2934

Anal. Chem. 1991, 63, 2934-2938

Near-Infrared Fluorescence Probe for pH Determination Jyh-Myng Zen and Gabor Patonay*

Department of Chemistry, Georgia State University, Atlanta, Georgia 30303

Naflon, a perfluorosunonated bn-exchange polymer, has been evaluated as a substrate for lmmobllltlng a near-Infrared absorblng fluorophore to determlne pH In solutions. Following slmple preparation procedures a cyanlne dye with a pH-senSnlve functlonal group (a Ms(carboxyl1c acid) derlvatlve) was entrapped withln the Naflon matrlx and fabrlcated Into thln fllm. The fabrlcatlon of a thin-fllm probe gives greater versatllity for the method. Thls dye exhibits great stablllty lnslde the Naflon matrlx and has relatlvely long absorptlon and fluorescence wavelengths whlch allow researchersto utlllze the lower Interference of thls reglon. Since the absorptlon maximum of the study system Is around 800 nm In aqueous solutions, semlconductor lasers can be used. I n the study the feaslbllity of thls approach was Illustrated and the analytlcal utility of near-Infrared laser dlodes for pH determlnatlon was evaluated.

Nafion in the solution is then formed into an immobilized solid polymer electrolyte surface by evaporating the solvent. The immobilized solid polymer electrolyte can then be further modified for some specific applications. The same concept can also be applied in the fabrication of optrodes. For example, Hieftje and co-workers reported a fiber-optic probe utilizing a fluorescent dye entrapped within a perfluorinated ionomer matrix for air humidity measurement (17). Our near-IR pH-sensitive dye may be entrapped inside the Nafon matrix for pH measurement in a similar manner. This study has been carried out as a first step to investigate the feasibility of this approach. We report here the fluorescence and spectroscopic characteristics of near-IR pH-sensitive dye entrapped within a Nafion thin film, which was coated on the inside wall of a polystyrene cuvette. Furthermore, since Nafion may be affected by ion-exchanging a cation for the proton associated with the sulfonic acid groups, interference from alkali-metal ions for p H measurement by this method was also evaluated.

INTRODUCTION The pH determination of aqueous solutions is an important aspect of analytical chemistry ( I ) . Many dyes have been evaluated in the past as potential acidlbase indicators for the determination of solution pH (2-4). The analytical detection method preferred for this purpose is fluorescence, which offers enhanced selectivity and sensitivity. Fluorescent indicators are particularly useful in determining the p H of biological samples which have significant absorbance (5,6). In particular, the near-infrared (near-IR) region of the spectrum has proven useful in the study of biological samples because the absorbance of biologicals in this region is minimal and does not interfere with the absorbance or fluorescence of an near-IR chromophore (7, 8). Our previous study reported a near-IR spectrometric method using a near-IR absorbing fluorophore to determine pH in solutions (9). Note that, in this method, the dye was dissolved in solution. Cyanine dyes with pH-sensitive functional groups were prepared following simple synthetic routes. A bis(carboxy1icacid) derivative was used as a near-IR pH probe. The near-IR dye acid has an absorption maximum at 795 nm in aqueous solution which is in the region where inexpensive consumer laser diodes have their output maximum (785-795 nm). By combination of laser diodes with near-IR pH-sensitive chromophores, a promising new technique emerges. Although this method was useful when low interference is desired, dye immobilized to some suitable substrates and fabricated into thin film on the surface of glass, polystyrene, etc., should give the method greater versatility. The thin-film probes offer two important advantages relative to solution fluorescence measurements: they can measure pH without significantly perturbing the sample, and they can be used for continuous sensing. Perfluorosulfonic acid ionomer electrolytes, such as Ndion, are gaining increasing attention as surface films on chemically modified electrodes (1&16). Nafion can be made to cast on an electrode surface by applying ita solution over the electrode.

EXPERIMENTAL SECTION Reagents and Chemicals. The chemical structure of the pH sensitive dye used in this study is depicted in Figure 1. It is a bis(carboxy1ic acid) derivative of 2-(4’-chlor0-7’-[2’~-(1’’-ethyl3”,3”-dimethylindoleninylidene)]-3’,5’(1”’,3’”-propanediyl)1’,3’,5’-heptatrien-l’-yl~-l-ethyl-3,3-dimethylindolenine bromide, which was prepared in our laboratory as reported earlier (9). Spectrophotometric grade methanol was obtained from the Adrich Chemical Co. (Milwaukee, WI). Nafion perfluorinated ion-exchange powder, 5 w t % solution in a mixture of lower aliphatic alcohols and 10% water, was also obtained from Aldrich. Hydrochloric acid, sodium hydroxide, sodium chloride, potassium chloride,and lithium chloride were obtained from Fisher Scientific (Pittsburgh, PA). Method. A low3M stock solution of the pH-sensitive near-IR dye was prepared in spectrophotometric grade methanol. The required amounts of dye stock solution and Nafion solution were pipetted into a small beaker and thoroughly mixed by sonicating to form the coating solutions. The total volume of the coating solution is always 0.075 mL except there are different ratios between Nafion and dye. The 0.075-mL thoroughly mixed coating solution was then placed on the inside wall of a polystyrene cuvette and air-dried. A Nafion thin film with the pH-sensitive dye entrapped within was formed after the solvent was completely removed. The thin film formed by this method was not very uniform; however, this did not affect our results. Solution pH was varied for all test systems by slowly adding appropriately diluted hydrochloric acid or sodium hydroxide before being transferred into the cuvette in order to evaluate the behavior of the dye-entrapped Nafion thin film at different ionic strengths. The solution pH was monitored continuously with glass pH electrodes. Since the sensitivity of a glass electrode to alkali-metal cations reduces its response below the theoretical level at a pH values above 10, calibration curves for the LiC1, NaCl, and KCl solutions were measured. The pH values reported in this paper were corrected according to the calibration curves. Instrumentation. A Perkin-Elmer Lamda 2 UV/vis/near-IR spectrophotometer was used for absorption measurements. The spectrophotometer was interfaced to a Zenith 286 computer to store spectra and control the spectrophotometer. Each spectrum was recorded using a PECSS program. Cell data were obtained at room temperature using solutions in equilibrium with air. Solution pH was measured with an Orion Research Model 701A digital ionalyzer. Fluorescence spectra were recorded on an SLM 8000

* To whom correspondence should be addressed. 0003-270019 110363-2934$02.50/0

0 199 1 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991

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Wavelength (nm) Flgwe 3. Typical absorption spectra of the pH-sensitive near-IR dye in aqueous solution. spectrofluorometer interfaced to an IBM PS/2 computer. Fluorescence intensities of dye-entrapped Ndion thin films were excited with a simple and inexpensive laser diode (30 mW, 780 nm) along with an amplifier and were measured by DVM,which was constructed from off-the-shelf components in our laboratory as reported earlier (1419). The Schott Model 250 autotitrator and Model T90/10 autoburet interfaced to a 286 computer was used to determine the pK, values.

RESULTS AND DISCUSSION Representative absorption spectra of the pH-sensitive near-IR dye entrapped within the Ndion matrix in aqueous solution are shown in Figure 2. The dye spectrum was similar to that in aqueous solution except for a minor spectral shift of the near-IR absorption maxima. The absorption spectra for the near-IR dye in solution is depicted in Figure 3. Obviously, the spectrophotoscopic property of the dye was not changed in the Nafon environment. The near-IR absorption maximum of the dye was in the same region as the output wavelength of several commercially available laser diodes. Moreover, the dye was found to be very stable inside the Nafion matrix. After soaking in aqueous solution for days,

no leaching of the dye was observed; the absorption spectrum was found to be exactly the same. Our previous study showed that pKa values for the dye acid are 4.90 and 6.80, respectively, for the two carboxylic groups (9). The relatively low pKa value of the first carboxylic group was explained by the positive charge of the dye molecule. Once the first carboxylic group is fully ionized, the neutral zwitterionic form will predominate. Ionization of the second carboxylic group is more difficult because of the lack of a net positive charge. However, the situation is very different in the presence of Nafion. The pK, values for the dye acid in the Nafion environment were determined with automated titrators using NaOH (pH 10.60) solution. Figure 4 shows the titration curve for the dye solution. The titration curves were evaluated using first and second derivatives. Only one pK, value (about 8.3) was observed for the dye acid in the Ndion environment. Apparently, most of the positive charge of the dye molecule was used for bonding to the negative-charge sulfonic group of the Nafon. Hence, the two carboxylic groups are in a negatively charged environment that results in only one pKa value. It is important to remember, however, that the pK, value determined this way does not necessarily indicate the pH region where the dye exhibits spectral changes. There may be several explanationsfor this difference. Cyanine dyes are known to change their spectra upon formation of dimers. The dimer/monomer equilibrium may also change with changing pH. However, not enough information was obtained from the experiments of this study to indicate the processes with respect to acid/base chemistry of the near-IR dye in these Nafion films. For practical applicability, at this point, we do not need to fully characterize dye acid/base chemistry. Nevertheless, future studies are planned with respect to these questions.

2936

ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991

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Typical absorption spectra of the near-IR dye acid entrapped within the Ndion matrix in solutions of different p H s at low ionic strengths are depicted in Figure 5. These changes in the absorption spectra were repeatedly observed when the pH of the solutions added into the cuvette was switched back and forth between pH 11and 13. This phenomenon indicates that the hydrogen ion concentration is the primary determining factor. The response time for the changes was within the limitation of the instrument response time. The higher ionic strength solutions resulted in almost identical spectra and therefore are not shown here. As can be seen from Figure 5, an absorption peak a t 450 nm appears at higher pH values a t the expense of the near-IR peak at 802 nm. Compared to the absorption spectra of the free dye acid in solutions of different pH's at low ionic strengths (9),the pH-sensitive region moved from pH 7-10 to p H 10-13. This change of pH-sensitive region is not surprising since the -SO3- sites of the Nafion can attract protons and hold them inside the polymer matrix, an effect which is apparently equal to lowering the pH of the solution. Both the 450-nm and near-IR peaks can be used for determining the pH of the environment around the dye molecule. Figure 6a shows calibration curves of three different cuvettes coated with different concentrations of dye for determining the pH using the 450-nm absorption peak. The volume ratios between Nafion and dye of the 0.075-mL coating solutions used to prepare the thin films on the cuvettes were 55:20, 60:15, and 65:10, respectively. As can be seen, the 450-nm absorption peak appears in basic solutions and the intensity of the peak increases with increasing basicity of the solution. The calibration curve is less accurate when a lower concentration of the dye was used to prepare the thin-film-coated cuvette. Although this phenomenon may be used for pH

determination, the 450-nm peak is in the region of higher interference that renders its use less feasible. The near-IR peak detected at 802 nm in aqueous solution is more important since it facilitates the determination of pH in the near-IR spectral region. Moreover, the molar absorptivity at 802 nm is much higher than a t 450 nm a t the same pH, which provides more accuracy for the pH determination. Figure 6b shows the calibration curves for the same three thin-film-coated cuvettes except using the near-IR absorption peak. The intensity of this near-IR peak becomes lower as the pH of the solution increases. The same trend of change was observed for all three cuvettes coated with Nafion thinf i containing different dye concentration. Interestingly, the difference in absorbance for these cuvettes generally follows Beer's law, which once again demonstrates the stability of the dye inside the Nafion matrix. Our previous study indicated that near-IR laser diode induced fluorescence can be used for determining pH where the free dye was dissolved in solutions (9). This aspect was evaluated using our modified SLM 8000 fluorometer. The optical arrangement of the near-IR laser diode modified fluorometer has been described earlier (20). We now extend the study into solid surfaces for the purpose of giving this method greater versatility. The thin-film system can be studied by the simple and inexpensive near-IR laser diode system which was constructed from off-the-shelf components in our laboratory, as reported earlier (18). The near-IR fluorescence intensity of the dye-entrapped Ndion system was determined using a laser diode excitation source of 780 nm 30 mW in aqueous solutions of different pH's. To avoid saturation of the detector, the concentration of dye inside the Ndion matrix was reduced until the maximum signal intensity was lower than 25% of full scale. Note that, under these conditions, the dye concentration used to prepare the thin f i i for fluorescence study is much lower than that for absorption study. The volume ratio of the 0.075-mL coating solution between Nafion and dye was 67% The calibration curve obtained is shown in Figure 7. The region where pH may be determined is fairly similar using either near-IR absorbance or fluorescence methods. Most important of all, however, is that this result indicates near-IR laser diode induced fluorescence may be used for determining pH using film probes. The possible interference from other cations for pH determination using this method was also studied. Since at this working pH region most of the cations will precipitate with the hydroxide ions, only the alkali-metal ions were chosen for study. Actually, the alkali-metal ions are the major interference for certain glass pH electrodes, the response of which reduces below the theoretical at pH values above 10, depending on the selectivity of the electrode. The calibration

ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991 -0-

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curves of the glass electrode used in this study (Fisher Scientific) with the interference of the alkali-metal ions are shown in Figure 8. As can be seen, the interference is in the order Li+ > Na+ > K+. The same experiments described in this paper were repeated for the LiC1, NaC1, and KCl aqueous solutions using both the absorption and fluorescence methods. Figure 9 shows the calibration curves for determining pH using the near-IR absorption peak at different concentrations of LiCl aqueous solutions. As can be seen, all four curves generally follow the same trend of change. The shift in the pH-sensitive region actually is not a real effect, because the pH values used in the figure were direct readings from the pH meter. After the pH values were corrected according to the calibration curve of LiCl (aq) measured for the glass electrode, all four curves were virtually identical. The same phenomenon was also found for the NaCl and KC1 aqueous solutions. Apparently, the interference from the alkali-metal ions is insignificant to the pH determination using the near-IR absorption peak, indicating an important advantage of this method. These experiments indicate that our near-IR pH probe is suitably selective and free of alkaline error. The near-IR fluorescence intensity in LiCl aqueous solutions of different pH's (corrected) was also measured, as shown in the calibration curves of Figure 10. Both the pH-sensitive region and the trend of change were found to be very similar in different concentrations of LiCl aqueous solutions. However, the fluorescence intensity decreased as the concentration of LiCl increased. The same phenomenon was again observed for both the NaCl and KC1 aqueous solutions except with a different degree of decrease in fluorescence intensity a t the

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Figure 11. Fluorescence spectra of 0.025 mL of lo3 M near-IR pH-sensitive dye in 2.5 mL of (a water, (b) 2 M KCI (aq), (c) 2 M NaCl (aq), and (d) 2 M LiCi (as) solutions. Excitation wavelength = 795 nm.

same ionic strengths. A comparison between Figures 9 and 10 reveals that the interference of the alkali-metal ions with the p H determination using near-IR absorbance and fluorescence methods is somewhat different. This difference is as expected since fluorescence is characteristic of the excited state of the molecule while absorption is characteristic of its ground state. In order to confirm the above expectation, 0.025 mL of dye stock solution was added to 2.5 mL of water, 2 M LiCl (as), 2 M NaCl (aq), and 2 M KCL (aq), respectively, and both the absorption and fluorescence spectra were taken for comparison. The absorption spectra were found to be very similar for all solutions with almost identical absorption maxima. Using the same excitation wavelength of 795 nm, the fluorescence spectra for all solutions are shown in Figure 11. As can be seen, the fluorescence intensity decreases in the order LiCl > NaCl > KC1. This result confirms our expectation. Apparently, the hydrolysis reactions of the alkali-metal ions cause the change of the environment around the molecule and result in the difference in fluorescence intensity. The order of decrease in fluorescence intensity follows exactly the order of increase in hydration energy, which is Li+ > Na+ > K+. Due to the ion-exchange property of Ndion, the alkali-metal ions should be able to exchange to some extent with H+ and an ion-exchange equilibrium will be established. The equilibrium constant of alkali-metal ions in cation-exchange resin (Rel-SO,-H+) is in the order K+ > Na+ > H+ > Li+ (21,22). The same order is also expected for the Nafion polymer

2938

ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991 -0-

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Experimental Sequence Figure 12. Fluorescence intensity measured using the dye-entrapped Nafion thin-film probe and near-IR laser diode excitation In H,O (pH 2.65), 2 M LiCl (aq), 2 M NaCl (aq),and 2 M KCI (as) under sequences (a) LICI-H,0-NaCI-H,0-KCI-H20 and (b) KCI-H,O-NaCI-H,O-LiCIH2O.

matrix, which also consists of the sulfonic acid groups. A similar phenomenon was observed when Nafion-coated electrodes were studied (23,24). The interference of the alkalimetal ions with the fluorescence intensity of the dye-entrapped Nafion system through the effects of hydrolysis and the ionexchange process can be very clearly demonstrated by the following experiment. Two freshly prepared film-coated cuvettes with the same dye concentration were used to measure the fluorescence intensity in HzO (pH 2-65), 2 M LiCl (aq), 2 M NaCl (aq), and 2 M KCl (aq) with two different sequences. Sequence a was LiC1-HzO-NaC1-HzO-KCl-HzO. More specifically,the fluorescence intensity was f i t measured in LiCl (aq) solution. Then after being rinsed three times with HzO, the fluorescence intensity was measured in HzO. The same procedure was repeated for the NaCl (as) and KCl (as) solutions. Sequence b was KC1-HzO-NaC1-HzO-LiCl-H,O, which is in reverse order of sequence a. The results for both sequences are shown in Figure 12. Comparison of the first point for both sequences reveals that the fluorescence intensity is higher in LiCl (as) than in KC1 (aq). This result seems to be in contrast to the order of decrease in fluorescence intensity due to hydrolysis, which is Li+ > Na+ > K+. However, in light of the fact that K+ has a higher equilibrium constant for the ion-exchange process than Li+, the amount of K+ exchanged into the Nafion matrix apparently is much higher than that of Li+. Therefore, the results of the first point for both sequences actually is a compromise result of these two effects. The results of the second point for both sequences confirm the above explanation. Since K+ has a higher equilibrium constant for the ion-exchange process than He, hardly any K+ was moved out from the matrix. Hence, only a slight increase in fluorescence intensity was observed. While the equilibrium constant of H+ is higher than that of Li+, a much bigger increase in fluorescence intensity is observed. The same explanation is also true for the rest of the points in both

sequences; therefore, it is not mentioned here. Note that the use of pH 2.65 HzO is only for the purpose of demonstration; the fluorescence intensity can always be recovered to a similar value despite which alkali-metal ions exist, provided a much stronger acid is added. This study demonstrates that Nafion can be used as a substrate for immobilizing a near-IR pH-sensitive dye to determine pH in solutions. The region where pH may be determined is fairly similar using either near-IR absorbance or fluorescence methods. The absorbance method does not exhibit any ionic strength problems and the fluorescence method can be used a t lower ionic strengths. Future work will concentrate on studying the effect of different dyes with different absorption maxima and pK, values in the Nafion matrix. The ultimate goal of these studies is the development of a near-IR pH sensor that can be used in the physiological pH range. Hopefully, a large pH range may be covered if multiple-laser diode excitation is utilized. Moreover, the fabrication of optrodes by attaching the products of this study system to the end of fiber optics is also underway. Registry No. H+, 12408-02-5; bis(carboxy1ic acid) dye, 137008-57-2.

LITERATURE CITED (1) Guilbault, G. G. Practical fluorescence; Marcel Decker Inc.: New York, 1973. (2) Bosch, E.; Casassas, E.; Izquierdo. A.; Roses, M. Anal. Chem. 1984, 56, 1422. (3) Woods, E. A.; Ruzicka, J.; Christian, G. D.; Charlson, R. J. Anal. Cbem. 1986, 58, 2496. (4) Isreai, Y. Anal. Chim. Acta 1986, 206,313. (5) Mashimo, T.; Kamaya, H.; Udea, I.Mol. Pharmacol. 1986, 29, 149. (6) LaManna, J. C.; McCracken, K. A. Anal. Biocbem. 1984, 742,117. (7) Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 6 3 , 321A. (8) Imasaka. T.; Ishibashi, N. Anal. Cbem. 1990, 62,363A. (9) Boyer, A. E.; Devanathan, S.;Hamilton, D.; Patonay, G. Ta/anta, in press. (IO) Murray, R. W.; Ewing, A. G.; Durst, R. A. Anal. Chem. 1987, 59,379. (11) Gough, D. A.; Leypoldt, J. K. Anal. Cbem. 1979, 57,439. (12) Buttry, D. A.; Redepenning, J.; Anson, F. C. J. Phys. Cbem. 1988, 90,6227. (13) Buttry, D. A.; Anson, F. C. J. Am. Cbem. SOC. 1984, 706, 59. (14) Andrieux, C. P.; Dumas-Bouchiat, J. M.; Saveant, J. M. J. Nectroanal. Chem. 1981, 723, 171. (15) Szentirmay, M. N.; Martin, C. R. Anal. Chem. 1984, 56, 1898. (16) Fan, F. F.; Bard, A. J. J. Nectrocbem. SOC. 1986, 733, 310. (17) Zhu, C.; Bright, F. V.; Wyatt, W. A.; Hieftje, G. M. J. Nectrochem. SOC. 1989, 736 (2), 567. (18) Unger, E.; Patonay, G. Anal. Chem. 1989, 67,1425. (19) Hicks, J.; Patonay, G. Anal. Chem. 1990, 62,1543. (20) Hicks, J.; Andrews-Wilberforce, D.; Patonay, G. Anal. Instrum. 1990, 79,29. (21) Meites, L. Handbook of Analytical Chemistry; McGraw-Hill Book Co.: New York, 1963. (22) Yeager, H. L.; Steck, A. Anal. Cbem. 1979, 57,862. (23) Naegeli. R.; Redepenning, J.; Anson, F. C. J. Pbys. Chem. 1986, 90, 6227. (24) Redepenning. J.; Anson, F. C. J. Pbys. Cbem. 1967, 97,4349.

RECEIVED for review May 21, 1991. Accepted September 23, 1991. This work was supported in part by a grant from the National Science Foundation (CHE-890456). Acknowledgment is also made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research.