Anal. Chem. 1990, 62, 1528-1531
1528
ISE is present. The ISE will generally react to added ligand, although the response may not be Nernstian. Simply because no metal has been added does not imply "the absence of metal ion". C. Difficulties Obtaining FQ Parameters. Numerous reviews have been published on the various models used to represent metal binding to humic materials, and we will not add to them here (11,13-20). Determination of binding parameters like K and CL is useful only insofar as they can accurately predict metal speciation; it is not a goal in itself. Binding parameters that accurately predict the change in fulvic acid fluorescence as a function of added metal are useful only if they also accurately describe free metal and bound metal concentrations. The data analysis method described in ref 3 using FQ data alone can produce binding constants that fit the FQ data, but do not necessarily predict metal speciation well. An empirical model based on metal concentration measurements may not have real chemical meaning, but if it accurately predicts free and bound metal speciation in solution, then it serves an important purpose (11). Summary. In their discussion of our paper (1, 2 ) , the authors ignore the motivation for this work: how can we predict metal-organic binding in natural waters? Futhermore, they ignore the central uncertainty of the FQ technique: how can we know that the quenching in a complex mixture of fluorophores is proportional to metal bound? These questions are much more important than further refinements of nonlinear regression procedures and improvements in our ability to curve-fit FQ data. Further research with FQ might address these questions: What information can FQ contribute to improve our ability to predict free metal activities in natural waters? In what cases is the proportionality of quenching Q to [CuL] (constant A ) a valid assumption? Can FQ be used to distinguish betweeen different binding sites, different binding stoichiometries? We hope this approach will permit the advantageous use of fluorescence quenching to study the complex ligand mixture in metal-fulvic binding.
LITERATURE CITED (1) Ryan, D. K.; Ventry, L. S. preceding comment. (2) Cabaniss, S. E.; Shuman. M. S. Anal. Chem. 1988, 6 0 , 2418.
(3) Ryan, D. K.; Weber, J. H. Anal. Chem. 1982, 54. 986. (4) Fish, W.: Morel, F. M. M. Can. J . Chem. 1985, 63, 1185. (5) Cabaniss, S. E.; Shuman, M. S. Anal. Chem. 1988. 58, 398. (6) Ryan, D. K.: Weber, J. H. Envlron. Scl. Techno/. 1982, 16, 866. (7) Ryan, D. K.; Thompson, C. P.; Weber, J. H. Can. J . Chem. 1983. 61,
1505. (8) Berger, P.; Ewald, M.; Liu, D.: Weber, J. H. Mar. Chem. 1984, 14, 289. (9) Newell, A. D. M.S. Thesis, University of North Carolina at Chapel Hill, 1983; 87 pp. (10) Saar, R. A.; Weber, J. H. Anal. Chem. 1980, 52, 2095. (11) Cabaniss. S. E.; Shuman, M. S. Geochim. Cosmochim. Acta 1988, 52, 185. (12) Wilkinson, L. SYSAT: The System for Statistics; SYSTAT, Inc.: Evanston, IL, 1986; 817 pp. (13) Sunda, W. G.; Hanson, P. J. I n Chemical Modeling in Aquatic Systems; ACS Symposium Series 93; Jenne, E. A,, Ed.: American Chemical Society: Washington, DC, 1979; pp 147-180. (14) Pungor, E.; Toth, K. Precipitate-based Ion-Selective Electrodes: In Ion-Selective Electrodes ln Analytical Chemisfty: Freiser, H., Ed.; Plenum Press: New York, 1979; pp 143-210. (15) Cabaniss, S. E.; Shuman, M. S.; Collins, 8. J. In Complexation ot Trace Metals in Natural Waters; Kremer, C. J. M., Duinker, J. C., Eds.; Martinus Nijhoff: The Hague, 1984; pp 165-179. (16) Buffle, J. In Metal Ions in 6iolcgical Systems; Sigel, H., Ed.; Marcel Dekker: New York, 1984; pp 165-221. (17) Perdue, E. M.; Lytle, C. R. In Aquatlc and Terrestrial Humic Materials: Christman, R. F., Gjessing, E. T., Eds.: Ann Arbor Science: Ann Arbor, M I 1983; pp 295-314. (18) Turner, D. R.; Varney, M. S.; Whitfiild, M.; Mantoura, R. F. C.: Riley, J. P. Geochim. Cosmochim. Acta 1986, 50, 289. (19) Dzomback. D. A.: Fish, W.; Morel, F. M. M. Environ. Sci. Technol. 1988, 20,669. (20) Fish, W.; Dzomback, D. A.; Morel, F. M. M. Environ. Sci. Technol. 1988, 20, 676. (21) Cabaniss, S. E. Environ. Sci. Technol. 1987, 21, 209.
' To whom correspondence should be addressed.
Stephen E. Cabaniss* Department of Chemistry Kent State University Kent, Ohio 44242
Mark S. Shuman Department of Environmental Science and Engineering University of North Carolina Chapel Hill, North Carolina 27599-7400 RECEIVED for review February 8,1990. Accepted March 12, 1990.
Fiber-optic Potassium Ion Sensor Using Alkyl-Acridine Orange in Plasticized Poly(viny1 chloride) Membrane Sir: Optical measurement of chemical species through an optical fiber is one of the most advanced sensing techniques. Various fiber-optic sensors have been developed, especially for measurements of pH (1-31, oxygen ( 4 , 5 ) ,and metal ions (6,7). Some suitable indicators specific to the chemical species of interest have been used as transducing materials in these studies. However, these is difficulty in construction of a fiber-optic sensor for alkali-metal ions, since few chromogenic dyes interact with these ions. The first sensor sensitive to an alkali-metal ion was reported by Zhujun et al. (8). The sensing mechanism is based on fluorescence quenching by a copper ion; a weak quencher, Na+, competitively binds to a fluorescent dye with a strong quencher, Cu2+. Wolfbeis and his co-workers have developed an optical K+ sensor, in which the membrane potential between an aqueous sample solution and a Langmuir-Blodgett
layer is measured by using a potential-sensitive fluorescent dye (9, 10). The sensor has a logarithmic response to the analyte concentration and has a wide dynamic range. Alder et al. have constructed a different type of K+ sensor using a chromo-ionophore consisting of a crown ether and a dye molecule (11). For these sensors, the preparation of the optically sensitive membrane is rather difficult, since the structure of the membrane is highly sophisticated and should be carefully controlled. Furthermore, it is necessary to synthesize a new chromogenic dye sensitive to a specific alkalimetal ion. For determination of alkali-metal ions, an ion-selective electrode with a plasticized poly(viny1 chloride) (PVC) membrane is currently used (12). This plasticized PVC membrane consists of poly(viny1 chloride), a neutral ionophore, a hydrophobic ion exchanger, and a plasticizer. Optical sensors
0003-2700/90/0362-1528$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
n
Beam Splitter
AtLaser
1520
50t
Filter1
Reference
Optical Fiber
Figure 1. Schematic diagram of fiber-optic potassium ion sensor.
using plasticized PVC membranes have been developed elsewhere (13, 14). However, an indicator that directly interacts with a specific cation must be synthesized to construct a fiber-optic sensor. In this study, we report a general approach to constructing a fiber-optic sensor using the plasticized PVC membrane. The fluorescence intensity of the hydrophobic probe, dodecyl acridine orange (dodecyl-AO+),is known to be dependent on the polarity around the probe molecule. Dodecyl-AO+ is incorporated in the membrane containing valinomycin, and it moves to an aqueous phase by ion-exchange with the potassium ion in a sample solution. If so, the fluorescence intensity of dodecyl-AO' decreases with increase of the polarity of the microenvironment around the probe molecule. Dodecyl-AO+,used as a chromogenic indicator, does not directly interact with the potassium ion. Thus this technique can be generally applied to fiber-optic sensors responding to other cations by changing the plasticized PVC membrane with a different neutral ionophore.
EXPERIMENTAL SECTION Apparatus. The apparatus of the fiber-optic sensor system is shown in Figure 1. The major system is identical with that in the previous report (3),and the difference is briefly described here. The output power of an argon ion laser is adjusted to 10 pW, which provides a sufficient fluorescence intensity from the sensor. The laser beam is modulated to square waves at 85 Hz by a mechanical chopper (NF Circuit Design Block, CH-353). The synchronized signal from the chopper is fed to a lock-in amplifier (NF Circuit Design Block, 5600) as a reference. The modulated laser beam is focused by lens 1 (focal length, 70 mm) onto the front surface of a quartz optical fiber (Showa Electric Wire and Cable, NI/SF-400/500-UV). The core and cladding diameters are 400 and 500 pm, respectively. Fluorescence from the sensing head is collected by the same optical fiber. A fluorometer (Hitachi, MPF-4) is used for recording the fluorescence spectra of dodecyl-A0+. Reagents. A hydrophobic probe of 3,6-bis(dimethylamino)-10-dodecylacridiniumbromide (dodecyl-AO+Br-)and a cation exchanger of sodium tetrakis[3,5-bis(trifluoromethyl)phenyllborate (Na+TFPB-)were purchased from Dojindo Laboratories. A neutral ionophore of valinomycin was obtained from Aldrich and a plasticizer of dibutyl sebacate from Nakarai. A Trizma preset pH crystal (pH 7.5) was purchased from Sigma. A sample of potassium chloride and other chemicals were supplied from Kishida. All reagents were used as received. The water was distilled and deionized. The buffer solution was prepared by dissolving Trizma in water, and the concentration was adjusted to 0.05 M. All measurements were performed in the buffer solution. Membrane Preparation. A plasticized PVC membrane is obtained according to the preparation procedure of the membrane for an ion-selective electrode (15). Valinomycin (0.01 g) and
I 0
50
100
Concentration of Kf / rnM Figure 2. Sensor response to various potassium ion concentrations.
Na'TFPB- (0.001 g) were dissolved in dibutyl sebacate (0.5 g), and the solution was mixed with tetrahydrofuran (5.0 mL). Poly(viny1 chloride) (0.2 g) was gradually dissolved in this mixed solution, which was poured into a petri dish (diameter, 67 mm). By vaporization of tetrahydrofuran, a plasticized PVC membrane sensitive to K+ was obtained. The thickness of the membrane was 200 pm. The end surface of the optical fiber was soaked with tetrahydrofuran, and a small piece (1mm square) of the membrane was fixed to this end surface. The hydrophobic probe of M in the buffer solution, dodecyl-A0+ was dissolved at 1 X and the plasticized PVC membrane attached on the distal end was immersed into the dodecyl-A0+ solution for 10 s. Dodecyl-A0+ was ion-exchanged with Na+ and dissolved into the plasticized PVC membrane. The sensor was further washed with a copious amount of the buffer solution.
RESULTS AND DISCUSSION Sensor Response. The calibration curve of the sensor to the potassium ion concentration is shown in Figure 2. The fluorescence intensity decreases linearly with increase of potassium ion concentration in the sample solution. The response of the sensor was reversible, and the response time was 10 s to reach the (1- l / e ) level of the maximum signal. The detection limit for the potassium ion was 0.5 mM, which was determined by fluctuation of the signal intensity caused by an unstable output power of the argon ion laser (*0.5%). In the ion-selective electrode, the potential of the plasticized PVC membrane is proportional to the logarithmic analyte concentration. However, the observed sensor response is linear to the potassium ion concentration. This result implies that the sensor response is not ascribed to the change in the membrane potential but is ascribed to the hydrophobicity around dodecyl-A0+ as discussed below. Response Mechanism. The excitation and emission spectra of dodecyl-AO+ in the plasticizer of dibutyl sebacate and in the aqueous buffer solution are shown in Figure 3. The fluorescence intensity of dodecyl-A0+ in dibutyl sebacate is 7 times larger than that in the aqueous buffer solution. It implies that dodecyl-AO+ is useful as a hydrophobic probe such as 8-anilino-1-naphthalenesulfonate (16). The response mechanism assumed in this study is schematically illustrated in Figure 4. When the plasticized PVC membrane containing dodecyl-AO+ is immersed into the sample solution, the potassium ion selectively forms a complex with valinomycin. Dodecyl-AO+ concomitantly moves from the membrane phase to the boundary of the aqueous phase. The microenvironmental polarity a t the boundary is much higher than that in the membrane. Thus the fluorescence intensity may decrease with increase of the potassium ion
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
the membrane, respectively. The extraction constants of the K+-valinomycin complex and the K+-TFPB- ion-pair, K,,, and Kex2,are defined by Kex,
= [K+valIm/[K+la[vallm
K,,, = [K+TFPB-],/ [K+],[TFPB-] ,
(4) (5)
where [K+], and [TFPB-1, are the concentrations of the potassium ion and TFPB- in the aqueous solution and [val], is the concentration of valinomycin in the membrane. From these equations, the fluorescence intensity from the sensor is calculated to be F = kl[dodecyl-AO+]o, k l ( 1 - kz)(K,,,[valI, + Kex,[TFPB-lJ[K+]a (6)
1
1
Buffer Solution (pH7.5)
I
Wavelength /nm
Flgure 3. Excitation and emission spectra of dodecyl-AO' in organic and aqueous solvents. The excitation and emission wavelengths are 493 and 525 nm, respectively. The concentration of dodecyl-AO' is M. adjusted to 2 X Solution, Membrane
Valinomycin
Flgure 4. Response mechanism of fiber-optic sensor using dodecyl-AO+ and plasticized poly(viny1 chloride) membrane.
concentration in the sample solution. Since dodecyl-AO' has a long alkyl chain, the chromophore part of acridine orange may go out from the membrane, but a long alkyl chain may remain inside the membrane. When the concentration of the potassium ion in the sample solution becomes low, dodecyl-A0+ may go back again into the membrane phase. This movement of dodecyl-AO+ results the fast and reversible response of the sensor. Based on the mechanism described above, the fluorescence intensity, F, from the sensor is represented by eq 1
F = kl([dodecyl-AO+],
+ k,[dodecyl-AO+],)
(1)
where [dodecyl-AO+]is the concentration of dodecyl-AO', k , is the instrumental constant, and k2 is the ratio of the fluorescence intensity of dodecyl-AO+in the aqueous solution (a) to that in the membrane (m). A mass balance equation can be written as follows: [ d ~ d e c y l - A O + ] ~=, [dodecyl-AO+],
+ [dodecyl-AO+],
(2) where [d~decyl-AO+]~, is the initial concentration of dodecyl-AO+ in the membrane. Since the potassium ion forms a complex with valinomycin and an ion-pair with TFPB- in the membrane, a charge balance is given by [dodecyl-AO+I, = [K+val],
+ [K+TFPB-],
(3)
where [K+val], and [K+TFPB-1, are the concentrations of the K+-valinomycin complex and the K+-TFPB- ion-pair in
The extraction constants are dependent on the kind of the organic solvent in the membrane phase. But, the value of K,,, is on the same order of that of K,,, (17,18). Since the concentration of valinomycin in the membrane, [val],, is nearly equal to the initial concentration of valinomycin, [val], is 1.9 x M in the prepared membrane. The initial concentration of TFPB- in the membrane is 2.4 X lV3M. The concentration of TFPB- in the aqueous solution, [TFPB-I,, depends both on [K+ TFPB-1, and on [K+],. However, TFPB- is highly lipophilic, and [TFPB-1, is much lower than 10" M between the aqueous and dichloromethane phases when [K+TFPB-1, and [K+], are on the order of M, for example (18). Thus the value of K,,,[TFPB-], is negligibly small in comparison with that of K,,,[val],. Therefore, the slope of eq 6 against [K+], is considered to be almost constant. This result shows that the fluorescence intensity from the sensor decreases linearly with increase of the potassium ion concentration. The experimental result shown in Figure 2 qualitatively agrees with the present model. Numerous cyanine dyes and oxanol dyes are well-known as potential-sensitive probes (19). Rhodamines are also useful as fluorescent indicators for the membrane potential ( 9 , I O ) . The potential of the ion-selective electrode using the prepared membrane is ascertained to obey the Nernstian response, i.e. a logarithmic response to the potassium ion concentration. The response of the present optical sensor is linear (not logarithmic) to the analyte concentration. Selectivity. The prepared membrane contains valinomycin as a neutral ionophore, and the potassium ion in the sample solution is selectively extracted into the membrane. This optical sensor is considered to be selective to the potassium ion. When the sensor was immersed into a 0.1 M Na+ solution and a 0.1 M Ca2+ solution, no change in the fluorescence intensity was observed. Since the present sensor is sensitive to 0.5 mM of the potassium ion solution, the optical selectivity coefficient defined in ref 14 is at least less than 5 X (0.5 mM/O.1 M) for both Na+ and Ca2+. From eq 6, the selectivity coefficient is represented by the ratio of the terms, K,,,[val], + K,,,[TFPB-],, for the interference ion and the potassium ion. The calculated values for Na+ obtained by using the extraction constants reported in refs 17 and 18 are 1.8 X lo", assuming no TFPB-, and 0.24, assuming no valinomycin in the membrane. Selectivity to the potassium ion significantly increases with decrease of the concentration of TFPB-. However, TFPB- is necessary in the membrane to prevent extraction of an anion into the membrane. It limits the cation selectivity in the present sensor. The sensitivity of the sensor to 0.05 M K,SO, solution is almost identical with that of 0.1 M KNO, solution. This result implies that the sensor response is not affected by anions in the sample solution. Reproducibility and Lifetime. The concentrations of the K+-valinomycin complex and the K+-TFPB- ion-pair formed in the membrane are varied by the chemical composition of the membrane. The initial concentration of dodecyl-AO+ is
Anal. Chem. 1990, 62, 1531-1532
also affected by the time to immerse the membrane into the dodecyl-AO+solution. Thus the fluorescence intensity from the sensor depends on the preparation procedure of the plasticized PVC membrane. However, the ratio of the signal intensities for the sample and blank solutions was ascertained to be almost identical for several sensors constructed by the similar procedure. The signal intensity from the sensor gradually decreases with time, and it restricts the long time operation of the sensor. Due to a low output power of the laser, photobleaching of dodecyl-A0+is negligible. As shown in Figure 3,dodecyl-A0+ is slightly soluble in water. Thus the signal decrease is considered to be due to leaching of dodecyl-A0+ from the plasticized PVC membrane. The more lipophilic acridine orange, 3,6-bis(dimethylamino)-l0-hexadecylacridinium bromide, was synthesized according to the procedure reported in ref 20,and this molecule was used as fluorogenic chromophore instead of dodecyl-AO+. The constructed sensor responded to the potassium ion selectively, and the signal intensity was stable at least for several hours. It implies that the lipophilic alkyl chain is essential for a long-term operation of the sensor.
Application to Other Plasticized PVC Membrane. There are many ionophores that extract only a specific cation into a membrane (21). Various ion-selective electrodes sensitive to specific cations have been developed by using the plasticized PVC membrane containing such ionophores. All these membranes might be modified to the optically sensitive membranes by immersing those into the solution containing dodecyl-A0+. The ion-selective electrodes using the plasticized PVC membranes are also useful for the measurement of anionic species (12). In a similar manner, optically sensitive membranes to specific anions might be obtained by incorporating the hydrophobic probes with a negative charge (16) in the membrane. However, such a probe currently used is highly soluble in water and leaches from the membrane. Thus it will be necessary to develop a fluorescent hydrophobic probe with a long alkyl chain. It is noted that high solubility of the chromogenic probe in the plasticized PVC membrane and presence of a charge in the probe are essential to obtain a fast and reversible response of the sensor.
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LITERATURE CITED Zhujun, 2.; Zhang. Y.; Wangbai, M.; Russell, R.; Shakhsher, A. M.; Grant, C. L.; Seitz, W. R.; Sundberg, D. C. Anal. Chem. 1888, 67, 202. Wolfbeis. 0.S.; Offenbacher, H. Sens. Actuators 1886,9 . 85. Kawabata, Y.; Tsuchida, K.; Imasaka, T.; Ishibashi, N. Anal. Sci. 1887,3 , 7. Zhujun, 2.; Seitz, W. R. Anal. Chem. 1888, 58, 220. Wolfbeis, 0.S.; Weis, L. J.; Leiner, M. J. P.; Zlegler, W. E. Anal. Chem. 1888,60, 2028. Zhujun, 2.; Seitz, W. R. Anal. Chlm. Acfa 1885, 777, 251. Kawabata, Y.; Tahara, R.; Irnasaka, T.; Ishibashi, N. Anal. Chlm. Acta l888*272, 287. Zhujun, 2.; Muliin, J. L.; Seitz, W. R. Anal. Chim. Acta 1888, 784, 251. Wolfbeis, 0.S.; Schaffar, B. P. H. Anal. Chlm. Acta 1887, 798, 1. Schaffar, B. P. H.; Wolfbeis. 0.S.; Leltner, A. Analyst 1988, 173, 693. Alder, J. F.; Ashworth, D. C.; Narayanaswamy. R.; Moss, R. E.; Sutherland, I. 0.Analyst 1887, 772, 1191. Covington, K. Ion -Selective flechode Methodology; CRC Press: Boca Raton, FL, 1979; Volume 1. Suzuki, K.; Tohda, K.; Tanda, Y.; Ohzora, H.; Nlshihama, S.; Inoue, H.; Shirai, T. Anal. Chem. 1889. 61, 382. Seiler, K.; Morf, W. E.; Rusterholz, B.; Simon, W. Anal. Sei. 1888,5 , 557. Pick, J.; Toth, K.; Pungor, E.; Vasak, M.; Simon, W. Anal. Chim. Acta 1873,64,477. Turner, D. C.; Brand, L. Blochemisby 1888, 7 , 3381. Eisenman, G. Membranes; Marcel Dekker: New York and Basel. 1975; Volume 2. Nishida, H.; Takada, N.; Yoshimura, M.; Sonoda, T.; Kobayashi. H. Bull. Chem. SOC.Jpn. 1884, 57, 2600. Waggoner. A. S. Annu. Rev. Biophys. Bioeng. 1878,8 , 47. Yamagushi, A.; Masui, T.; Watanabe, F. J . Phys. Chem. 1981, 85, 281. Dietrich, B. J . Chem. fduc. 1885, 62, 954. * Author to whom correspondence should be addressed.
Yuji Kawabata Ryuichi Tahara Toshito Kamichika Totaro Imasaka Nobuhiko Ishibashi* Faculty of Engineering Kyushu University Hakozaki, Fukuoka 812,Japan
RECEIVED for review January 17,1990.Accepted April 2,1990. This research was supported by Grant-in-Aid for Scientific Research from the Ministry of Education of Japan.
Multiway Analysis of Variance for the Interpretation of Interlaboratory Studies Sir: In a recent paper in this journal, Thompson (1) advocates the use of robust estimators to mitigate the effects of nonnormality in the statistical analysis of interlaboratory studies. To demonstrate these tools, he reanalyzes a large-scale study comparing two different catalysts for the Kjeldahl protein determination in feeds which was organized by Kane (2)in 1984. Thompson concludes that there is no difference between the two catalysts, just as Kane concluded, for most feeds, by a series of one-way analyses of variance after deleting outliers. The purpose of this communication is to point out that the same conclusion can also be reached, and presented in a concise form, by a single three-way analysis of variance (anova) before identifying outliers. It was, after all, to describe the insensitivity of the anova to mild departures from normality that the term “robust” was re-coined for circulation in the realm of statistics (3). Kane sent blind duplicates of 26 different feeds, which were grouped into 13 pairs (“Youden pairs”) of near-duplicates to 0003-2700/90/0382-1531$02.50/0
discourage data censoring, to each of 22 laboratories. He requested two Kjeldahl protein determinations on every sample, one using a mercury catalyst and one using a copper catalyst. In all, 2288 results, ranging from 10 to 90% crude protein, were reported to him. To carry out an anova on this body of data for present purposes, a linear model incorporating the main factors of the study, and all the interactions among them, was postulated Yijkl= M si + L j + Ck SLij + SCik + LCjk +
+
+
SLC,,
+ eijkl
where Yijklis the value of Ith replicate, using the kth catalyst (Ck),in the j t h laboratory (Lj),on the ith sample (Si). The term SLij is the interaction of the ith sample with the j t h laboratory, etc., eyklis the random error of measurement, and M is the mean of all values. The sample effects can be regarded as fiied and the laboratory effects, random; the catalyst effects are, of course, fixed. 0 1990 American Chemical Society
