J . Phys. Chem. 1989, 93, 1464-1413
7464 Conclusion
This method of observation of the diffraction diagrams of phases in equilibrium with the crystalline state over small temperature intervals enabled accurate localization of the appearance and disappearance of the various phases and the transitions between them. Mesophases of identical symmetry were observed in formamide, glycerol, and water with closely similar lattice parameters. Thus these three solvents have similar capability to form lyotropic mesophases. This last result confirms the idea that the polymorphism of amphiphilic molecules is dominated by their ability to build interfaces organized in symmetric films, not by the effects of solvation and the dipolar repulsions.28
The similarity in the sequence of the ordered phases in the three solvents with CTAB cannot, however, be generalized to a wide range of organic solvents5 or other surfactant^.^^ Investigation of the influence of solvation forces, dipolar interactions, the role of the counterion and hydrogen bonding in lyotropic phases, and micelle formation in nonaqueous solvents is in progress.
Acknowledgment. It is a pleasure for us to thank J. Charvolin for fruitful comments concerning this study. Registry No. CTAB, 57-09-0; formamide, 75-12-7; glycerol, 56-8 1-5. (29) Auvray, X.; Danoix, F.; Petipas, C.; Anthore, R.; Rim, I.; Lattes, A,, to be published.
Lipoidal Eosin and Fluorescein Derivatives as Probes of the Electrostatic Characteristics of Self-Assembled Surfactant/Water Interfaces Janine Kibblewhite,t Calum J. Drummond,*,* Franz Grieser,t and Peter J. Thistlethwaitet Department of Physical Chemistry, The University of Melbourne, Parkville, Victoria 3052, Australia, and CSIRO, Division of Chemicals and Polymers, Private Bag 10, Clayton, Victoria 31 68, Australia (Received: January 10, 1989; In Final Form: April 14, 1989)
Lipoidal eosin and fluorescein derivatives have been investigated in order to determine whether or not they can be used to probe the electrostatic characteristics of surfactant monolayers spread at the air/water interface. The visible absorption spectra, fluorescence emission spectra, and acid-base behavior of 5-(N-hexadecanoy1)aminoeosin (HAE) and 5-(N-octadecanoy1)aminofluorein (OAF) have been examined at aqueous/surfactant interfaces. For comparison the spectral properties and acid-base behavior of closely related water-soluble analogues of HAE and OAF in 1,4-dioxane/water mixtures have also been studied. Lipoidal fluorescein derivatives were assessed to be the most suitable lipoidal indicators to probe the electrostatic characteristics of surfactant monolayers spread at the air/water interface. A preliminary examination of the pH-dependent (DPL) monolayers is reported. fluorescence properties of OAF in L-P,y-dipalmitoyl-a-lecithin
Introduction
It has been comprehensively demonstrated that lipoidal acidbase indicators can be employed to measure the mean field electrostatic potential at or in the vicinity of the aqueous/surfactant interfaces of micelles,l-10vesicles,+12 membra ne^,'^*^* soap films,15 and surfactant monolayers deposited onto either quartz or silica plates.I6'* When lipoidal acid-base indicators are used for this purpose it is generally assumed1-18 that
where \k, k , T, e, pK2, and pKaob"denote the mean field electrostatic potential at the average site of residence for the prototropic part of the acid-base indicator, the Boltzmann constant, the absolute temperature, the elementary electrostatic charge, the intrinsic interfacial pKa, and the apparent pKa of the prototropic part of the acid-base indicator residing at or in the vicinity of the charged aqueous/surfactant interface, respectively. As a result of recent advances, it is now possible to obtain the steady-state fluorescence spectrum of some fluorophores residing in surfactant monolayers spread at the air/water At present the experimental constraints are such that only fluorophores with a high fluorescence quantum yield can be utilized. Nevertheless, in principle, one should be able to use lipoidal acid-base indicators that have a pH-dependent fluorescence spectrum to probe the electrostatic characteristics of surfactant monolayers spread at the air/water interface. One can then, via a Langmuir trough facility, control intermolecular spacing and
hence surface charge density. Surfactant monolayers spread at the air/water interface are therefore ideally suited for studying (1) Fernandez, M. S.; Fromherz, P. J . Phys. Chem. 1977,81, 1755. (2) Frahm, J.; Diekmann, S.; Haase, A. Ber. Bunsen-Ges. Phys. Chem. 1980, 84, 566. (3) Lovelock, B.; Grieser, F.; Healy, T. W. J. Phys. Chem. 1985, 89, 501. (4) Drummond, C. J.; Grieser, F.; Healy, T. W. Faraday Discuss. Chem. SOC.1986, 81, 95. (5) Drummond, C. J.; Grieser, F. Phofochem. Phofobiol. 1987, 45, 19. (6) Hartland, G. V.; Grieser, F.; White, L. R. J . Chem. Soc., Faraday Trans. I 1987,83, 591. (7) Drummond, C. J.; Grieser, F.; Healy, T. W. Chem. Phys. Left. 1987, 140, 493. ( 8 ) Kibblewhite, J.; Drummond, C. J.; Grieser, F.; Healy, T. W. J . Phys. Chem. 1987, 91, 4658. (9) Drummond, C . J.; Grieser, F.; Healy, T. W. J . Phys. Chem. 1988,92,
2604.
J. J . Phys. Chem. 1988, 92, 5580. ( 1 1) Fernandez, M. S. Biochim. Biophys. Acra 1981,646, 27. (12) Kramer, R. Biochim. Biophys. Acfa 1983, 735, 145. (13) Drummond, C. J.; Grieser, F. Lungmuir 1987, 3, 855. (14) Fromherz, P. Volume on Biomembranes, Biological Transport; Fleisher, S., Fleisher, B., Eds.; Methods in Enzymology; Colowick, S . P., Kaplan, N . O., Eds.; Academic: New York, in press. (15) Fromherz, P.; Kotulla, R. Eer. Bunsen-Ges. Phys. Chem. 1984, 88. (10) Grieser, F.; Drummond, C.
1106.
(16) Fromherz, P. Biochim. Biophys. Acta 1973, 323, 326. (17) Fromherz, P.; Masters, B. Biochim. Biophys. Acfa 1974, 356, 270. (18) Lovelock, B.; Grieser, F.; Healy, T. W. Langmuir 1986, 2, 443. (19) Subramanian, R.; Patterson, L. K. J . Phys. Chem. 1985,89, 1202. (20) Agrawal, M. L.; Chauvet, J. P.; Patterson, L. K. J. Phys. Chem. 1985, 89. - , 2979. - -
(21) Teissie, J.; Prats, M.; Soucaille, P.; Tocanne, J.-F. Proc. Nafl.Acad. Sci. USA 1985, 82, 3217. (22) Prats, M.; Tocanne, J.-F.; Teissie, J. Eur. J. Biochem. 1985, 149,663.
'The University of Melbourne. ICSIRO.
0022-3654/89/2093-7464$01 S O / O
0 1989 American Chemical Society
The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 7465 supplied. The derivatives were stored in a dessicator at 0 OC and were shielded from light by wrapping their glass containers in aluminum foil. Surfactants. N-Dodecyl octaoxyethylene glycol monoether (CI2EB)was obtained from Nikko Chemical Co. and was stored in a refrigerated desiccator. Dodecyltrimethylammonium chloride (DTAC), and dodecyltrimethylammonium bromide (DTAB) were purchased from Tokyo Kasei Kogyo Co. Sodium dodecyl sulfate (SDS) was supplied by Sigma Chemical Co. L-@,y-Dipalmitoyl-u-lecithin (DPL) was puriss grade from Fluka AG Buchs and was stored at 0 "C. The surfactants were not further purified. Solvents and Inorganic Reagents. Milli-Q filtered water (conductivity < 1 X 10" R-I cm-' at 25 "C and air/water surface tension = 72.0 m N m-l at 25 "C) was used to prepare all the aqueous solutions. The 1,4-dioxane was of UV spectroscopic grade from Fluka and was passed through an aluminum oxide column O NHCoCYl C O O H (active neutral Brockmann grade 1, BDH) prior to use to remove NHCOCHJ any peroxides or water present. The chloroform and methanol were all spectroscopicgrade reagents obtained from Ajax Chemical Co. and were used as supplied. HCl and HC104 were analytical (JAF) (IAE) grade reagents supplied by May and Baker and by Ajax, reFigure 1. Molecular structures of the neutral quinonoid form of 54Nspectively. NaBr, NaC104, and N a O H were analytical grade octadecanoy1)aminofluorescein (OAF), 5-(N-hexadecanoyl)aminoeosin reagents obtained from Ajax. (HAE), 5-iodoacetamidofluorescein (IAF), and eosin-5-iodoacetamide UV/ Vis Absorption Spectra. The UV/vis absorption spectra (IAE). of the eosin and fluorescein derivatives in water and aqueous the electrostatic characteristics of aqueous/surfactant interfaces. micellar solutions were recorded on a 150-20 Hitachi UV/vis Fluorescence measurements on lipoidal molecules with a spectrophotometer, while those of the derivatives in 1,Cdioxfluorescein skeleton that might be suitable for surface potential ane/water mixtures were recorded on a Varian Cary Model 210 studies in air/water monolayers have already been r e p ~ r t e d ? ' ~ ~ UV/vis ~~ spectrophotometer. All UV/vis spectra were obtained However, in this earlier work, no consideration had been given at 25 "C. In the micellar solutions probe concentrations ranged to the complications that may arise from the presence of multiple from 2 X 10" to 5 X 10" mol dm-3. In the 1,4-dioxane/water molecular acid-base groups. mol dm-3. mixtures probe concentrations were ca. 1 X The purpose of the present work was to make a complete Fluorescence Emission Spectra. The emission spectra of the characterization of the spectral and acid-base behavior, in surderivatives in aqueous micellar solutions and water were recorded factant self-assembly systems, of the lipoidal probes 5-(N-hexaon a microprocessor-controlled Perkin-Elmer LS-5 spectrofluodecanoy1)aminoeosin (HAE) and 5-(N-octadecanoyl)aminorometer with emission and excitation bandwidths set at 2.5 nm. fluorescein (OAF) (see Figure 1). This is a necessary precursor Probe concentrations were typically 5 X lo-' mol dm-3. The to using these probes in air/water monolayer studies. Accordingly, wavelength of excitation was varied (vide infra). The emission the spectral properties (visible absorption and fluorescence spectra of the derivatives in water and aqueous micellar solutions emission) and acid-base behavior of HAE and O A F in wellwere collected a t 25 OC. characterized aqueous micellar systems (simple model interfaces) Optical fibers were employed to transport exciting light to the were investigated. For comparison the spectral properties and surfactant monolayers spread at the air/water interface and to acid-base behavior of eosin-5-iodoacetamide (IAE) and 5-iodotake the monolayer emission back to the Perkin-Elmer LS-5 acetamidofluorescein (IAF), closely related water-soluble anaspectrofluorometer. A more comprehensive description of the logues of H A E and OAF (Figure l ) , in l,Cdioxane/water mixfiber-optic system has been given e l s e ~ h e r e . ~A~ schematic +*~~~~ tures were also studied. Based on the knowledge gained from these diagram of the experimental arrangement is shown in Figure 2. systems, O A F was considered to be the most suitable lipoidal The optical fibers were maintained at a constant distance from indicator to probe the electrostatic characteristics of surfactant the surface (7 mm). A neutral density filter was placed directly monolayers spread at the air/water interface. Preliminary pH under the optical fibers to reduce scatter from the base of the (DPL) titration results for OAF in L-P,y-dipalmitoyl-a-lecithin trough, and the spectra were recorded in the dark to minimize monolayers are reported. stray light. The fluorescence emission spectra were signal averaged over 10 scans to increase the signal to noise ratio. The excitation Experimental Section and emission bandwidths were 15 and 10 nm, respectively. Eosin and Fluorescein Derivatives. 5-(N-Octadecanoyl)Monolayer emission spectra were obtained at ambient temperature. aminofluorescein (OAF), 5-(N-hexadecanoyl)aminoeosin (HAE), A monolayer of 20 mol % OAF in DPL was prepared by mixing 5-iodoacetamidofluorescein (IAF), and eosin-5-iodoacetamide the requisite quantity of 1.6 mmol dm-3 OAF in methanol with (IAE) were obtained from Molecular Probes and were used as that of 1.6 mmol dm-3 DPL in chloroform and spreading 36 pL of the resulting solution at the air/water interface. The subphase contained 0.1 mol dm-3 NaC104. (23) Grieser, F.; Thistlethwaite, P.; Triandos, P. J . Am. Chem. Soc. 1986, 108, 3844. In a typical experiment, the solid Teflon trough (60.00 X 16.50 (24) Prats, M.; Tocanne, J.-F.;Teissie, J. Nature 1986, 322, 756. X 1.5 cm) was thoroughly cleaned and filled with the desired (25) Grieser, F.; Thistlethwaite, P.; Triandos, P. Langmuir 1987, 3, 1173. subphase solution. The surface was swept repeatedly with the (26) Boulu, L. G.;Patterson, L. K.; Chauvet, J. P.; Kozak, J. J. J . Chem. barriers and cleaned by suction. The barriers were placed in the Phys. 1987,86, 503. (27) Bohorquez, M.; Patterson, L. K.; Brault, D. Photochem. Photobiol. middle region of the trough at a separation that would yield an 1987, 46, 953. area per surfactant molecule of 0.70 nm2 (i.e. 15 cm), and a (28) Prats, M.;Tocanne, J.-F.;Teissie, J. Eur. J. Biochem. 1987,162,379. "blank" spectrum of the subphase was recorded. The monolayer (29) Grieser, F.; Thistlethwaite, P.; Urquhart, R.; Patterson, L. K. J. Phys. was then spread between the barriers and left for approximately Chem. 1987, 91, 5286. (30) Grieser, F.; Thistlethwaite, P.; Urquhart, R. Chem. Phys. Lett. 1987, 10 min to allow both the spreading solvent to evaporate and the 141, 108. monolayer to equilibrate. Throughout this 10-min period the (31) Bohorquez, M.; Patterson, L. K. J. Phys. Chem. 1988, 92, 1835. cabinet enclosing the trough was flushed with nitrogen. The total (32) Dick, H. A.; Bolton, J. R.; Picard, G.; Munger, G.; Leblanc, R. M. Langmuir 1988, 4, 133. fluorescence emission spectrum was then recorded and the mon-
bcooH
7466
The Journal of Physical Chemistry, Vol. 93, No. 21, 1989
Kibblewhite et al.
emission from
excitation light from fluorimeter
pH meter
magnetic stirrer bar
magnetic 'stirrer bar
-
7
adjustable feet
9
magietic solid 'steel stirrer block
I
neutral density filter
solid teflon trough
J
maglietic stirrer
ad] stable feet
Figure 2. Schematic representation of the experimental arrangement for the measurement of the fluorescence emission from surfactant monolayers spread at the air/water interface.
olayer fluorescence emission spectrum obtained by subtracting the "blank" spectrum. The pH value of the subphase was then changed, and after the pH equilibration period (vide infra) the process of obtaining a monolayer fluorescence emission spectrum was repeated. This procedure of changing the subphase pH and taking a spectrum was continued until all the requisite monolayer fluorescence emission spectra were acquired. p H Titrations. The pH values were determined by using a combined glass pH electrode and an Orion Research microprocessor ionanalyzer/901 which was calibrated by employing Merck buffer solutions. For the aqueous micellar solutions and the monolayer subphase solutions it was assumed that the pH-meter readings accurately reflected the pH values of the solutions. The method of Van Uitert and H a a was ~ ~ employed ~ to obtain the proton activities from the pH-meter readings in the 1,4-dioxane/water mixtures. A detailed description of this method has been given elsewhere.34 pH Titrations were performed in dioxane/water mixtures that contained 20, 40, 60, and 80 wt % dioxane. The monolayer subphase pH values were measured in situ with the aid of two glass pH electrodes and two pH meters. The pH electrodes were immersed in the subphase and situated at opposite ends of the trough behind the Teflon barriers that enclosed the spread monolayer. Two magnetic stirrer beads, one at each end of the trough, were employed to gently stir the subphase. The experimental arrangement is depicted in Figure 2. The pH equilibration time after the addition of either acid or base to the subphase, Le., the elapsed time before the pH-meter reading at each end of the trough coincided, was typically 20 min. All the acid-base equilibria of the eosin and fluorescein derivatives can be treated as being of the form HA2
H+ + AZ-1
(2) where HA, A, H+, and z represent a conjugate acid form of the derivative, the associated conjugate base form of the derivative, the proton, and the overall charge of the relevant conjugate acid form of the derivative, respectively. The thermodynamic equilibrium constant for equilibrium 2 is given by (3)
where square brackets denote concentrations and Y~ and YHA denote the activity coefficients of the relevant conjugate base and conjugate acid forms of the derivatives, respectively, referred to the particular solvating medium at infinite dilution. Complex organic resonance hybrid ions are involved in the acid-base (33) Van Uitert, L. G.; Haas, C:G. J . Am. Chem. SOC.1953, 75, 541. (34) Drummond, C. J.; Grieser, F.; Healy, T. W. J . Chem. SOC.,Faraday Trans. 1 1989, 85, 537.
equilibria of the fluorescein and eosin derivatives, and it is not easy to see how one can approximate their activity coefficients in the media examined. Therefore the activity coefficient term had to be neglected in the pKa determinations. The pKa values of the eosin and fluorescein derivatives in the various media were determined from UV/vis absorption and fluorescence emission spectra by utilizing the relationship (4) where A is the absorbance/fluorescence emission intensity measured at a select wavelength for the pH value being examined, and A H A and A A are the absorbance/fluorescence emission intensity measured at the same select wavelength when only the conjugate acid and conjugate base species are present, respectively. In general A values relating to at least six different pH values, where there was a mixture of conjugate acid and conjugate base species, were obtained. The select wavelength for the pKa calculations depended upon the particular system under investigation (vide infra). For the pKa determinations based on UV/vis absorption spectra NaOH and HC1 were used to adjust the pH. For the pKa determinations based on fluorescence emission spectra, unless stated otherwise, NaOH and HC104 were used to adjust the pH. The pH titrations were carried out in both directions, from low to high pH and vice versa, to check for any hysteresis effects. In each experiment great care was taken to ensure that the intrinsic ionic strength of the medium was not significantly altered during the pH titration. All the quoted pKa values are the average values obtained from the analysis of the pH titration data. The error margin associated with each pKa value indicates the range of deviation from the average value.
Results and Discussion In aqueous solutions, xanthene dyes that possess the fluorescein skeleton (e.g. IAF, IAE, OAF, and HAE) can exist in many different forms. Depending on the pH of the aqueous solutions, these particular xanthene dyes can occur in dianionic, X2-, monoanionic, HX-, neutral/zwitterionic, H2X, and cationic, H3X+, f o r m ~ . ~ ~ - ~Their O acid-base equilibria can be represented as Ka( 1)
HX- '
Ka(2)
' H2X -
+ H+ HX- + H+ X2-
(5)
(6)
(35) Zanker, V.; Peter, W. Chem. Ber. 1958, 91, 572. (36) Lindquist, L. Ark. Kemi. 1960, 16, 79. (37) Rozwadowski, M. Acta Phys. Pol. 1961, 20, 1005.
(38) Leonhardt, H.; Gordon, L.; Livingston, R. J . Phys. Chem. 1971,75, 245.
The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 1461
Eosin and Fluorescein Derivatives as Probes
TABLE I: Visible Absorption Data for IAF in Water and Dioxane/Water Mixtures and for OAF in Aqueous Micellar Solutions species A,, f 1 nm isosbestic point, f l nm deriv system X2HX-O H2X" H3Xt X2-/HXHX-/H2X H2X/H3Xt H,O 49 1 483 IAF 436.6 48lC 438 46 1 436 463 J IAF 26% Dd 494 487 443ibse483c 442 464 475 -# J 497 IAF 40% Dd 495 443 464 482 -# J 60% Dd 498 IAF 497 444 460 490 -h -8 f 500 80% Dd IAF 501 445 460 -# f 498 OAF 495 447 465 482 C12Es' -k -# -k f DTACl 499 OAF 494 466 -# f DTAWJ 500 448 OAF 496 465 486
OAF
SDSl
496
483
443,b.'" 482c
443
465 438 468 O 1 2 nm. The HX- form also has another absorbance band maximum at about A , - 30 nm. bZwitterionicmodification. cQuinonoid modification. Weight percent of 1,4-dioxanein the 1,4-dioxane/water mixture. CMolarextinction coefficient 0.25 of that found in H20. 'No isosbestic point in the 330-800-nm region. #Lactonic modification; no absorption maximum for this species in the 330-800-nm region. hSpectra for the transition from H2X to H3Xt not obtained (see text for details). '[Surfactant] = 0.01 mol dm-3. '[Surfactant] = 0.05 mol d d . kH3X+species present at immeasurably low pH value (see text for details). '4 mol dm-3 NaBr. '"Molar extinction coefficient 0.68 of that found for IAF in H20.
t
01
00
c361 Wavelength InmJ
Figure 3. UV/visible absorption spectrum of 1.3 X mol IAF in water as a function of pH. The values given in the figure indicate the
pH values at which certain spectra were recorded. where Ka(l), Ka(2), and K,(3) denote the acid-base equilibrium constants for the first, second, and third protonation of the xanthene dyes, respectively. Frequently, both the spectral properties and acid-base behavior of species residing at aqueous interfaces are substantially different from those of the same species in bulk water.1° However, before any significance can be attached to these differences it is necessary to characterize the spectral properties and acid-base behavior of the species in a series of reference solvents and preferably also interfacial microenvironments. I ,4-Dioxane/ Water Mixtures and Micelles. To characterize the influence exerted by the solvating medium on the spectral properties and acid-base behavior of O A F and HAE, IAF and IAE were examined in 1,Cdioxane-water mixtures. In addition, as interfacial regions provide a unique form of solvating microenvironment, OAF and HAE were examined in aqueous micellar solutions. The long hydrocarbon chain substituents ensure that OAF and HAE are situated in micelles. The hydrophilic nature of the xanthene moieties should result in them being located, on average, at the micelle/water interface. To date, aqueous micellar interfaces are the best characterized surfactant/water interfaces.1° ( a ) Visible Absorption Spectra. (i) IAFIOAF. Figures 3 and 4 show the effect that changing the pH can have on the UV/visible absorption spectra of IAF and OAF. Table I summarizes the spectral data obtained for IAF and OAF in all the systems studied. The acid-base equilibria scheme for fluorescein, originally proposed by Zanker and Peter35and subsequently confirmed by many other researchers,3640 can be adopted to explain the visible absorption data for IAF and OAF in the various systems examined. Figure 5 depicts the species considered to participate in the acid-base equilibria associated with IAF and OAF.
M
5x
100
WAVELEffiTH
550
W
inml
Figure 4. UV/visible absorption spectrum of 4 X IO" mol OAF in an aqueous 0.01 mol d m 3 CI2E8solution as a function of pH. The values given in the figure indicate the pH values at which certain spectra
were recorded. 0
-0
. H'
;l
cH*
0
HO
-H'Il+H'
HO
OH
Figure 5. Species considered to be involved in the acid-base equilibria of the fluorescein derivatives (R = NHCOCH21 for IAF and R =
NHCO(CH,),,CH, for OAF). (39) Martin, M. M.; Lindquist, L. J . Lumin. 1975, 10, 381. (40) Fompeydie, D.; Onur, F.; Levillain, P. Bull. SOC.Chim.Fr. 1979, 9-10, 1-375.
From Table I it is evident that the different species undergo a small bathochromic shift in their long wavelength UV/visible
7468 The Journal of Physical Chemistry, Vol. 93, No. 21, 1989
Kibblewhite et al. ' 0 9 3 497 Oianion Monoanion
19n
02t
0O L
I
L50
500 h'AVELENGTH I n n 1
550
Mi0
Figure 6. UV/visible absorption spectrum of 1 X mol d m 3 IAE in water as a function of pH. The values given in the figure indicate the pH values at which certain spectra were recorded.
absorption band maxima as the polarity of the l,Cdioxane/water mixtures is progressively decreased. For all the species, this can be interpreted in terms of the charge distribution in the electronic ground state being slightly less delocalized than in the electronic excited state. As well, the shift can be interpreted in terms of the ground state being more stabilized by solventsolute interaction (hydrogen bond formation) than the excited ~ t a t e . ~ ' .The ~~ relatively weak solvatochromism of IAF, and by inference OAF, coupled with the fact that solvent polarity is not the only factor values, means that the A,, values that may influence the A, cannot be employed to gauge interfacial solvent properties. In all the solvating media investigated the lactonic modification of the H2X form predominates. The lactonic modification does not absorb in the visible region. In some of the solvating media the quinonoid and zwitterionic modifications are also present (Table I). Since conjugation does not extend between the xanthene and phenyl parts of the fluorescein sekelton, the quinonoid modification is expected to have a spectrum similar to that of monoanionic form and the zwitterionic modification is expected to have a spectrum similar to that of the cationic form. The spectrum of IAF in water (Figure 3) exemplifies the presence of the quinonoid and zwitterionic modifications of the H2X form. The spectrum of OAF in aqueous micellar C12Essolution (Figure 4) is devoid of the quinonoid and zwitterionic modifications of the H2X form. Interestingly, the quinonoid and zwitterionic modifications of the H2X form are found only in water, aqueous micellar SDS solution, and the 20 wt % 1,4-dioxane/water mixture. This suggests that the SDS interfacial microenvironment is much more aqueous in nature than the C12E8,DTAC, and DTAB interfacial microenvironments. This finding is consistent with other evidence which indicates that the micelle/water interfacial region of SDS micelles may be inherently more aqueous in nature than that of CI2E8,DTAC, and DTAB micelle^.^*'^^^^ (ii) IAEIHAE. Figures 6 and 7 illustrate the effect that varying the pH can have on the UV/visible absorption spectra of IAE and HAE. The spectral data for IAE in the 1,4-dioxane/water mixtures and HAE in the aqueous micellar solutions are given in Table 11. Clearly, the spectral response of IAE and HAE to pH changes (Figures 6 and 7) is dissimilar to that of IAF and O A F (Figures 3 and 4). This can be attributed to the eosin derivatives having a different mode of protonation from that of the fluorescein derivatives (vide infra).@ The surmised sequence of protonation for IAE and HAE in aqueous solutions is shown (41) Martin, M. Chem. Phys. Len. 1975, 35, 105. Fleming, G. R.; Knight, A. W. E.; Morris, J. M.; Morrison, R. J. S.; Robinson, G.W. J. Am. Chem. SOC.1977, 99, 4306. (43) Drummond, C. J.; Grieser, F.; Healy, ?. W. J . Chem. Soc., Faraday Trons. I 1989, 85, 561. (42)
Figure 7. UV/visible absorption spectrum of 2 X 10" mol dm-3 HAE in an aqueous 0.01 mol dme3CI2E8solution as a function of pH. The values given in the figure indicate the pH values at which certain spectra were recorded. A,, for the X2' species is at 526 nm. As the pH is decreased toward the value where the HX- species (A, = 535 nm) predominates, the absorbance at A, for the X2- species decreases. The X2-/HX- isosbestic point is at 531 nm. As the pH is further decreased the absorbance at A,, for the HX- species approaches zero. TABLE II: Visible Absorption Data for IAE in Water and Dioxane/Water Mixtures and for HAE in Aqueous Micellar Solutions
deriv JAE IAE IAE IAE
IAE HAE HAE HAE HAE HAE HAE
svstem H20 20% Db 40% Db 60%Db 80% Db C12EsC DTACd DTAB"' SDSd
CIZEB/SDS'K C12E8/SDsh
'pecies f l nm X2HX-
519 526 530
517 522 522 523
531
526 526
534 535
524 526 525 525 526
533
538 525 533 531
isobestic point, k1 nm
X2-/HX-a
525
526 528 530 531 528 532 -a
530 530
"No obvious isosbestic point in the 330-800-nm region. Weight percent of 1,4-dioxane in the 1,4-dioxane/water mixture. [Surfactant] = 0.01 mol dm-'. d[Surfactant] = 0.05 mol dm-'. c 4 mol dm-3 NaBr. fTotal [surfactant] = 0.05 mol dm3. g 1 : l molar mixture. 1:4 molar mixture.
in Figure 8. The third protonation of the eosin skeleton occurs only in very acidic media (vide infra), and in the pH range investigated (0-14) the H3X+ form of the eosin derivatives was not observed. The trend is for the absorbance maximum of the different IAE species to undergo a small bathochromic shift as the polarity of the dioxane/water mixtures is progressively decreased. This behavior is analogous to that of IAF in dioxane/water mixtures. Hence, analogous reasons can be given to account for the IAE A,, shifts. The H2X form appears to exist almost exclusively in the lactonic modification in all the media examined. On the basis of the fact that the xanthene and phenyl parts of the eosin derivatives are not conjugated, one would have predicted a priori that the spectra of the X2- and HX- forms of IAE, and HAE, would be very similar. This is the case only when water and SDS micelles are the solvating medium (Table I1 and compare Figures 6 and 7). The most likely explanation for this Occurrence is that in the less "aqueous-like" media the xanthene chromophore's specific solute-solvent interactions are highly dependent on whether or not the benzoate group is protonated. (b)pK, Datafrom the Visible Absorption Spectra. Information about interfacial solvent characteristics can sometimes be acquired by comparing the intrinsic interfacial pK, values of prototropic
Eosin and Fluorescein Derivatives as Probes Er
The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 7469 4
Br
-0
0 Br
Er
coo. H'
11
+H+
HO Er
-H'l!,
+H*
0
10
20
30 Dielectric
HO
50
40
60
70
80
Constant
Figure 9. ApKam(0)and ApK,' (0)values for IAF in 1,4-dioxane/water mixtures as a function of the dielectric constants of the mixtures. The data are joined by lines of best fit. The lines do not represent any kind of theoretical fit.
Er
R
Figure 8. Species surmised to be involved in the acid-base equilibria of
the eosin derivatives (R = NHCOCHJ for IAE and R = NHCO(CH2)&H3 for HAE).
Br H
O
\
W
0
/
H
Br
O
!r W
/
0
Br
TABLE 111: pK, Data for IAF in Water and for OAF in Aqueous Micellar Solutions Obtained from the Visible Absorption Spectra deriv system pK.Ob(l) pKSob(2) pKaob(3) 6.89 f 0.03" 4.05 f 0.08b 2.08 f 0.03c H20 IAF 0.70 f 0.10 OAF CIZEsd 8.04 f 0.07 6.34 f 0.05 5.78 f 0.05 3.71 f 0.05 CO.5' OAF DTAC' 7.47 f 0.07 5.66 f 0.08 OAF DTAB'J 0.82 f 0.06 OAF SDS' 8.84 f 0.02 5.97 0.03 3.98 f 0.06 " P K , ~ ( I ) .bpK,w(2). cpK,w(3). [Surfactant] = 0.01 mol dm-3. '[Surfactant] = 0.05 mol dm". /See text for details. 84 mol dm-3 NaBr.
moieties (Le., pK2 values) with their pK, values in organic solvent/water mixtures.1,io~13,34,43~ This is conditional upon (i) the apparent interfacial pK, values being unperturbed by specific molecular interactions and/or interfacial "salt effects" and (ii) the organic solvent/water pK, values being unperturbed by specific solute-solvent interactions. It is assumed that pK2 values can be directly compared with pK; values here^,^^-^^,^^,^^^^ PK,' = pKam- log ,YH+ (8) and pKamis an organic solvent/water pK, value and ,yH+ denotes the primary medium effect on the proton. In practice, it is assumed that the mean primary medium effect on HC1, ,y*, approximates ,yH+. In the present study the ,y* values given in ref 44 have been used. Figure 9 displays the ApKam(Le., pKam- pKaW)and ApK,' (Le., pKai ;pKaW)values for IAF in dioxane/water mixtures as a function of the dielectric constants of the mixtures. Table I11 contains the pKaWvalues. Fluorescein pKaw(I), pKaw(2),and pKaw(3)values of 6.7,4.4, and 2.2, respectively, have been reported e l ~ e w h e r e . ~ ~IfJ one * keeps in mind the minor structural differences between IAF and fluorescein, the IAF pKaWvalues (Table 111) are consistent with the literature values. Table I11 also (44) Drummond, C. J.; Grieser, F.; Healy, T. W. J . Chem. SOC.,Faraday Trans. I 1989, 85, 521. (45) Drummond, C . J.; Grieser, F.; Healy, T. W. J . Chem. SOC.,Faraday Trans. I 1989, 85, 551. (46) Drummond, C . J.; Warr, G. G.; Grieser, F.; Ninham, B. W.; Evans, D. F. J . Phys. Chem. 1985, 89, 2103.
(PR) (BPB) Figure 10. Molecular structures of the quinonoid form of phenol red
(PR) and bromophenol blue (BPB). contains the OAF pKaobSvalues obtained in the aqueous micellar solutions. For each system investigated, the IAF or OAF pK,(l) value was determined by analyzing the absorbance values at the X2A, value. The spectrum of the HX- form was assumed to be the last spectrum to pass through the X2-/HX- isosbestic point when the pH was lowered by small increments. The pK,(2) and pK,(3) values were determined by analyzing the absorbance values at the XZ-/HX- isosbestic point and the H3X+ A, value, respectively. Accurate pKam(1) and pKam(3)values could not be obtained for IAF in the 80 wt % dioxane/water mixture. In the micellar DTAC solution the pKaobS(3)value was shifted to an immeasurably low value ( < O S ) . It has not always been recognized that (i) the X2- and HXforms of fluorescein derivatives have similar,,A values and that (ii) the concomitant decrease in the absorbance value at the X2& value as the pH is decreased must be ascribed to the sequential protonation of two distinct types of acid-base groups. This oversight has led to erroneous pK, determination^.^' To our knowledge, there has been no previous report of a detailed study of the acid-base behavior of fluorescein or a closely related derivative in organic solvent/water mixtures. Therefore, we note that the ApKamdata for IAF (Figure 9) provide support for the acid-base equilibria scheme depicted in Figure 5 . As one would expect, the IAF ApKam(1) behavior in dioxane/water mixtures closely corresponds to the ApKam behavior of the structurally similar phenol red molecule (Figure lo)." Moreover, despite the added complication introduced by the lactonic modification, the IAF pKaw(2)value is in accord with that expected (47) Thelen, M.; Petrone, G.; O'Shea, P. S . ; A n i , A. Eiochim. Eiophys. Acta 1984, 766, 161.
7470 The Journal of Physical Chemistry, Vol. 93, No. 21, 1989 11
TABLE V: p K 2 Data for OAF and HAE in Aqueous Micellar Solutions deriv system q ,mV pK;(l) pK,0(2) p&"(3) OAF Cl2Es 0 8.04 6.34 0.70
10
9 I
OAF OAF OAF OAF HAE HAE HAE HAE HAE
= . a Y
a
Kibblewhite et al.
7
E 5
DTAC DTAB" SDS SDS C12E8 DTAC DTAB" SDS SDS
+129
7.96
+18
7.77 6.47
-140
5.89 5.96 3.60 4.03 2.98 2.98 2.62 2.26
-115
2.69
-140 -1 15
0
+129 +18
6.90 5.96 5.05 4.83
