important that I and H202 solutions should be freshly prepared using deionized, distilled water. Such solutions can be stored in a refrigerator no longer than 12 hours. Aged solutions show a decreased CL response. The increase of I and H202 concentration by 10%and NaOH by 3% does not affect either the CL level of background or the signal over the limit of the experimental error, Le., 1%.
13. NOVEMBER 1975
SUMMARY Data reported here indicate that CH2O is the reagent involved in the radical C L reaction probably generating '&*. Of all factors studied, [CH20] has the most significant affect on CL intensity. In the optimal range of the initial pH = 11-12, the reaction order is observed to remain the first order throughout five orders of [CHzO]. From an analytical point of view, this feature of the Trautz-Schorigin reaction CL is very favorable for CH20 assay. The results obtained in this work are much better than those in previous ones (22). The CL flow technique and high-standard electronic equipment made it possible to increase the sensitivity by two orders of magnitude and to shorten the time of analysis to 2 minutes. In comparison with other recently elaborated methods for CHzO determination (27-30), the CL technique offers several advantages. As the CL reaction is essentially instantaneous, CH20 analysis is not delayed by long reaction times. Because the temperature coefficient for CL reaction is not very high, no precise temperature control is needed. The pump system maintains uniform, continuous flow and time control is not needed. Instrumentation is minimal; only a pump system and a photomultiplier with basic electronic equipment are required. The reagents used are normally found in the laboratory and their cost is very low. This method is expected to be especially useful for continuous, routine determination of CH20 and offers possibilities of automation. The interference of some carbonyl compounds and inorganic ions present in some waters is the only disadvantage. The extension of the CL system for the determination of other compounds is anticipated. Any compounds that react with CHz0, e.g., proteins (23), amines (29), with peroxides such as HO-CH2-OOH (25) or substitute gallic acid, e.g., tannins (24), may be assayed. Therefore, the modified CL Trautz-Schorigin reaction may extend the list of compounds determined by CL methods.
ACKNOWLEDGMENT The authors would like to thank D. M. Hercules and W. R. Seitz for encouragement and helpful discussion. We are also grateful to J. Lee, D. E. Brabham, and I. B. C. Matheson for their stimulating discussion and many valuable suggestions on singlet oxygen experiments.
LITERATURE CITED (1) W. R. Seitz and D. M. Hercules in "Chemiluminescence and Bioluminescence", M. J. Cormier, D. M. Hercules, and J. Lee, Ed., Plenum Press, New York, N.Y., 1973, p 427. (2) B. L. Strehler, Methods Biochem. Anal.. 18, 99 (1968). (3) A. Fontijn, P. Golomb. and J. A. Hodgeson in "Chemiluminescence and Bioluminescence." M. J. Cormier, D. M. Hercules, and J. Lee, Ed., Plenum Press, New York, N.Y., 1973, p 393. (4) W. R. Seitz and M. P Neary. Anal. Chem., 48, 188A (1974). (5) J. Lee, C. L. Murphy, G. J. Faini, and T. L. Baucom in "Liquid Scintillation
Counting: Recent Developments", P. E. Stanley and B. A. Scoggins, Ed., Academic Press, New York, N.Y., 1974, p. 403. (6) P. E. Stanley in "Liquid Scintillation Counting", Vol. 3, M. A. Crook and P. Johnson, Ed., Heyden. London, 1973. (7) H. H. Seliger in Ref. 3, p 461. (8) D. T.Bostick and D. M. Hercules, Anal. Chem., 47, 447 (1975). (9) D. C. Williams and W. R. Seitz, submitted to Clin. Chem. (10) M. P. Neary, W. R. Seitz, and D. M. Hercules, Anal. Left.,7, 563 (1974). (11) A. K. Babko, L. I. Dubovenko, and N. M. Lukovskaya, "Chemlluminescent Analyze", Kiev, USSR, 1986. (12) T. G. Burdo and W. R. Seitz, Anal. Chem., 47, 1639 (1975). (13) J. P. Auses, S.L. Cook, and I. T. Moloy, Anal. Chem., 47, 244 (1975). (14) L. I. Dubovenko and E. Ya. Khotines, Ukr. Khim. Zh., 37, 1154 (1971). (15) E. L. Wehry and A. W. Varnes, Anal. Chem., 45, 348 (1973). (16) 0. Shimomura, F. H. Johnson, and Y. Salga, Science, 140, 1339 (1963). (17) J. M. Petrusevitch and J. Slawinski, Biofizika, 14, 750 (1969). (18) M. Trautz and P. Schorigin, 2. Wiss. Photogr. Photochem. 3, 121 (1905). (19) E. J. Bowen and R. A. Lloyd, Proc. Chem. SOC.(London),305 (1963). (20) E. J. Bowen, Pure Appl. Chem., 9, 473 (1964). (21) E. McKeown and A. W. Waters, J. Chem. SOC.B, 1040 (1966). (22) D.Slawinska, D. Golebiowska, and J. Slawinski, Chem. Anal. (Warsaw), 11, 1117(1965). (23) D. Balcerowicz, K. Balcerowicz, D. Slawinska, and J. Slawlnski, Chem. Anal. (Warsaw), 15, 479 (1970). (24) I. Milczarek, E. Grabikowski, A. Murkowski, and J. Slawinski, Chem. Anal. (WWSaW), 17, 31 (1972). (25) A. R. Shoaf and R. H. Steele, Biochem. Biophys. Res. Commun., 81, 1363 (1974). (26) A. P. Altshuller and J. J. Bufalini, Photochem. Photobiol., 4, 97 (1965). (27) I. F. Walker, "Formaldehyde, ii", Ed., Am. Chem. SOC.Monograph Series, New York, N.Y., 1953. (28) B. K. Afghan, A. U. Kulkarni, R. Leung, and J. F. Ryan, Envlron. Lett.,7, 53 (1974). (29) B. K. Afghan, A. U. Kulkarnl, and J. F. Ryan, Anal. Chem. 47, 488 (1975). (30) S.Ikeda, Anal. Chem., 48, 1567 (1974). (31) K. Lichszteld. T. Michalska, and D. Slawinska, "11 Pollsh Conference on Luminescence, Vol. 2, Institute of Physics, N. Copernicus University, Torun, 1974, p 113. (32) D. Slawinska and I. Kruk, Ref. 31, 207. (33) D. Slawinska, Zesz. Nauk. Politech. Szczecin., 107, Pr. Monogr. 48, 117 (1968). (34) R. F. Vassil'ev and G. B. Meluzova, Mol. Photochem.. 2, 251 (1970). (35) J. Slawinski, Photochem. Photo6iol., 13, 489 (1971). (36) J. Slawinski, B. Szczodrowska, and M. Wlodarczyk-Graetzer, Acta Biochem. Pon.. 20, 119 (1973). (37) J. Stauff and G. Rummler, 2. Phys. Chem. (Frankfurta m Main), 34, 67 (1962). (38) V. Ya. Shiyapintokh, 0. N. Karpukhin, L. M. Postnikov, V. F. Tsepalov, A. A. Vichutinskii, and I. V. Zakharov," Chemilurnlnescent Techniques in Chemical Reactions," Ed. Science, Moscow, 1966, pp 261, 266 and 276. (39) i. B. L. Matheson. N. U. Curry, and J. Lee, J. Am. Chem. Soc., 98, 3348 (1974). (40) E. A. Ogryzlo in "Chemiluminescence and Bioluminescence," M. J. Cormier, D. M. Hercules and J. Lee, Ed., Plenum Press, New York, N.Y., 1973. (41) R. S.Becker, "Theory and Interpretation of Fluorescence and Phosphorescence," J. Wiley, New York, London, Sydney, Toronto, 1969, p 243. (42) L. J. Durham, C. R. Wurster, Jr., and H. S. Mosher, J. Am. Chem. SOC., 80, 332 (1958). (43) K. Lichszteld, Technical University, Szczecin, personal communication, 1975. (44) D. E. Brabham, Dissertation, The Florida State University, 1973. (45) D. E. Brabham and M. Kasha, Chem. Phys. Left.,29, 159 (1974). (46) D. R. Kearns, J. Am. Chem. SOC.,91, 6554 (1969). (47) D. Slawinska and J. Slawinski, Nature, 213, 902 (1967). (48) D. Slawinska and J. Slawinski, Rocz. Chem., 44, 2415 (1970). (49) J. Slawinski, Rocz. Chem., 44, 2187 (1970).
RECEIVED for review June 10, 1975. Accepted July 25, 1975. This work was supported in part by the National Institute of General Medical Sciences under Grant GM17913.
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