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Anal. Chem. 1991, 63, 688-692
LITERATURE CITED (1) Von Michaelis, Hans. Cyan- and the Environment-Proceedings of a Conference; Geotechnical Engineering Program, Dept. of Civil Engineerlng, CSU: Fort Collins, CO, 1985; Vol. 1, pp 51-66. (2) Duodoroff, Peter. Toxicity to Fish of Cyanides and Related Compounds, A Review. EPA-440/5-80-37, 1976. (3) Cyanides in Water. 1987 Annual Book of ASTM Standards, ASTM: Philadelphia, PA, 1987; Vol. 11.2, D-2036-82, D4282-83, pp 118-129. (4) Qwllty Criteria for Water 1986 (Gold Book); US.EPA Office of Water Regulations and Standards: Washington, D.C., 1986; EPA-440/5-86001. (5) Tarasankar, Pal; Ashes, Ganguly; Durga, S. Maity. Anal. Chem. 1986, 58, 1564-1566. (6) Roy, Ram B. Am. Lab. 1988, 2 0 , 104-112. (7) Sekerka, I.; Lechner, J. F. Water Res. 1976, 7 0 , 479-483. (8) Pohlandt, Chrlstel. S.Afr. J . Chem. 1984, 3 7 , 133-137. (9) Nonomura, Makoto. Anal. Chem. 1987, 5 9 , 2073-2076.
(IO) Pohlandt, Christel. S . Afr. J . Chem. 1985, 3 8 , 110-114. (11) Jungreis, E. Israel J . Chem. 1969, 7 , 583-584. (12) Jungreis, Ervin; Ain. Fanny. Anal. Chim. Acta 1977, 88, 191-192. (13) Goulden, D. Peter; Afghan, Badar: Brooksbank, Peter. Anal. Chem. 1972, 59, 1845-1849. (14) Kelada, P. Nabih. J . Water Pollut. ControlFed. 1989, 61, 350-356. (15) Pihlar, B.; Kosta, L. Anal. Chim. Acta 1980, 88, 751-281. (16) Zaidi, S.A.; Carey, J. Cyanide and the Environment-Proceedings of a Conference : Geotechnical Engineering Program, Dept. of Civil Engineering, CSU: Fort Collins, CO. 1985; Vol. 2, pp 363-377. (17) Ohno, S. Bull. Chem. Soc. Jpn. 1967, 4 0 , 1765-1775. (18) Huiatt, J. L. Cyanide and the Environment-Proceedings of a Conference; Geotechnical Engineering Program, Dept. of Civll Engineering, CSU: Fort Collins, CO, 1985; Vol. 1. pp 65-81.
RECEIVED for review August 10, 1990. Accepted January 4, 1991.
Kinetic Titration Method To Determine the Excited-State Concentration of a Photochemical Sensitizer P. E. Poston and J. M. Harris* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
A noncomparative method to determine the excited-tripietstate concentration of a photosensitizer is described. The method Is based on the kinetics of reaction or quenching of the excited state and avoids many of the limitations of current techniques. Since the lifetimes of excited triplet states of molecules in fluid solution are generally microseconds or longer, a diffusiontontrolledquencher in less than millimolar concentrations can significantly influence the decay rate of the triplet population. I f this small concentration of quencher is comparable to the initial concentration of excited states, then the kinetics of the triplet-state decay are no longer pseudo first order. This kinetic behavior is exploited to provide a simple, noncomparative titration method for determining excited-triplet-state concentrations. The rate constant for quenching need not be known in advance, and any method of monitoring the quenching kinetics can be used. The technique is evaluated in the present study for determining the excited-triplet-state concentration of benzophenone by measuring the decay kinetics of phosphorescence quenched by biacetyi.
In order to accurately determine the quantum yields of photoinitiated reactions or molar properties of excited states, it is necessary to know the concentration of photoexcited molecules. Concentrations of excited states can be estimated from the absorption cross section, excited-state lifetime, and ground-state concentration of a species together with the excitation optical power, beam spot size, and pulse duration. These estimates can be inaccurate, however, due to spatial inhomogeneity of the excitation beam or depletion of the ground-state population, neither of which are easily characterized. Determining excited-triplet-state concentrations is particularly difficult, because yields of intersystem crossing are generally not known with accuracy. Techniques for obtaining concentrations, molar absorptivities, or quantum yields of formation of triplet excited states fall generally into two classes, comparative and noncompa-
rative methods. Comparative methods to estimate triplet-state molar absorptivities ( I ) are typically based on triplet-triplet energy transfer from a standard excited triplet donor; the decrease in the optical absorption by the donor (having a known T, T, absorptivity) is correlated with the increase in absorption by the acceptor to obtain the acceptor molar absorptivity (2). The method assumes that all of the acceptor population arises from energy transfer from the donor, which may be difficult to assure. Uncertainties also arise from the molar absorptivity of the donor or its photoproduct and from any yield of donor photoproduct that is not quenched by the acceptor ( I ) . By use of the values of T-T molar absorptivity thus obtained, comparative methods can also be used to determine intersystem crossing yields ( 3 ) . Here, the triplet concentration of the unknown is estimated from its T-T absorption and compared to that of a standard having known triplet yield and absorptivity. For the results to be valid, the triplet states must not absorb the exciting light ( 4 ) and depletion of the ground state must be negligible (5). To avoid some of the uncertainties and assumptions of comparative methods, noncomparative methods for determining triplet-state populations and molar absorptivities have been developed (6). These methods do not rely on intermolecular interactions such as energy transfer nor do they require knowledge of the molar absorptivity or triplet yield of a standard. For example, triplet-state concentrations and molar absorptivities can be determined by direct photolysis of an unknown while simultaneously measuring the triplet-state absorption and the loss of ground-state absorption. In order to obtain excited-state concentrations, the method requires either a spectral region where the ground state absorbs free of any triplet-state absorption or an isobestic point where the molar absorptivities of the ground and excited states are equal (7). Other noncomparative methods are based on the photolysis kinetics for generating excited triplet states (6, 8). Methods that do not require prior knowledge of the triplet yield or molar absorptivity are based either on the intensity dependence of saturation by excitation pulses that are longer than the singlet lifetime (9) or on the time dependence of triplet-state saturation using a chopped continuous excitation
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0003-2700/91/0363-0688$02.50/00 199 1 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
kisc
Dl I I
\
03
I
101
I kds
I
I
I
I
Do
I//
Figure 1. Kinetic scheme employed in the excked-state titration study. The ground state of a photoinitiator or donor, Do, is pumped at a rate of Iu, by an excitation pulse. Following rapM Internal conversion and intersystem crossing to the lowest triplet state, D,, these states can decay naturally at a rate of k, or can be quenched at a rate of k,[Q,], where [ Q,] is the ground-state quencher concentration. The quencher triplet state of photoproduct, Q3, decays at a rate, k,, that is negligible compared to k,.
source (IO). Both methods require modest excitation power so that the excitation rate does not exceed the excited singlet decay rate (6). This paper describes a new, noncomparative method to determine excited-triplet-state concentrations that avoids many of the limitations of the above techniques. The method is independent of the excitation kinetics of the photosensitizer but is instead based on the triplet-state kinetics, being dependent upon the quencher concentration. When the quencher concentration is varied over a range that includes the initial excited-state concentration, one has the basis for a "kinetic" titration. Since lifetimes of excited triplet states of molecules in fluid solution are generally microseconds or longer, a diffusion-controlled quencher at less than millimolar concentrations can significantly influence the decay rate of the triplet population. If this small concentration of quencher is comparable to the initial concentration of excited states, then the kinetics of the triplet-state decay are no longer pseudo first order. This kinetic behavior is exploited in the present work to provide a simple, noncomparative titration method for estimating excited-triplet-state concentrations. The rate constant for quenching need not be known in advance, and any method of monitoring the quenching kinetics (T-T absorption, phosphorescence, or photothermal measurements) could be used. The technique is evaluated in the present study for determining the excited-triplet-state concentration of benzophenone by measuring the decay kinetics of phosphorescence as it is quenched by biacetyl. THEORY The scheme for the kinetic titration method for determining photoexcited triplet-state concentrations of a photoinitiator is outlined in Figure 1. Upon pulsed optical excitation at a rate given by la,, the product of excitation intensity and the cross section for absorption, the excited singlet population of the donor produces triplet states, D3, with an intersystem crossing efficiency, &c = ki,,/(kds + kist). For the case of benzophenone used to test the method, ki, is very large, and triplet states are formed with unity quantum yield in approximately 20-30 ps (IO,11). The triplet-state population of the donor decays with a rate, kdt, that may include both nonradiative and weak radiative (phosphorescence) processes. Again for the particular case of benzophenone, the triplet lifetime in CC1, a t room temperature is approximately 100 ~.tswith a quantum yield of phosphorescence of approximately 2% (12, 13). The addition of a molecule, Qo,that reacts with or quenches the donor triplet population has the effect of increasing the rate of the decay of population, D3, according to
Q(t)/dt
=
+ kqQo(t)l&(t)
(1)
680
Under pseudo-first-order kinetic conditions most commonly used to study the rates of quenching of excited states, the concentration of quencher is much larger than the concentration of excited states so that one can assume Qo does not change over the lifetime of the donor decay. For efficient quenchers of long-lived triplet states produced at significant concentrations by pulsed laser excitation, this assumption can break down, causing the kinetics to depart from pseudofirst-order conditions. This kinetic behavior is the basis for determining the donor triplet concentration as developed in this work. The significant departure from first-order kinetics occurs whenever the initial concentration of triplet donors, D3(0), equals or exceeds the initial quencher concentration, Qo(0). The concentration of quencher required to initially double the decay rate of the donor is Qo(0)= kdt/kq. Thus, for triplet states of molecules having lifetimes in excess of 10 ~.ts(kdt < lo59-l) quenched at diffusion-controlled rates (kq = 1O'O M-l s-l), excited states produced in concentrations equal to or greater than M can be easily quantified by the departure of efficient quenching kinetics from pseudo-first-order conditions. To understand excited-state decay data produced under these conditions, the rate of change of the quencher concentration must also be added into the model: To simplify the kinetic analysis and reduce the number of unknown parameters that must be determined, quenchers are chosen that produce triplet states or photoproducts that are much longer lived than the excited triplet state of the donor. This allows the process of regenerating quenching ground states, Qo, by the decay of Q3 to be neglected, which simplifies eq 2:
(3) In the example chemical system used to test the method, this approximation is valid because the excited triplet states of the biacetyl quencher decay a t a rate that is an order of magnitude smaller than the unquenched decay of the benzophenone donor. Since the quenching process further decreases the lifetime of the excited donor, the regeneration of ground-state quencher molecules over the lifetime of the donor becomes even smaller as more quencher is added to the solution. Equations 1and 3 are a simple pair of coupled, differential equations that can be evaluated to determine the initial excited-state concentration, D3(0),by measuring the time decay of the donor triplet population, D3(t),for a series of known quencher concentrations, Qo(0). The only unknowns in the model are the intrinsic decay rate of the donor, k d t (which is obtained by fitting the unquenched, first-order decay kinetics of the donor), and the quenching rate constant, kq, and excited-state concentration, D3(0)(which are obtained from the dependence of D3(t)on quencher concentration). The latter process is analogous to an excited-state titration, where the mix of first- and second-order behavior in the quenching kinetics is modified by the relative concentration of quencher. The "equivalence point" of such a titration, where the concentrations of excited state and quencher are the same, produces decay kinetics that are purely second order when the intrinsic decay rate of the donor is small in comparison to the initial quenching rate. EXPERIMENTAL SECTION Sample excitation was provided by a Quanta-Ray pulsed dye laser pumped by the second harmonic output from a Q-switched, Quanta-Ray Nd:YAG laser with a filled-in beam operated at 20 Hz. The dye laser was operated with DCM,tuned to a wavelength
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
of 644 nm, and frequency doubled to 322 nm. The excitation pulses had 50-pJ energy at the sample where they were focused to a 180-pm radius spot, measured by translating a knife edge through the beam. Samples were irradiated at a 1-Hz repetition rate controlled by a shutter to allow ample time for excited-state relaxation and to allow diffusion and convection to clear the beam volume of any photoproducts produced before the next pulse. The triplet-state decay kinetics of benzophenone were determined by measuring phosphorescence collected with an F/1.1 lens, band-pass filtered at 430 nm (Ditrich Optical), and focused onto a fast photomultiplier wired for high current, pulsed signals (14). Signals were digitized by using a LeCroy 9400 oscilloscope and sent via an IEEE-488 interface to a personal computer for analysis. The benzophenone (Baker) samples were prepared at a concentration of 1.04 X lo-‘ M in carbon tetrachloride (Omnisolve). Biacetyl (Aldrich)concentrations of 4.56 X lo-’, 1.14 X lo4, 4.56 X lo”, and 9.12X lo4 M were used to investigate the quenching kinetics. These concentrations were chosen to cover the range below and above the expected excited-state concentration. At lower quencher concentrations, only 5-10 laser shots at a time were averaged because some photolysis of the quencher was observed as indicated by lengthening donor lifetimes with longer irradiation times. This greater rate of photolysis at lower quencher concentrations was likely due to the larger fraction of quencher molecules that were excited by energy transfer. For these samples, the sample cell was agitated at intervals between 5 laser shots. Twenty to 50 transients were averaged together, and 3 such averages were taken for each quencher concentration. The unquenched decay rate of triplet benzophenone was determined by a nonlinear least-squares fit of a single exponential decay model to the phosphorescence data weighted for the shot noise that dominates the signal variance (15). To fit the quenching results, the coupled differential rate equations, eqs 1and 3, were solved numerically by using a finite-difference method where dt is approximated by a small finite time interval, At = 5.6 ps. This assumption allows the equations to be written in the following form, where at time t ’ = t + At: hD3(t+At) = -k&3(t)& - kP3(t)Qo(t)At (4) D3(t+At)
D3(t)
+ AD,(t+At)
AQo( t +At) = -k&(
t)Qo(t )At
(5) (6)
This process was continued for each point in time t ” = t ’ + At, etc. The method is very easy to program and is amenable to spreadsheet manipulations on a personal computer. By use of this form for the solution to the coupled differential equations, the best tit model of the data was determined by using nonlinear least squares. Since the values for Qo(0)and kdt are known in advance of the analysis,the only fitting parameters were D3(0) and k,. These parameters were chosen to give the best fit to the phosphorescence data determined by minimizing the weighted sum of squares of the residuals or x2. All four phosphorescence decay curves at four different quencher concentrations were fit simultaneously to increase the precision of the results (16). Uncertainties in the parameters were evaluated by fitting of the x 2 error surface near its minimum to a parabolic model to estimate the curvature of the surface with respect to each parameter (15,17).
RESULTS AND DISCUSSION The procedure of varying the quencher concentration over a range that includes the excited-state concentration is, in effect, an excited-state titration, where the equivalence point occurs where the quenching kinetics shift from mixed second and first order to purely second order. This can be seen quantitatively by examination of eq 1. At low values of Qo, the right-hand side of the equation reverts to -kd$3(t), which is a pure first-order decay. As the quencher concentration is increased but is still less than the donor excited-state concentration, both terms in eq 1 need to be considered. The number of donor excited states is greater than the number of available quencher molecules. Therefore, the kinetics of
1.5~
I
-2ilA24
- 2 . 50
20 40 60 80 100 120 140
Time (microseconds)
Model time dependence of the quencher concentration, Q,(t), relative to the initial concentration of donor triplet states, D3(0). The initial concentrations of quencher relathre to the donor exclted state are (a)3.2, (b) 1.6, (c)0.40, and (d) 0.16; the concentrations are plotted on a logarithmic scale to cover the large range. Rates and relative concentrations for this example are drawn from the benzophenone/ biacetyl quenching results (see below). Figure 2.
excited-state decay arise from both quenched and residual unquenched donor molecules, because the ground-state quencher population, QO(t),decays along with the triplet donor population, as shown by eq 3. These kinetics are observed up to the point where the initial concentration of quencher molecules is equal to the initial population of donor excited states. After this equivalence point is reached, the kinetics become increasingly more dominated by pseudo-first-order quenching. The expected time dependence of a quencher concentration and its sensitivity to the relative concentration of donor excited states is illustrated in Figure 2. For a quencher that is 7 times smaller in concentration than the initial donor excited state, a large fraction (about 50%) of the quencher population is consumed during the lifetime of the donor population. At concentrations of quencher only 3 times higher than the excited donor, the relative change in quencher concentration is only 2070,and the concentration remains well above that of the donor even a t times greater than l/(kqQo). At sufficiently high quencher concentrations, therefore, time-dependent changes in the quencher concentration are smaller so that a more constant value of k,Qo yields decay kinetics of the donor that are closer to first order. The sensitivity of excited-state decay kinetics to small and changing quencher concentrations is exploited in this study to determine concentrations of excited states. As a particular example, the decay of the triplet-state population of benzophenone in carbon tetrachloride was measured by detecting phosphorescence emission. Biacetyl was chosen as a quencher since its triplet energy is about 12 kcal/mol below the triplet state of benzophenone, which leads to efficient triplet energy transfer (18). In addition, the triplet lifetime of biacetyl is an order of magnitude larger than that of benzophenone, which allows the return of triplet biacetyl to its ground state to be neglected over the time scale of the benzophenone decay. Quencher concentrations in the range 0.46-9.1 p M were chosen to bracket the expected excited-state concentration of benzophenone. The phosphorescence decay data from all four quencher concentrations, shown in Figure 3, were simultaneously fit by the kinetic model (eqs 4-7). The value of the unquenched triplet decay rate of benzophenone, kdt = 1.62 (f0.05) X lo4s-l, was obtained from a nonlinear least-squares fit of the phosphorescence decay in the absence of biacetyl. By use of t,his results, the two unknown parameters that best fit the quenching data are an excited-triplet-state concen-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 7, APRIL 1, 1991
: l
; 106
6
107
4
109 Quenching Rate, kq, (H-1 s-1) 108
801
4 1010
Flgure 4. Lowest excited-state concentrations that can be determined by kinetic titration versus the quenching rate constant, k,. The values of D3(0)that can be optimally quantified are pbtted for a family of triplet decay rate values, k, = (a) 1.0 X 108s-', (b) 1.0 X lo7 s-', (c) 1.0 X loss-', (d) 1.0 X lo5 s-', and (e) 1.0 X lo4 s-'. 0
60
120 180 240
Time (microseconds) Flgure 3. Benzophenone phosphorescence intensity (points) and fit to eqs 4-7 (lines),where (a) [O,] = 0.5 pM, (b) [O,]= 1.14 pM, (c)
[O,] = 4.56 pM, and (d) [O,] = 9.12 pM. Note that increasing phosphorescence intensity is downward. tration of benzophenone, D3(0)= 2.8 (f0.5) X lo4 M, and a bimolecular quenching constant, k , = 3.43 (i0.14) X lo9 M-l s-l, which is close to the limit of diffusion control. The fit of the data by the model is excellent, as shown in Figure 3. The validity of these results can be checked for the case of benzophenone, since ki, is very large, producing a quantum yield of triplet-state formation that is indistinguishable from unity (10, 11,18). Furthermore, the profile of the excitation laser beam a t the sample was mapped with photothermal beam deflection (19,20) and found to be nearly Gaussian with only slight asymmetry. Under these conditions, the theoretical excited-triplet-state population immediately following the laser pulse can be estimated by integrating the excitation rate over the pulse duration ( t = to):
D,(t=to) = Do(0)[l- exp(-Za,to)]
(8) For the laser pulse energy, beam spot size, and pulse duration, the average intensity of radiation is I = 2.0 (*0.3) X photons s-l cm-2. An advantage of a Gaussian profile is that the intensity falls off rapidly with radial distance such that, in the absence of saturation, 75% of the excited molecules are illuminated at an intensity that is within a factor of 2 of this average; more uniform concentration profiles can be generated by using an aperature to select the central intensity region of the Gaussian beam. The absorption cross section of benzophenone at the laser wavelength calculated (21) from the molar absorptivity if u1 = 3.85 X cm2,and the laser pulse duration is 4.0 ns. Using these data in eq 8 results in a predicted triplet concentration, D3(t=to)= 3.1 (f0.5) X lo* M, which agrees within the experimental error with the estimate obtained by the kinetic titration method. While the application of eq 8 is reasonable when the intersystem crossing yield, laser beam profile, and pulse energy are known, the kinetic titration method requires virtually no prior information about the excitation source profile or energy, the photophysics of the triplet donor, or the quenching reaction. These asects are unique advantages of the kinetic titration approach over other noncomparative methods. Since the evolution in the apparent order of the quenching kinetics depends only on the limited concentration of quencher molecules, the method does not require a precise estimate of k,. The quenching reaction should be fast in order to maximize sensitivity, but it need not be diffusion limited when
the excited-state concentration is sufficiently high and its unquenched lifetime is long. The range of k , and its impact on the concentrations of excited states that can be determined are illustrated in Figure 4. Here, the lowest triplet concentration that can be optimally determined (where k,Qo(0) = k,D,(O) = k d t ) is plotted versus k , for a family of kdt values. From this plot, it is clear tha longer lived excited states result in lower measurable concentrations, where for diffusion-controlled quenching ( k , = 1O'O M-' 8) of triplet states that live for loops submicromolar concentrations of excited states should be readily quantified. This predicted level of sensitivity is only a factor of 2 better than that achieved in the results of Figure 3. Some concern about the effects of mass diffusion on the concentrations of longer lived excited states could be raised. This is a minor concern in liquids since diffusion distances of small molecules on time scales of 100 ps are less than 1 pm, much smaller than the typical excitation beam spot size. For short-lived excited states with lifetimes as short as 10 ns, excited-state concentrations as low as 10 mM should be able to be determined if an efficient (diffusion-controlled) quencher can be used. This opens up the possibility of applying the method to the determination of excited-singlet-state concentrations. Precautions must be taken in applying the method under these conditions because the optical gain exhibited by such a large singlet population could result in stimulated emission dominating the excited-state decay (22-24). At the other end of the k , range, fairly inefficient photochemical reactions such as the generation of free radicals by H abstraction, which exhibit rates of the order of 106-107 M-' s-l (20), are shown in Figure 4 to be viable for kinetic titrations of excited triplet states that live for 10-100 ps and at initial concentrations in the 10-100 mM range. Higher concentrations of excited states can always be determined, in each of the examples discussed above, by increasing the concentration of quencher into the range where it is comparable to the donor concentration. The only challenge of this situation is that the kinetics of the excited-state decay become faster, dominated by a larger second-order rate term, kqQo; the kinetics may also be more complicated at higher excited-state concentrations due to triplet-triplet annihilation (18), for example. The excited-state kinetic titration technique is not restricted to photoluminescence detection of the donor decay kinetics. Any method of detecting the excited-state population decay would be applicable, including the time-resolved measurement of T, T, optical absorption and commonly employed flash photolysis experiments (18,25). Since the method only utilizes changes in the form of the triplet decay kinetics with quencher
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Anal. Chem. 1991, 63,692-699
concentration, the T-T molar absorptivity of the donor need not be known. One could similarly measure the kinetics of heat released by the excited triplet states on a time scale from nanoseconds to microseconds by using photothermal calorimetry methods (20, 26-28). More structurally selective spectroscopies, such as time-resolved ESR (29) and Raman spectroscopy (30),could also be used to discriminate between the decay kinetics of the excited donor and products of the quenching process.
LITERATURE CITED Bensasson, R.; Land, E. J. I n Photochemical and Photobiologlcal Reviews; Smith. K. C., Ed.; Plenum Press: New York. 1978: Vol. 3. Chapter 5. Land, E. J. Roc. R. Soc. London, Ser. A 1988, 305, 457-471. Richards, J. T.; West, 0.; Thomas, J. K. J. Phys. Chem. 1970, 74, 4137-4141. Speiser, S.;Van der Wed, R.; Kommandeur, J. Chem. Phys. 1973, 1 , 287-305. Bensasson, R.; Goldschmldt, C. R.; Land, E. J.; Truscott, T. G. Photochem. Photobbl. 1978, 26, 277. Carmichael, I.; Hug, G. L. Appl. Spectrosc. 1987, 4 1 , 1033-1038. Pavlopoulos, T. G. J. Opt. SOC.Am. 1973, 63, 180. Carmichael, I.; Hug, G. L. J. Phys. Chem. 1985, 69, 4036-4039. Lachlsh. U.; Shafferman, A.; Stein, G. J. Chem. Phys. 1976. 64, 4205. Rentzepis, P. M. Science 1970, 169, 239-247. Greene, B. I.; Hochstrasser, R. M.; Weisman, R. 6. J. Chem. Phys. 1979, 70, 1247-1259. Sattiel, J.; Curtis, H. C.; Metts, L.; Mlley, J. W.; Winterle, J.; Wrlghton, M. J. Am. Chem. SOC. 1970, 92, 410-411. Schuster, D. I.; Weil, T. M. Mol. Photochem. 1972, 4 , 447-452.
(14) Harris, J. M.; Lytle, F. E.; McCain, T. C. Anal. Chem. 1976, 48, 2095-2098. (15) Bevington, P. R. Data Reduction end Enor Ana/ys/s for the Physicel Sciences; McGraw-Hill: New York, 1969; Chapters 6, 11. (16) Phillips, G. R.; Harris, J. M; Eyring, E. M. Anal. Chem. 1982, 5 4 , -2053-2058 - - - - - - -. (17) Phillips, G. R.; Eyring, E. M. Anal. Chem. 1988, 60, 738-741. (18) Turro, N. J. Modern Molecular Photochemistry; Benjamln/Cummings: Menlow Park, CA, 1978; Chapters 5-6. (19) Gagne. M. C.; Galarneau. P.; Chin, S. L. Can. J . Phys. 1988, 64. 11 16-1 120. (20) Poston, P. E.; Harris, J. M. J. Am. Chem. SOC.1990, 112, 644-650. (21) Schifer, F. P. I n Dye Lasers, 2nd ed.;Schifer, F. P., Ed.; Springer. Verlag: Berlin, 1977; p 33. (22) Porter, G.; Topp, M. R. Roc. R . SOC. London, Ser. A 1970, 315, 163. (23) Lesslng, H. E.; Lippert, E.; Rapp, W. Chem. Phys. Lett. 1970, 7 , 247. (24) Busch, G. E.; Greve, K. S.; Olsen. G. L.; Jones, R. P.; Rentzepis, P. M. Chem. Phys. Lett. 1975, 33, 417. (25) Labhart, H.; Heinzelmann, W. I n fhotophysics of Oganic Mo/ecuies I ; Birks, J. B., Ed.; Wiley: New York, 1973; p 297. (26) Rossbroich, G.; Garcia, N. A.; Braslavsky, S. E. J. Photochem. 1985, 31, 37-47. (27) Terazima, M.; Azumi, T. Chem. Phys. Lett. 1987, 141, 237-240. (28) Isak, S. J.; Komorowski, S. J.; Merrow, C. N.; Poston, P. E.; Eyring, E. M. Appl. Spectrosc. 1989, 43. 419-422. (29) Yamauchi, S.; Hirota, N.; Takahara, S.; Sakuragi, H.; Tokumaru, K. J. Am. Chem. Soc.1985, 107. 5021. (30) Beck, S. M.; Brus, L. E. J. Chem. Phys. 1981, 75, 4934.
RECEIVED for review August 31, 1990. Accepted January 11, 1991. This work was supported in part by the National Science Foundation under Grants CHE85-06667 and CHE90-10319 and by Dow Chemical, U.S.A.
Development of Catalytic Photometric Flow Injection Methods for the Determination of Selenium Paul M. Shiundu and Adrian P. Wade* Laboratory for Automated Chemical Analysis, Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1 Y6
A sensitive spectrophotometric flow InJectlon method has been developed for the determination of selenium. I n thls, selenium catalyzes the oxldatlon of phenyihydrazlne by POtassium chlorate. A red complex is obtalned by coupllng the product of the redox step with 1,841hydroxynaphthalene3,6-dlsulfonlc acld (chromotroplc acld) under acldlc conditions. The reaction follows pseudo-firstorder klnetlcs, and for 5.0 ppm Se( IV) at 60 OC, a pseudo-first-order rate constant of 2.05 X s-' was obtalned. Chemlcal Interference studles showed that most metal Ions could be tolerated at the 100 ppm concentration level while others could be effectively masked with EDTA. Method development Included opthnizatbn of reaction temperature, pH, reagent concentrations, and flow condltlons. The performance of the optimized conventional flow InJectlonmanifold Is contrasted with that of stopped-flow and flow-reversal conflguratlons. The conventlonal manifold yielded a sample throughput rate of at least 60 h-', a detection limit of 0.52 ppm Se(IV), and a ilnear range of 0.0-50.0 ppm. A method in which the sample plug was stopped for 30 s Increased sensltivfty by a factor of 2.5 while malntaining a sample throughput rate of 45 h-'. At 45 h-', the flow-reversal method provlded a signlflcant further Improvement in sensitivity and a detection limit of 0.15 ppm. The reverse trend (conventional > stopped flow > flow reversal) was observed for the linear dynamic range.
* To whom correspondence should be addressed.
INTRODUCTION In recent years, there has been a growing interest in understanding the physiological role of trace selenium in domesticated animals and man. Selenium is essential to life; its deficiency is toxic to animals ( I , 2) and has been correlated with diseases such as cardiomyopy in man (I, 3). Excessively high levels have been associated with cancer (4), and a small difference is thought to separate the toxic and essential levels (1-4). The essential and beneficial roles of selenium in animal and human physiology are documented; selenium is a component of the enzyme glutathione peroxidase (5), which scavenges the traces of peroxides generated during cellular metabolism before such peroxides can induce oxidative changes deleterious to health. The ability of selenium to reduce the toxicity of heavy metals has been the subject of much study in recent years (6, 7). There are suggestions that selenide combines with mercury(I1) and cadmium(I1) to give metabolically inert metal selenides. Selenium has also been known to prevent several types of chemically induced cancer in animals (8, 9). Shamberger et al. (10, 11) noted that human cancer death rates are lower in countries where more selenium occurs in the environment and that human mortality from heart diseases is also lower in the high-selenium areas. The complexity of the above studies calls for the development of precise and accurate methods for selenium determination in biological, environmental, and/or food product samples.
Instrumental Methods for the Determination of Selenium. Analytical methods already used include gas chro-
0003-2700/91/0363-0692$02.50/00 1991 American Chemical Society
