Anal. Chem. 1994,66,919-923
Usefulness of the Stopped-Flow Mixing Technique for Micelle-Stabilized Room-Temperature Liquid Phosphorimetry S. Panadero, A. Gbmez-Hens, and D. P6rez-Bendlto' Department of Analytical Chemistty, Faculty of Sciences, University of Cbrdoba, E- 14004 Cbrdoba, Spain
The stopped-flow mixing technique was applied for the first time to micelle-stabilized room-temperaturephosphorimetry by measuring the fast appearanceof the phosphorescentsignal yielded by carbaryl in the presence of sodium dodecyl sulfate and thallous ion. The slope and amplitudeof the kinetic curves obtained are directly proportional to the analyte concentration, which allows one to develop very simple, fast, automaticmethods for the phosphorimetric determination of dissolved carbaryl without the need for solid substrates. Kinetic and equilibrium data can be obtained within only 2-3 s with a greater precision than that provided by solid-surface room-temperature phosphorimetry. The calibration graphs obtained for the proposed kinetic and equilibrium stopped-flow method were linear over the range 0.030-2.0 and 0.050-3.0 pg mL-l carbaryl, respectively, and the detection limits achieved were 0.010 and 0.014 pg mL-', respectively, Le., lower than those provided by solid substrates. Kinetic methodology is widely used in phosphorimetry to determine the lifetime of the excited triplet state, a characteristic property of molecules and their environment. The phosphorescence lifetime is a major parameter since compounds having very similar spectral features may have greatly different triplet lifetimes. However, kinetic methodology has not been used for analytical purposes by measuring the rate of formation of a phosphorescent system. Similar to other analytical techniques, this can be accomplished by obtaining a kinetic curve reflecting the variation of phosphorescence with time and using its slope as an analytical parameter. Such measurements cannot be made when the phosphorescent technique used involves immobilization of the phosphorescent analyte in a low-temperature glass or adsorption into an inert substrate such as filter paper. However, micelle-stabilized room-temperature phosphorimetry (MSRTP) in aqueous solution,' which involves using a heavy atom, enables application of kinetic methodology. This is especially useful when the phosphorescent signal is unstable or takes a long time to stabilize, which hinders application of a phosphorescence method to routine analyses. The rate of formation of a phosphorescent system is usually very fast and kinetic data cannot be obtained by using batch technique. Instead, the stopped-flow mixing technique is the most commonly used whenever fast systems are involved. Its usefulness for studying the kinetics and mechanism of fast reactions and developing kinetic determination methods has been widely employed.* It can equally be used to develop methods by measuring the amp1itudeof the kinetic (1) ClincLovc, L. J.;Skrilcc, M.;Habarta, J.G.A M / . Chem. 1980,52,754-759. (2) Gomcz-Hens, A.; P€rez-Bendito, D. Anal. Chim. Acra 1991,242, 147-177.
0003-2700/94/0366-0919$04.50/0 0 1994 Amerlcan Chemlcal Soclety
curves obtained. This approach is particularly attractive for routine determinations where automatic instrumentation and a high sample throughput are required. This paper reports the first analytical application of the stopped-flow mixing technique in MSRTP and shows its advantages over conventionalMSRTP and solid-surfaceroomtemperature phosphorimetry (SSRTP). The dynamic signal provided by the pesticide carbaryl (1-naphthyl N-methylcarbamate) in the presence of sodium dodecyl sulfate (SDS) and thallous ion was used for this purpose. The system had not thus far been studied in solution, although the phosphorescence of carbaryl has been widely investigated by SSRTP using filter ~ a p e r and ~ - ~determination methods have been r e p ~ r t e d . ~In, ~fact, this phosphorescent system was selected to demonstrate a specific instance of the assets of the stoppedflow mixing technique in RTP. The use of solid supports in RTP has two majors limitations, namely, (1) the presence of background signal and (2) critical sample preparation and measurement requirements. All suitable supports for RTP have background phosphorescence,*~~ which limits application of SSRTP to trace analysis. Several attempts have been made to decrease such background signals. Thus, Campiglia and De Lima7 obtained the best results by using water extraction followed by ultraviolet exposure, which provided a detection limit of -one-tenth for carbaryl. The use of sodium iodide previously spotted on paper as the heavy atom source allowed the detection limit to be lowered even further. Although the analyte phosphorescence enhancement was smaller with iodide ion than it was with thallous ion, these authors found the background signal to be also enhanced by thallous ions in such a way that the detection limit was affected, so they chose iodide ion as the heavy atom. In spite of these results, the substrate pretreatment is timeconsuming and the precision quite poor (1 1-1 3% as relative standard deviation). In addition, the method7 has not been applied to the analysis of real samples. Regarding the second above-mentioned limitation, there are a number of critical variables in sample preparation including the drying temperature, which affect the results ~ b t a i n e d .Su ~ et al.5 developed a sampling system for SSRTP based on ion-exchange filter paper, which was applied to the determination of various pesticides including carbaryl. A (3) Kirkbright, G. F.; Shaw, E. S.Can. J . Specrrosc. 1983, 28, 100-103. (4) Vanelk J. J.; Schulman, E. N.AMI. f2hem. 1% 56, 1030-1033. ( 5 ) Su,S.Y.;Asafu-Adjayc, E. B.; O a k , S.Amlysr 1984, 109, 1019-1023. ( 6 ) De Lima, C. G.; Andino, M.M.;Wincfordncr, J. D. Anal. Chem. 1986,58, 2867-2869. (7) Campi@, A. D.;De Lima, C. G . A M / . Chem. 1987, 59, 2822-2827. (8) Batch, R. P.;Wincfordncr. J. D. Talanta 1982, 29, 713-717. (9) Scharf, G.; Smith, B. W.; Wincfordncr, J. D. AMI. Chem. 1985, 57, 12301237.
Analytcal Chemistry, Vol. 66,No. 6,March 15, 1994 QiQ
relative standard deviation of 12-2 1% ' was obtained, which was ascribed to differences in the position of the samples. Another limitation ofthis technique is the long time the system takes to reach equilibrium, Thus, the determination of carbaryl5 requires -10 min in order to obtain stable measurements. These drawbacks hinder application of SSRTP to routine analyses and their automation. However, the usefulness of the stopped-flow mixing technique for fast acquisition of analytical data in MSRTP using kinetic and equilibrium measurements is clearly shown in this work, where it was readily applied to routine determinations.
EXPERIMENTAL SECTION Apparatus. A Perkin-Elmer LS-50 luminescence spectrometer in the phosphorescence mode was used. The delay and gating times used were 0.03 and 5 ms, respectively. The instrument was fitted with a stopped-flow module*Osupplied by Quimi-Sur Instrumentation and controlled by a HewlettPackard Vectra computer. Reaction rate data were obtained by using the Kinetic Obey application program. The observation cell of the stopped-flow module had a path length of 1.O cm, and the excitation and emission slits were adjusted to provide a 15-nm band pass. The temperature of the solutions in the stopped-flow module and the cell compartment was kept constant at 20 OC by circulating water from a thermostated tank. Reagents. All chemicals used were of analytical-reagent grade. A 100 pg mL-I stock solution of carbaryl (Riedel) was prepared in 0.25 M aqueous sodium dodecyl sulfate (SDS); 0.25 M thallium(1) nitrate (Merck) and 0.5 M sodium sulfite (Fluka) solutions were also made, and an ammonium acetate buffer solution (0.1 M, pH 7.5) was employed. Procedures. Kinetic Method. A solution containing 0.8 mL of 0.25 M thallium(1) nitrate, 0.5 mL of ammonium acetate buffer, and 0.6 mL of 0.5 M sodium sulfite in a final aqueous volume of 10 mL was used to fill one of the two IO-mL reservoir syringes of the stopped-flow module. The other syringe was filled with 10 mL of a premixed micellar solution consisting of 3 mL of 0.25 M SDS, 0.5 mL of buffer, 0.6 mL of 0.5 M sodium sulfite, and a volume of carbaryl standard solution at a final concentration between 0.03 and 2.0 p g mL-l. After the two 2-mL drive syringes had been filled, 0.15 mL from each solution was mixed in the mixing chamber in each run. The variation of the phosphorescence intensity (Aex 284, A,, 488 nm) was monitored for -4 s, and the data obtained were processed by linear regression by the microcomputer, running software for application of the initial rate method (Kinetic Obey). The slopes of the phosphorescence-time curves were determined within 1.O s, and each sample was assayed in triplicate. The blank signal was found to be negligible. All measurements were made at 20 "C. Equilibrium Method. One of the two reservoir syringes was filled with 10 mL of an aqueous solution containing 0.8 mL of 0.25 M thallium(1) nitrate, 0.5 mL of ammonium acetate buffer, and 0.2 mL of 0.5 M sodium sulfite. The other syringe was filled with 10 mL of a premixed 10-mL solution consisting of 3 mL of 0.25 M SDS, 0.5 mL of buffer, 0.2 mL of 0.5 M sodium sulfite, and a volume of standard
-
solution of carbaryl at a final concentration between 0.05 and 3.0 pg mL-I. After the driver syringes were filled, equal volumes (0.15 mL) of both solutions were mixed in the mixing chamber in each run. The analytical parameter used was the amplitude of the kinetic curve, which was measured (Lx284, Lm488 nm) 3 s after the reagents were mixed. Each sample was assayed in triplicate. Measurements were made at 20 OC,and the blank signal was found to be negligible. Determination of Carbaryl in Irrigation Water. No sample pretreatment was required for these analyses. Water samples were spiked with appropriate amounts of carbaryl, and 3 mL of each sample was treated as described above.
RESULTS AND DISCUSSION Obtaining a phosphorescent signal from a dissolved substance entails the following: (1) removing dissolved oxygen from the solution, (2) avoiding collisional quenching by (usually) exploiting the protective screening effect of micelles, and (3) using heavy atoms to increase the phosphorescence yield by effectively promoting the SI TI intersystem crossing via spin-orbit coupling. Bearing these requirements in mind, we studied the phosphorescence of dissolved carbaryl in order to check the applicability of the stopped-flow mixing technique to a phosphorescent system. One of the most troublesome aspects of MSRTP is oxygen removal from the micellar solution. The use of an inert gas for this purpose gives rise to the formation of foam and is quite time-consuming. In addition, it is poorly effective with flowing systems such as those of the stopped-flow mixing technique because oxygen may penetrate through the walls of Teflon tubes. On the other hand, the chemical deoxygenation method using sulfite ion reportedly provides satisfactory results." A previous study of the phosphorescence of carbaryl adsorbed on filter paper6 in the presence of different surfactants and heavy atoms showed the maximum phosphorescent signal is obtained when the surfactant salt thallous dodecyl sulfate (TlDS) was used. The way in which the reactants are mixed in solution has a marked effect on the kinetics of the phosphorescence process. Thus, a solution prepared batchwise by sequentially mixing the carbaryl solution in SDS, thallous nitrate and sodium sulfite provided a phosphorescence signal at Lx284 nm and Ae, 488 nm that took 15 min to stabilize. On the other hand, by using the stopped-flow mixing technique and placing carbaryl dissolved in SDS in one syringe, thallous ion in the other, and sodium sulfite in both, a kinetic curve was obtained for which the equilibrium signal was achieved in only 2-3 s. Manual mixing of these two solutions allows the system to stabilize within -2 min. Even though this time is shorter than that achieved by sequential mixing, it is still much longer than that afforded by the stopped-flow technique. This can be ascribed to the fast, thorough mixing of the streams from the syringes in the flow cell resulting from the pressure exerted by the instrumental system, which favors interaction of carbaryl with the heavy atoms. This dynamic effect does not affect the phosphorescent signal since measurements start to be made after the solution in the flow cell has reached steady state. As shown in Figure 1, the slope and amplitude of the
-
-
~
(10) Loriguillo, A.; Silva, M.; PCrez-Bendito, D. Awl. Chim. Acfa 1987, 199, 29-40.
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Analytical Chemistry, Vol. 66,No. 6, March 15, 1994
(11) Diaz Garcia, M.
E.;Sanz-Medcl, A. A w l .
Chem. 1986, 58, 1436-1440.
IP
I
2
L
6
8
Time (s) Figure 1. Kinetic curves obtained at different carbaryl concentrations (pg mL-I): (1) 0.1, (2) 0.5, (3) 1.0, and (4) 2.0. [SDS] = 7.5 X lo-' M. M; [TI(I)] = 2 X M; [Na2S03]= 3 X
kinetic curves obtained at different carbaryl concentrations were directly proportional to such concentrations. Cetyltrimethylammonium bromide (CTAB) was used instead of SDS,but no phosphorescence signal was obtained at the characteristic wavelengths of carbaryl in the presence of M CTAB. This is consistent with the results obtained by SSRTP,6 where the bromine atom in the CTAB molecule was seemingly observed not to exert an efficient heavy-atom effect on the carbaryl phosphorescence. No phosphorescence was obtained from the solution when thallous ion was replaced with iodide ion, which also enhances the phosphorescence of carbaryl in SSRTP,' because iodide ions are repelled by the negative chargeof SDSmicelles. Accordingly, the heavy atom must be on the micellar surface (Stern layer) in order to promote intersystem crossing in the carbaryl molecule and the subsequent phosphorescence emission. When carbaryl dissolved in SDS,held in one of the syringes of the stoppedflow module, and thallous solution, held in the other, is combined in the mixing chamber (which also acted as the observation cell), the slope of the kinetic curve obtained should correspond to the rate of interaction of thallous ions with carbaryl inside the micelles, which would give rise to efficient spin-orbit coupling. Although the heavy-atom effect in phosphorescent molecules has been extensively studied by measuring phosphorescence lifetimes, the stopped-flow mixing technique would offer additional information in fundamental studies of this luminescence phenomenon as this technique has been used to study the reaction kinetics and mechanism of a host of other chemical systems. Effect of Variables. Experimental variables were optimized by the univariate method in order to obtain the maximum possible slope and amplitude in the kinetic curves. All concentrations given are initial concentrations in the mixing chamber, and each reported result is the average of three measurements. Increasing temperatures in the range 20-50 O C resulted in a decreased slope and amplitude of the kinetic curve. This is quite logical since an increase in the temperature must result in increased vibronic quenching of the triplet state via radiationless decay to the ground state. A temperature of 20 O C was thus chosen. Low concentrations of organic solvents
2 L 6 8 1 [Naz SOJ] x 10' / M
0
Effect of pH (A) and the sodium sulfite concentratlon (E)on and amplitude (- -) of the kinetic curves provided by the phosphorescent system: [carbaryl] = 1 pg mL-? [SDS] = 5 X M; [TYI)] = 1.25 X M; [Na2S03]= 5 X lo-' M. In (E), a 5 X 10" M ammonium acetate buffer, pH 7.5, was used. Flgwe 2.
the slope (-)
-
such as ethanol and dimethylformamide had an adverse effect on the phosphorescence signal. Thus, the presence of 0.1% ,ethanol decreased the phosphorescence by 50%, while that of 0.2% caused the signal to desappear altogether. Organic solvents are known to alter and eventually destroy SDS micelles. In addition, they may slightly increase the solubility of carbaryl in the bulk solution, thereby decreasing its concentration on the SDS micelles and diminishing the signal. The effect of pH on the system was studied by adjusting the pH value of each solution in the syringes, which allowed the same value to be obtained in the mixing chamber as checked in the wastes. Thevariation of both measurement parameters with this variable is shown in Figure 2A; as can be seen, the optimum pH range was 7.5-8.0 for the slope and 7.8-8.3 for the signal amplitude. Three buffer solutions (Tris, sodium dihydrogen phosphate, and ammonium acetate) were assayed. No phosphorescence signal was obtained in the presence of Tris buffer, and the signal obtained with the phosphate buffer was -8 times lower than that obtained with ammonium acetate. The slope and amplitude of the kinetic curve were constant in the ammonium acetate buffer concentration range 4 X 10-3-10-2M. Both parameters decreasedslightly at higher buffer concentrations. A 5 X M buffer concentration was chosen. The effect on the system of the sodium sulfite concentration, which was used to scavenge dissolved oxygenll in both syringe solutions, is shown in Figure 2B. The decrease in both parameters at sodium sulfite concentrations above the optimum upper limit can be ascribed to two facts: (1) the high concentration of sodium ions, which can displace thallous ions from the micellar surface,l1J2 and (2) the inner filter effect due to the formation of the thallous sulfite complex,13 (12) Kim, H.; Crouch, S.R.;Zabik, M.J.; Sclim, S.A. And. Chem. 1990,62,
2365-2369.
Analytical Chemistry, Vol. 66,No. 6,March 15, 1004
921
Table 1. Featurea of the Proposed Methods
dynamic method kinetic
[TI NO31 x 102/M 4
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- 60 -LO
- 20
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,
,
I
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5
,
(
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[SDS] x 10' IM Flgure 9. Effectof the thallous ion (A) and surfactant (B) concentratlon on the slope (-) and amplitude (- -) of the kinetic curves proby the phosphorescent system. Experimental conditions as in Procedures, except 5 X lo-* M SDS In (A): [carbaryl] = 1 pg d-'.
-
which absorbs at the excitation wavelength of carbaryl. The curve reflecting the variation of the kinetic parameter is shifted with respect to the curve obtained for the equilibrium parameter, which can be ascribed to the fact that measurements were obtained at the start of thereaction and the negative effect of sodium ions became apparent when the concentration exceeded that of equilibrium. As shown in Figure 3A, the variation of the thallium(1) concentration had a similar effect on both the slope and amplitude of the kinetic curve. A 2 X M concentration was chosen. The optimum range for amplitude measurements was 2 X 10-3-10-2 M, whereas the slopes of the kinetic curves were constant and independent of this variable over the range 2 X 10-2-4 X M. The effect of the SDS content on the system was studied by placing this surfactant in the syringe containing carbaryl. Figure 3B shows the variation of both parameters with this variable; as can be seen, no phosphorescence signal was obtained in the absence of SDS,thereby showing the significance of using an organized medium for the phosphorescent system. The slope and amplitude of the kinetic curve remained constant above 5 X and 7.5 X M SDS,respectively. These concentrations are higher than the critical micelle concentration for SDS, which is reportedI4 to be 8.1 X M. Analytical Figures of Merit. The phosphorescence-time curves obtained for different amounts of carbaryl at excitation and emission wavelengths of 284 and 488 nm, respectively, were processed by using a kinetic (initial rate) and an equilibrium (signal amplitude) method. Some of the features of these methods are summarized in Table 1. Both methods feature a wide linear range, and standard errors and correlation
11.6 & 0.2 0.64* 0.27 0.437 0.999 (8-1 pg-1 mL) 33.9 & 0.3 2.10 0.52 1.017 0.999 (PIT' mL)
Standard error of the estimate. Correlation coefficient, n = 6.
Table 2. Effect of Varlow Pestkidas on the DetMmlnatkn of 0.5 pg mL-I Carbaryl
species added Propoxur,carbofuran aldrin, dimethoate malathion dicrotophos warfarin parathion malachite green bromadiolone diphacinone diquat
tolerated concn ratid (epecieslanalyte) A B 1006
lOO*
100 50 10 5 1 1 1
10 1 1 5 1
A, kinetic method; B, equilibrium method. b Maximum ratio assayed.
coefficients indicative of very good calibration linearity. The detection limits, obtained according to IUPAC,I5 were 0.0 10 pg mL-l for the kinetic method and 0.014 pg mL-l for the equilibrium method, i.e., - 5 times lower than those achieved by using SSRTP'. The precision was determined at two carbaryl concentrations, 0.5 and 1.5 pg mL-'; the relative standard deviation (n = 11) was 2.9 and 2.5%, respectively, for the kinetic method, and 2.9 and 2.1% respectively, for the equilibrium method; hence the precision was very similar in both cases and better than for the SSRTPmethod for ~ a r b a r y l . ~ The effect of various pesticides on the determination of carbaryl was investigated in order to study the selectivity of both methods. Table 2 summarizes the results obtained; as can be seen, the kinetic method is somewhat more tolerant to the pesticides assayed than is the equilibrium method. Applications. In order to check the usefulness of the proposed stopped-flow methods, three irrigation water samples were spiked with two different amounts of carbaryl to obtain an analyte concentration of 1 and 3 pg mL-l. Each sample (3 mL) was analyzed as described above. Table 3 lists the analytical recoveries obtained, which ranged from 88.0 to 108.0% (mean, 97.7%). CONCLUSIONS Use of a stopped-flow mixing technique in combination with MSRTP provides an alternative to SSRTP. We chose to use the phosphorescenceof carbaryl in order to demonstrate the potential of the proposed approach by contrast with a well-documented SSRTP determination, the figures of merit of which are no quite satisfactory. Compared to conventional MSRTP, where a phosphorescent system takes a relatively long time to reach equilibrium, the special features of the stopped-flow mixing technique allow the fast acquisition of
(13) Nugara, N. E.; King, A. D. Anal. Chem.1989,61,1431-1435. (14) ClineLove,L.J.;Dorscy,J.G.;Habarta,J.G.AMl.Chem.1984,56,1132A(15) Long, G.L.; Winefordner, J. D. AM/.Chem.1983, 55, 712A-724A. 1148A.
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AneWG9l Chemistry, Vd. 66, No. 6, March 15, 1994
+
intercept
0.03-2.0
equilibrium 0.05-3.0 a
SEEa
(piZf-1)
Tabla S. Romvwy of Carbaryl in Ilrlgrtlon W a t r
Mmple
added MmL-9
1
1.0 3.0 1.0 3.0 1.0 3.0
2 3 a
foundaa bg mL-9 A B 0.98 2.98 1.06 3.24 1.02 3.16
0.98 2.90 0.88 2.80 0.88 2.70
% re&
A
B
90.0 99.3 106.0 108.0 102.0 106.3
98.0 98.7 88.0 93.3 88.0 90.0
Mean of three detad"inatin8. b A, kineticmethod, B,equilibrium
method.
kinetic and quilibrium data and provide a very simple means of accomplishing automation in routine analyses involving phosphorescent species. In addition, the proposed approach is free from the background signal encountered in SSRTP, which affects the detection limits achieved, and allows one to
improve the precision of the analytical results obtained. Moreover, the use of the stopped-flow mixing technique in MSRTP can be a simple means for obtaining essential data on phosphorescent systems such as thoae rquired for study of the interaction of heavy atoms with phosphorescentspecies. In any case, we are currently studying other phosphorescent systems in order to determine the actual potential of the proposed approach as an alternative to RTP techniques.
ACKNOWLEWMENT This work was supported by a grant from the Comisidn Interministerialde Ciencia y TecnologSa (Grant PB9 1-08440). Recehmd for review September 16, 1003. Accepted December 16,
[email protected] Ab8trrct publiahed in Advance ACS Abstracts, February 1, 1994.
AM-1
W b Y , V d 66, Ilk. 6. klbrch 75, 1994
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