Langmuir 1996, 12, 6717-6720
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Total Internal Reflection Fluorescence Measurements of Protonation Equilibria of Rhodamine B and Octadecylrhodamine B at a Toluene/Water Interface Hitoshi Watarai* Department of Chemistry, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan Fumiko Funaki Department of Chemistry, Faculty of Education, Akita University, Akita 010, Japan Received July 2, 1996. In Final Form: October 1, 1996
Introduction Chemical reactions at liquid-liquid interfaces are stimulating subjects in various research fields including colloidal chemistry,1 separation chemistry2 and biochemical sciences.3 However, the techniques to measure reactions at liquid-liquid interfaces are still limited in comparison with those for air-liquid and solid-liquid interfaces. Previously, we have developed a high-speed stirring method, which is an indirect photometric method to measure an interfacial adsorptivity and interfacial reaction rate of a dilute solute in liquid-liquid systems and by using this method investigated the role of interface in the extraction equilibria and extraction kinetics of various metal ions.4 In the present work, we demonstrated a total internal reflection fluorescence (TIRF) spectrometry as a direct method to detect interfacially adsorbed fluorescent molecules and its acid-base equilibria at the liquid-liquid interface. The TIRF method was introduced initially for the measurement of the adsorption phenomena at a solidliquid interface, but study of the liquid-liquid interface has rarely been reported.5,6 The advantages of TIRF are (1) fluorescent molecules in a very thin layer on the order of 100 nm just outside the interface of the organic phase is observable at high sensitivity by an evanescent wave excitation, provided that the refractive index of the organic phase is higher than that of the aqueous phase as it usually is, (2) the penetration depth of the evanescent wave can be controlled by the wavelength and incident angle of the excitation beam, and (3) information concerning the species, the amount, and the orientation of the interfacially adsorbed molecules can be obtained. Rhodamine B is a typical fluorescent dye used as an ion-association extractant in solvent extraction photometry or fluorimetry of anionic species.7 The chemical forms depend on the polarity of solvent: a colorless lactone form in less polar solvents (e.g., benzene and toluene) and a violet zwitterion in polar solution (e.g., alcohol and aqueous solution). A violet cationic (protonated) form can be formed in acidic solutions as shown in Figure 1.8 In the present study, the adsorption and the protonation of rhodamine B (RB) and octadecylrhodamine B (C18RB) at a toluene/ (1) Szymanowski, J.; Tondre, C. Solv. Extr. Ion Exch. 1994, 12, 873. (2) Danesi, P. R. Principles and Practices of Solvent Extraction; Rydberg, J., Musikas, C., Choppin, G. R., Eds.; Dekker: New York, 1992; Chapter 5, p 157. (3) Nelson, A. Langmuir 1996, 12, 2058. (4) Watarai, H. Trend. Anal. Chem. 1993, 12, 313. (5) Watarai, H.; Saitoh, Y. Chem. Lett. 1995, 283. (6) Okumura, R.; Hinoue, T.; Watarai, H. Anal. Sci. 1996, 12, 393. (7) Ueno, K.; Imamura, T.; Cheng, K. L. Handbook of Organic Analytical Reagents, 2nd ed.; CRC Press: Boca Raton, 1992; p 525. (8) Ramette, R. W.; Sandell, E. B. J. Am. Chem. Soc. 1956, 78, 4872.
S0743-7463(96)00654-3 CCC: $12.00
Figure 1. Principal forms of rhodamine B and C18-rhodamine B in toluene and aqueous solutions.
Figure 2. Optical arrangements for the TIRF measurements at the toluene/water interface.
water interface were investigated by means of TIRF and interfacial tension measurements. Experimental Section Materials. Rhodamine B (RB, Merk, G. R.) and octadecylrhodamine B (C18RB, Nihonkankoshikiso, NKX731) were used without further purification. A colorless solution of C18RB in toluene was prepared by shaking a pale red toluene solution of NKX731 with a 0.01 M NaOH aqueous solution three times, successively by 0.1 M Na2SO4 aqueous solution two times. Toluene and heptane (Wako, G. R.) were purified by distillation. Sulfuric acid, sodium sulfate, and sodium hydroxide were all of reagent grade. Water was purified by Milli-QII system. Measurements. TIRF measurements was carried out by Hitachi 650-40 spectrofluorometer with a laboratory-made total internal reflection cell (Figure 2) at 25.0 ( 0.1 °C. The cell contained 1.2 mL of each phase, and the inside wall in contact with the organic phase was made hydrophobic by treating with 2% dichlorodimethylsilane in benzene in order to produce a flat liquid-liquid interface. Through a rectangular prism, an excitation beam illuminated the interface at the incident angle of θ ) 72° which was sufficiently greater than the critical angle of 63° in a toluene/water pair, and the fluorescence from the interface was measured through another prism placed at right angles to the incident beam. The penetration depth of the
© 1996 American Chemical Society
6718 Langmuir, Vol. 12, No. 26, 1996
Notes
Figure 3. Distribution equilibria of rhodamine B in heptane/ water (b) and toluene/water (O) systems. KD(heptane) ) 3.60, KD(toluene) ) 670, and pKa ) 3.22 were obtained.
Figure 5. Variation of TIRF intensity at toluene/water interface with rhodamine B (a) and C18-rhodamine B (b) concentrations. Aqueous phase (y) 0.10 M H2SO4, (b) 0.010 M H2SO4, (O) 0.10 M Na2SO4.
Figure 4. Equilibrium scheme for the distribution and interfacial adsorption of rhodamine B and C18-rhodamine B which are abbreviated by R. excitation beam at λ ) 554 nm was calculated as 184 nm by9
dp ) (λ/2π)(n12 sin2 θ - n22)1/2
(1)
where n1 and n2 are the refractive indexes of toluene (n1 ) 1.494 13 at 25 °C) and water (n2 ) 1.332 87 at 25 °C), respectively. Interfacial tension in toluene/water systems was measured by a drop-volume method at 25.0 ( 0.1 °C.9 Distribution experiments of RB in heptane/water and toluene/water systems were carried out by a centrifuge-tube method. Ten mL of a RB aqueous solution (1.0 × 10-5 M) in the pH range 1-13 was shaken with the same volume of heptane or toluene by a reciprocating shaker SR-II (Taiyo Kagaku, Japan) for 30 min. After 1 h for phase separation, an aliquot of the aqueous phase was taken for spectrophotometry at 554 nm by photodiode array spectrophotometer HP8452A, and for pH measurement by Horiba F-14 pH meter. The ionic strength of the aqueous phase was maintained at 0.3 by sodium sulfate.
Results and Discussion Liquid-Liquid Distribution. Two-phase equilibria of RB in heptane/water and toluene/water systems were shown in Figure 3 as a function of pH. Distribution ratio D of a dye R can be represented using the distribution constant, KD ) [R]0/[R] and the acid dissociation constant of the protonated form Ka ) [H+][R]/[HR+]:
D ) KD/(1 + [H+]/Ka)
(2)
From the results in heptane/water system, KD ) 3.60 and pKa ) 3.22 were determined. The KD in toluene/water system was calculated as 670 using eq 2. These analysis confirmed the distribution scheme as depicted in Figure 4; (1) a pH-independent distribution equilibrium between the lactone form in organic phase and the zwitterion in aqueous phase and (2) the protonation of the zwitterion (9) Hirschfeld, T. Can. Spectrosc. 1965, 10, 128. (10) Watarai, H.; Horii, Y.; Fujishima, M. Bull. Chem. Soc. Jpn. 1988, 61, 1159.
in the aqueous phase. The pKa value determined here was in excellent agreement with the literature value of 3.22.8 Interfacial Protonation Measured by TIRF. The TIRF intensities of RB and C18RB were dependent on the concentration of the dyes, pH, and the Na2SO4 concentration as shown in Figure 5. In RB, the TIRF intensity at 588 nm showed a maximum at 3.0 × 10-7 M in 0.01 M H2SO4 (pH ) 2.2), while in 0.1 M Na2SO4 the maximum shifted to higher RB concentration. As for C18RB, a maximum appeared at 3.0 × 10-7 M in 0.1 M H2SO4 (pH ) 1.1) shifted toward a higher C18RB concentration in 0.01 M H2SO4 and 0.1 M Na2SO4. The increase in the TIRF intensity must be due to the increase in the interfacial concentration of the dye, while the decrease of TIRF in the higher dye concentration is attributable to a decrease of the excitation intensity by absorption, a selfabsorption of the fluorescence and a quenching by an intermolecular energy transfer.11 These results suggested that the toluene/water interface is almost saturated when the total concentration of RB or C18RB was over 3 × 10-7 M in highly acidic aqueous conditions. This concentration corresponds to the interfacial concentration of 3 × 10-10 mol/cm2 at the interface in the cell, assuming a quantitative adsorption of the dyes. At the concentration of 3.0 × 10-7 M, we examined the pH dependence of TIRF intensity of RB and C18RB. As shown in Figure 6, the TIRF intensity was independent of pH in the region 4-6, while it increased with the decrease in pH below around 3, then it reached a constant value in the lowest pH examined. These results proved that the protonated form is more adsorbable at the interface than the zwitterion form. Therefore, the increase in TIRF intensity in the acidic region should reflect the protonation at the interface. The pH dependence of TIRF intensity was analyzed by the following consideration. The TIRF intensity, IF, can be represented by
IF ) φ1[R]i + φ2[HR+]i
(3)
where φ1 and φ2 refer to TIRF efficiencies of R and HR+ under the present experimental conditions, respectively, and [ ]i represents interfacial concentration. Assuming (11) Koizumi, M.; Kato, S.; Mataga, N.; Matsuura, T.; Usui, Y. Photosensitized Reactions; Kagakudojin: Kyoto, 1978.
Notes
Langmuir, Vol. 12, No. 26, 1996 6719
Figure 6. Dependence on the subphase pH of TIRF intensity at 588 nm of rhodamine B (b) and C18-rhodamine B (O), [RB] ) 3.0 × 10-7 M, [C18RB] ) 3.0 × 10-7 M, I ) 0.3.
that the interfacial concentrations of HR+ and R are shown by the Langmuir isotherm, the TIRF intensity is written by
IF )
aK′[R]0(φ1 + φ2[H+]/Ka′ ) a + K′[R]0(1 + [H+]/Ka′ )
Figure 7. Polarized TIRF intensities of adsorbed C18RB at toluene/aqueous phase (0.01 M H2SO4, pH ) 2.19) relative to those of 1.2 × 10-6 M C18RBClO4 in toluene. Vertical and horizontal polarizations to the interface are represented by 0 and 90, respectively, and the combination of the polarizers by the pair of 0 and 90 for the excitation-detection polarizers.
(4)
where a is the saturated interfacial concentration occupied by HR+ and R changing its ratio depending on pH, and K′ and Ka′ are the adsorption constant of R defined by K′ ) [R]i/[R]0 and the acid dissociation constant of HR+ at the interface defined by Ka′ ) [R]i[H+]/[HR+]i, respectively, under infinitely diluted condition. The total concentration [R]total is represented by
(
)
K′ K′[H ] + V0 V0 Ka′ +
[R]total ) [R]0 1 +
(5)
From eqs 4 and 5
IF ) aK′[R]total(φ1 + φ2[H+]/Ka′ ) a(1 + K′/V0 + K′[H+]/V0Ka′ ) + K′[R]total(1 + [H+]/Ka′ ) (6) When [H+] , Ka′, eq 6 is
IFb )
aK′[R]totalφ1 a + K′[R]total
Figure 8. Interfacial tension lowering in toluene/water with rhodamine B (a) and C18-rhodamine B (b) concentrations.
Z K′[R]totalφ1
(7)
since a . K′[R]total and V0 . K′ which were concluded from the interfacial tension measurements as described in a later section. When [H+] . Ka′, the TIRF intensity is approximated by IFa ) φ2[HR+]i in eq 3, eq 6 is
IFa Z aφ2
(8)
Therefore, when [H+] ) Ka′, eq 6 is
IF* )
aK′[R]total(φ1 + φ2) a + 2K′[R]total
) IFb + K′[R]totalIFa/a
(9)
since K′[R]total is much smaller than a. The contribution of the second term in eq 9 is less than 1%. These considerations lead to the conclusion that pKa′ is the pH at which IF begins to increase in the plot of IF vs pH shown in Figure 6. According to this criteria, one can estimate pKa′ ) 3.2 for RB and pKa′ ) 3.5 for C18RB.
Orientation of C18RB at the Interface. Polarized TIRF intensity was observed by placing polarizers on both excitation and emission sides of the cell. Measurements were carried out with four different combinations of horizontally and vertically polarized beams to the interface. The polarized TIRF intensity was normalized with the fluorescence intensity of perchlorate salt of cationic C18RB in toluene under the same experimental conditions, which is nonpolarized intrinsically. As shown in Figure 7, among the four different combinations of the polarizers, a maximum intensity was obtained when both were horizontally polarized. This means that the C18RB fluorophore is oriented parallel to the interface. This type of orientation has been reported also in the adsorption of acridine orange at hexadecane/water interface.12 Interfacial Protonation Observed from Interfacial Tension. The interfacial tension in toluene/water system was decreased as the RB or C18RB concentration was increased and the pH was lowered as shown in Figure 8. The lowering of the interfacial tension came out at a (12) Piasecki, D. A.; Wirth, M. J. J. Phys. Chem. 1993, 97, 7700.
6720 Langmuir, Vol. 12, No. 26, 1996
Notes
Table 1. Interfacial Adsorption Parameters of Rhodamine B and C18-Rhodamine B Determined by Interfacial Tension Measurements at 25 °C solute rhodamine B C18-rhodamine B
pH
log a (mol/cm2)
log K′, log K′app
pKa′ a
5.27 2.18 5.36 2.19
-10.29 -10.26 -10.39 -10.29
log K′ ) -5.30 log K′app ) -4.00 log K′ ) -5.45 log K′app ) -4.49
3.48 3.15
pKa′ ) log K′app - log K′ + pH when [H+] . Ka′, where Ka′ ) [R]i[H+]/[HR+]i, K′app ) K′(1 + [H+]/Ka′ ) and K′ ) [R]i/[R]0. a
concentration of 3.0 × 10-7 M in both cases of RB and C18RB at pH ) 2.2. From this figure, it was found that RB and C18RB were more adsorbable when they were protonated. The pH dependence of the interfacial tension can be represented by10
(
γ ) γ0 - aRT ln 1 +
)
Kapp′ [R]total a
(10)
where γ and γ0 are the interfacial tensions in the absence and presence of the dye and Kapp′ is the apparent adsorption constant at a given pH defined by
Kapp′ ) K′(1 + [H+]/Ka′)
(11)
In the plot of γ vs ln [RB] or ln [C18RB], the intersection of the two lines of γ ) γ0 and γ ) γ0 - aRT ln Kapp′[R]total/a affords the relation of ln [R]* ) ln a/Kapp′ where [R]* is the concentration of the dye at the intersection. In the pH around 5, Kapp′ is equal to K′. The slope of γ vs ln [R]total gives the value of a. Thus, one can obtain the values of log a, log K′, and pKa′. These values for RB and C18RB are listed in Table 1. We can conclude from the values in Table 1 that the pKa′ are similar among those obtained
by TIRF measurements and are not largely different from pKa in bulk aqueous phase. Conclusion We demonstrated in the present study that the interfacial protonation of RB and C18RB could be successfully measured by total internal reflection fluorometry (TIRF). It was found from the TIRF polarization measurement that the transition dipole of the fluorophore was placed parallel to the interface. The interfacial protonation was confirmed also by the interfacial tension lowering measured by varying pH and the dye concentration. The estimated protonation constants at the toluene/water interface were not largely different from the dissociation constants in the bulk aqueous phase under the present conditions. This means that the protonation of the fluorophore takes place at the aqueous phase side of the interface where the dielectric constant is not lowered seriously.13 The study on the kinetics of protonation and the rotation at the interface are now ongoing in order to get more information about the microscopic environmental effect of the liquid-liquid interface. Acknowledgment. This work was supported by Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science, Sports and Culture, Japan (No. 07404042). LA960654E
(13) Fernandez, M. S.; Fromherz, P. J. Phys. Chem. 1977, 81, 1755.
