- Email: [email protected]
Limitations arising in the study of the fluorescence quenching of rhodamine 6G by iodides using cw-laser thermal lens spectrometry
Spectrochimica Acta Part A 54 (1998) 101 – 110
Limitations arising in the study of the fluorescence quenching of rhodamine 6G by iodides using cw-laser thermal lens spectrometry Marc Fischer, Joseph Georges * Laboratoire des Sciences Analytiques, UMR 5619, Uni6ersite´ Claude Bernard-Lyon 1, Baˆtiment 308, 43 Boule6arde du 11No6embre 1918, 69622 Villeurbanne Cedex, France Received 31 May 1997; accepted 15 July 1997
Abstract Fluorescence quenching of rhodamine 6G by iodides has been carried out in ethanol and aqueous micellar solutions using cw-laser thermal lens spectrometry. Comparison of the photothermal results with those obtained from conventional fluorescence has shown some limitations of the photothermal method. Depending on the medium and the quencher concentration, the quenching level calculated from the photothermal data is higher than that derived from fluorescence. In some cases, the thermal lens signal is even higher than that expected for a nonfluorescent compound. A detailed study of the thermal lens behaviour has shown a converging effect on the probe beam arising simultaneously with the normal diverging effect of the thermal lens. The time scale of this abnormal signal is longer than that generated by thermal relaxation of the excited states and could originate from an endothermic (photo)chemical reaction subsequent to the quenching reaction. © 1998 Elsevier Science B.V. Keywords: Rhodamine 6G; Quenching by iodides; Thermal lens spectrometry; Anomalous signal
1. Introduction Rhodamine 6G (R 6G) is commonly used as a fluorescent standard and laser dye. This is because it is photochemically stable, has a high fluorescence quantum yield and has an insignificant triplet-state population [1]. In ethanol, R 6G dissolves completely into monomers and its fluorescence quantum yield is independent of the * Corresponding author. Tel.: + 33 78949510; fax: + 33 472431078; e-mail: [email protected]
concentration [2,3]. In contrast, the dye tends to aggregate in aqueous solutions because of hydrophobic interactions between the alkyl substituents, which results in an significant decrease in the fluorescence efficiency of the dye in concentrated solutions [4,5]. Quenching can also originate from an external quencher and the knowledge of the efficiency of any quenching process is of theoretical and practical interest. Two kinds of techniques, based on the complementary nature of excited-state relaxation through radiative and nonradiative processes, can
1386-1425/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 6 - 1 4 2 5 ( 9 7 ) 0 0 2 0 5 - 9
102
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
be used. Usually, fluorescence quantum yields are determined by relative measurements with respect to a standard for which the absolute fluorescence quantum yield must be known. Similarly, fluorescence quenching experiments are carried out by recording the decrease in fluorescence intensity as the concentration of the quencher is increased. The second kind of technique is based on the measurement of the heat yield of the sample, i.e. the ratio of the power converted to heat over the power absorbed. The heat yield of the fluorescent compound can be obtained by comparing the photothermal signal of the fluorescent sample with that of a reference absorber under the same experimental conditions [6 – 8]. The reference absorber is a compound of known absorbance and heat yield. Then, the fluorescence quantum yield is determined from the knowledge of the heat yield for the fluorescent compound. The method is absolute only if some care is taken in order to avoid errors which may arise when the sample signal is compared with the reference signal [9]. Another method, based on fluorescence quenching, has been proposed to eliminate the need of a reference absorber, by using fluorescence quenching [10–13]. The photothermal signal of the fluorescent sample is compared with that obtained with the same compound, the fluorescence of which has been quenched with a quencher. The method is expected to be more accurate provided that the absorption features of the sample and the thermo-optical properties of the medium are not changed upon addition of the quencher. This study results from an attempt to determine the fluorescence quantum yield of R 6G using thermal lens spectrometry and complete fluorescence quenching. The decrease in fluorescence efficiency upon addition of the quencher should result in a concomitant increase in the thermal power released by the sample. When the fluorescence quantum yield is high, the photothermal signal is expected to vary more rapidly than fluorescence does [5,14]. However, preliminary results have shown that complete quenching of R 6G by iodide could lead to erroneous values of the fluorescence quantum yield. The aim of the present work was to investigate the quenching of R 6G as a function of the quencher concentration
using thermal lens spectrometry. The thermal lens experiments were carried out in various media including ethanol, water and aqueous micellar solutions and the results were compared with those obtained from conventional fluorescence measurements.
2. Experimental
2.1. Instrumentation The dual-beam photothermal experimental setup consisted of an air-cooled argon–ion laser as the excitation laser and a helium–neon laser as the probe laser. The excitation laser was operated at 488 nm with the power adjusted to :4 mW at the sample cell in order to prevent optical saturation. Also, a low excitation power allowed the formation of weak thermal lenses, ensuring a linear response of the thermal lens signal with respect to the amount of heat released. The excitation beam was modulated at 15 Hz by an optical chopper with symmetrical or dissymmetrical slot blades depending on the solvent [15]. Both beams were focused independently by two lenses (f= 80 mm) in order to operate in a mode-mismatched configuration. The thermal lens signal was detected through a 1 mm pinhole with a PIN silicon photodiode. The photodiode output, converted to a voltage, was fed into a lock-in amplifier. For the time-resolved experiments, the signal from the photodiode was processed with a numerical memory (Metrix, VK12-2). Steady-state fluorescence measurements were obtained with a spectrofluorometer (Jobin-Yvon, JY3) working with a spectral bandpass of 2 nm for both excitation and emission. A 1.5 mm cell was used in order to minimize inner-filter effects. Absorbances were measured with a conventional spectrophotometer using 0.1 and 1 cm pathlength cells depending on the concentration.
2.2. Reagents Solutions of R 6G were prepared from the rhodamine chloride (Exciton) in ethanol, distilled water or micellar solutions. Micellar solutions
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
were prepared by dissolving either sodium dodecylsulfate (SDS), cetyl N,N,N-trimethylammonium bromide (CTABr) or polyoxyethylene 23 lauryl ether (Brij 35) in distilled water. Surfactant concentrations are indicated in weight percent (w/v). The pH of aqueous and micellar solutions were set at :4.8 with 10 − 2 M acetate buffer in order to prevent any pH variation which would result in a change of the absorption spectra. The reference absorbers used for the determination of the fluorescence quantum yield of R 6G were m-cresol purple in its acidic form (HCl, 10 − 3 M) and the basic form of phenol red in ethanol and in the aqueous solution, respectively [9]. All experiments were performed at room temperature (22 – 23°C) except those in CTABr micelles for which the solutions were slightly heated in order to work above the Krafft solubility boundary.
2.3. Procedure The basic relationship which describes the effect of a quencher Q on the steady-state fluorescence of a sample is given by the Stern-Volmer equation [16]
verted into F0/F from the knowledge of F0. Absolute fluorescence quantum yields were previously calculated from photothermal experiments according to the following equation [6–8]:
Ff = 1−
A rS l( f , AS r la
(1)
where F0 and F are the fluorescence quantum yields in the absence and in the presence of the quencher, respectively, Ksv (l mol − 1) is the SternVolmer quenching constant, defined as the quenching rate constant, kq (l mol − 1 s − 1), multiplied by the intrinsic lifetime of the fluorophore, and [Q] is the quencher concentration. If the fluorescence spectrum of the fluorophore is not altered by the quencher, then the fluorescence intensity is directly proportional to F. In order to compare fluorescence and thermal lens experiments, both kinds of measurements were expressed as F0/F vs. the quencher concentration. The fluorescence intensity (F) was measured at 600 nm, i.e. outside the absorption spectrum of the fluorophore, in order to avoid reabsorption effects, and the ratio F0/F was con-
(2)
where l( f is the mean fluorescence wavelength determined from the emission spectrum, la is the absorption wavelength, S is the thermal lens signal of the sample with absorbance A, and S r the thermal lens signal of the reference absorber with absorbance A r. The F0 values are 0.95, 0.89, 0.91, 0.90 and 0.81 in ethanol, water, 2% SDS, 5% Brij 35 and 2% CTABr, respectively [5]. The photothermal data were also expressed as F0/F according to the following calculation. The photothermal signal is proportional to the amount of heat released, Pth. In the absence of quencher, Pth(0) can be defined as: Pth(0) = (1− 10 − A) Pexc (1−F0x),
F0 = 1+Ks6 [Q], F
103
(3)
where Pexc is the excitation power and x= n¯ f / na accounts for the heat generated by the Stokes shift. In the presence of quenching, the same relation applies, defining a series of values for Pth and F. The thermal lens signals S0 and S, measured in the absence and in the presence of quencher, respectively, are therefore related by the following equation:
S0 1−F0x . = 1−Fx S
(4)
From the experimental values of S0/S and with the knowledge of F0, one can calculate F as a function of the quencher concentration. Conversely, the photothermal data can be expressed as a heat yield, Qth, defined as the ratio of thermal energy to energy absorbed:
104
Qth =
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Pth =1 − Fx. (1−10 − A) Pexc
(5)
When quenching of R 6G is complete, or more generally for a non-fluorescent compound, Qth = 1. The quenching experiments were performed as follows. A series of samples were prepared from the same diluted rhodamine solution. In each of them, graduated amounts of the quencher (NaI) were added, either by weighing small amounts of the salt or by adding small aliquots of a concentrated solution in order to minimise dilution effects. The addition of NaI had no effect on the absorption spectrum of the dye in ethanol and in the Brij 35, SDS or CTABr micellar solutions. However, in the Brij 35 micellar solution, absorbance at 488 nm was found to increase slightly upon the addition of NaI. This is because, in this solution, iodide has some tendency to form iodine which absorbs light at the excitation wavelength. On the contrary, in water, the absorption spectrum of R 6G was significantly altered upon the addition of NaI. This effect originates from the formation of nonluminescing complexes of the monoprotonated form of the dye with ionic halides, which results in a decrease of the absorbance at 488 nm and in static quenching of the dye solution [17,18]. Therefore, before each set of fluorescence and photothermal experiments, absorbances were measured in order to account for these variations in the experimental data. In water, the correction factor was applied with respect to the actual amount of energy absorbed by the solution. In the Brij 35 micellar solution, the correction factor also included the attenuation of the incident light due to the presence of small amounts of iodine absorbing at the working wavelength. In addition, the photothermal data were corrected for the amount of thermal energy released by iodine according to the relationship previously defined for a fluorescent compound in the presence of a concomitant [5]. Electrolytes have also been found to change the thermo-optical properties of solutions, leading to an enhancement or a decrease of the sensitivity of the thermal lens method depending on the solvent
[15,19]. For every solution studied, this effect has been measured and a correction factor was applied to the experimental photothermal data.
3. Results and discussion
3.1. Fluorescence data For a homogeneously fluorescing solution, the Stern-Volmer relation predicts a linear plot of F0/F vs. [Q]. The dynamic quenching process is diffusion controlled and requires collisional interactions between the excited fluorophore and the quencher [16]. When the Stern-Volmer relation is strictly obeyed, plotting F0/F should give a straight line with an intercept at unity and a slope equal to KSV which is a measure of the fluorophore’s sensitivity to the quencher. As shown in Figs. 1–5, quenching of R 6G by iodides depends greatly on the medium. In water, the Stern-Volmer plot is linear while deviations from the Stern-Volmer relation are observed in the other solutions studied when the iodide concentration increases. However, the first part of all the plots is linear and goes through unity, allowing the determination of KSV (Table 1). The sensi-
Fig. 1. Plots of F0/F for the quenching of R 6G by I − in ethanol using ( ) fluroescence and () photothermal data.
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Fig. 2. Plots of F0/F for the quenching of R 6G by I − in water using ( ) fluroescence and () photothermal data.
105
Fig. 4. Plots of F0/F for the quenching of R 6G by I − in water + 2% CTABr using ( ) fluroescence and () photothermal data.
tivity of R 6G to iodides is about twice as great in ethanol than in water. In the micellar solutions, it is observed that micelles have a different effect on the quenching efficiency of iodides depending on the surfactant charge. Iodides are more effective in micelles of opposite charge (CTABr) than in micelles of the same charge (SDS). In cationic CTABr micelles, the Stern-Volmer constant is about three times greater than in water, while in anionic SDS micelles the KSV value is only 0.7
M − 1, a factor of : 60-fold lower than in water. In a previous study, we have shown that the monoprotonated form of R 6G associates more or less with any kind of micelles thanks to the combination of hydrophobic and electrostatic interactions [5]. The increase in the extent of quenching of R 6G when the quencher is associated to the cationic micelles (CTABr) and, on the contrary, the high protection afforded by SDS, due to
Fig. 3. Plots of F0/F for the quenching of R 6G by I − in water+ 5% Brij 35 using ( ) fluroescence and () photothermal data.
Fig. 5. Plots of F0/F for the quenching of R 6G by I − in water + 2% SDS using ( ) fluroescence and () photothermal data.
106
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Table 1 Stern-Volmer constants for the quenching of rhodamine 6G by iodides in ethanol and in aqueous solutions Solvent
KSV (l mol−1)
EtOH
Water
5% Brij 35
2% CTABr
2% SDS
108
44
46
131
0.7
electrical repulsions between iodides and anionic micelles, reflect the importance of ions in the micellar electrical double layer [20]. In CTABr micelles, there is a competition between Br − and I − for counterion sites upon the addition of iodides. This results in a high local concentration of iodide ions which are much more efficient quenchers than bromides and in a ‘catalysis’ of the quenching reaction [21]. In the nonionic micellar solution (Brij 35), the Stern-Volmer constant determined at low quencher concentrations is of the same order as that determined in water, indicating that nonionic micelles have little effect on the quenching reaction. However, in all solutions except water, upward or downward curvings of the F0/F plots are rapidly observed, indicating a loss of the SternVolmer linearity. Similar plots have been found for many homogeneous liquid-phase systems [22]. Upward curving is generally accounted for by the presence of an additional quenching mechanism, usually referred to as static quenching. In addition to the possible formation of a nonfluorescent ground-state complex between the chromophore and the quencher, static quenching refers also to a very fast quenching reaction between a chromophore and a quencher, in close proximity, at the instant that the former becomes excited. This process can be observed in viscous solvents where the fluorophore, located within a spherical volume surrounding the quencher, is quenched instantaneously without collisional interactions. In contrast, downward curving of the Stern-Volmer plot can arise when the quenching efficiency (g) is less than 1. In this case, every encounter between the quencher and the excited fluorophore does not result in deactivation of the excited state and the quenching constant is a function of [Q].
In compartmentalised or microheterogeneous systems, such as micellar solutions, dynamic and/ or static quenching mechanisms have been proposed to explain the shape of the Stern-Volmer plots. The characteristics of these plots are also affected by the nature of the distribution of fluorophore and quencher species between the aqueous phase and micelles [22–25]. Especially, three kinds of associations have been considered for the quencher: partition, binding and the combination of both partition and binding [23]. In the case of partition, the distribution of the quencher is determined only by a partition equilibrium and the ratio of the quencher concentrations between the aqueous phase and micelles is constant. In the case of binding, where an ionic quencher can be bound by micelles with head-groups of opposite charge, the process implies that the interaction is saturable to a limited number of binding sites. As suggested by Blatt et al. [23], binding of I − to cationic CTABr micelles can enhance static quenching. In SDS solutions, upward curving of the Stern-Volmer plot can originate from the high quencher concentration necessary to decrease significantly the fluorescence. Such an increase of the quenching rate constant upon the addition of salts has been observed for nitrite quenching of Tb3 + bound to SDS micelles [26]. Upon the addition of Na + , the surface potential of SDS micelles is reduced and the repulsion between the anionic head-groups is partially shielded by the counterions. This change in surfactant–surfactant interaction makes the aggregation number and the micelle size increase [27]. Such changes in micelle size and structure would alter the interactions between the fluorophore and micelles as well as the distribution of iodide around the micelle surface.
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Therefore, the Stern-Volmer plots obtained in micellar solutions result from the combination of different kinds of quencher association processes and quenching mechanisms. In addition, it is likely that R 6G is also distributed and quenching can occur both in micelles and in the aqueous phase [20]. Understanding the processes involved in each medium studied would require computer simulations of the Stern-Volmer plots and is outside the scope of this paper.
3.2. Photothermal data Quenching experiments have also been carried out using thermal lens spectrometry. As suggested by previous works [5,14], recording the variation of thermal energy associated to a variation of fluorescence can be more sensitive, especially when the fluorescence quantum yield is high. Fig. 6 compares the relative variations of fluorescence, F, and of the photothermal signal, S, for quenching of R 6G by iodides in ethanol. As expected, S/S0 and F/F0 vary in the opposite direction, but the photothermal signal increases more rapidly than fluorescence decreases. In order to compare both kinds of experiments, the photothermal data were arranged as described in Section 2 and the results are combined in Figs.
Fig. 6. Quenching of R 6G by iodide in ethanol: relative () thermal lens signal and ( ) fluroescence intensity vs. quencher concentration.
107
Table 2 Values of the heat yield, Qth, for the quenching of rhodamine 6G by iodides in ethanol and in the Brij 35 micellar solution NaI (M)
Ff
Qth (fluo)
Qth (tls)
Ethanol 0 0.01 0.04 0.105
0.94 0.53 0.26 0.14
0.185 0.54 0.77 0.88
0.185 0.59 0.99 1.27
Water+5% Brij 35 0 0.18 0.32 0.61 0.98
0.90 0.13 0.08 0.05 0.04
0.23 0.89 0.93 0.96 0.97
0.23 0.93 0.99 1.08 1.10
(fluo), Values expected from fluorescence measurements; (tls), values derived from the thermal lens data
1–5 with those obtained by fluorescence. It can be seen that, in every medium studied except in water, the photothermal results are consistent with those obtained by fluorescence only over a limited range of quencher concentrations. Otherwise, the F0/F ratios obtained by thermal lens spectrometry deviate positively with respect to the fluorescence plots. These results are very surprising because they would mean that the amount of thermal energy produced by the quenching reaction is greater than the associate loss of radiative energy. The discrepancy between both experiments can be illustrated by comparing theoretical and experimental values of the heat yield. Calculations were made for high quenching levels in ethanol and the Brij 35 (Table 2). As previously noticed, Qth should maximise at unity for a non-fluorescent compound. However, it can be seen that the experimental values are greater than the expected ones and can be greater than unity, even when fluorescence quenching is not complete. Not only are these deviations detrimental when the photothermal method is used to investigate fluorescence quenching reactions, but also they are an important drawback for the determination of absolute fluorescence quantum yields using the completely quenched fluorophore as internal reference [10–12].
108
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Some experiments previously made with the same fluorophore but using pulsed-laser excitation did not show such abnormal results [28]. This suggested to us, that the difference could originate from the operating mode of the excitation laser and the time scale for the thermal lens to form. In cw-laser experiments, excitation occurs over a rather long time, typically in the order of ms or s, depending on whether the signal is sampled at the steady-state or before. In pulsed-laser experiments, sample irradiation occurs within a very short time ( : 10 ns) and the lens forms almost instantaneously (:ms). Therefore, the pulsedlaser method would be less sensitive to the presence of slow chemical and/or physical processes interfering with the formation of the thermal lens. Further evidence of such processes occurring in cw-laser thermal lens spectrometry came from the observation of abnormal signals when doing timeresolved experiments. These experiments were carried out with R 6G quenched by iodide in SDS micellar solutions, where the effects observed are the most typical. Three kinds of recordings were made using either a chopper or a mechanical shutter: the time dependence of the demodulated signal at the output of the lock-in amplifier, the time-profile of the ac signal during the first chopper cycles after the beginning of irradiation, and the build-up and relaxation of the thermal lens to steady-state. When the excitation laser is modulated with an optical chopper, the ac signal produced by the periodical build-up and relaxation of the thermal lens is converted into a dc signal through a lock-in amplifier. Since the formation of the thermal lens is of the order of the thermal time constant tc ( : ms), the signal risetime is limited by the time constant of either the lock-in amplifier or the recorder. Generally, the time required to get a steady-state value of the signal is some seconds after the beginning of irradiation (Fig. 7(a)). However, in most cases, when rhodamine is quenched by the addition of significant amounts of iodides, the time-dependent dc signal can be divided into two components that correspond to different time constants (Fig. 7(b)): a fast rising part, which is the typical signal induced by the thermal lens effect and a slower component with a
time constant of the order of minutes. Even though the signal was sampled at the top of the fast rising part, thus ignoring the slow component, the experimental values were generally too great. However, the demodulated signal does not account for the sign of the thermal lens formed and the time profile of the signal during the relaxation and the formation of the thermal lens. Fig. 8 shows the time dependence of the probe beam intensity during the first chopping cycles from the beginning of irradiation. When the chopper opens at the first cycle, the sample is irradiated and the probe beam intensity decreases as the thermal lens forms. At the end of the first ‘on’ period, when the excitation beam is blocked, the probe beam intensity not only increases due to the relaxation of the thermal lens, but also becomes greater than its initial value before the beginning of irradiation. Then, the probe beam intensity at the end of each successive relaxation period gradually increases until a steady-state. The ac component of the signal behaves as usually observed for the formation of a diverging lens, but its amplitude increases as the relative probe beam intensity increases. The slow rising component in Fig. 7 could originate from this effect and could explain
Fig. 7. Time-dependence of the demodulated signal for 2.5 × 10 − 6 M R 6G in 2% SDS micellar solution (a) in the absence of NaI and (b) in the presence of 0.22 M NaI; chopping frequency =15 Hz.
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Fig. 8. Time-profile of the ac signal during the first chopping cycles for R 6G quenched by iodides in 2% SDS micellar solution; [R 6G]=10 − 5 M; [NaI]=0.25 M; solution rested for some minutes before irradiation; chopping frequency= 15 Hz.
the errors in the measurement of the ‘true’ thermal lens signal. The third recording shows the build-up of the signal over a longer irradiation time (:10 s) and its subsequent relaxation (Fig. 9). When the excitation laser is turned on, using a mechanical
Fig. 9. Time-resolved build-up and relaxation of the thermal lens signal for 2.5 ×10 − 6 M R 6G quenched by 0.22 M NaI in 2% SDS micellar solution (a) immediately after refilling the sample cell and (b) after the solution was let to rest for 10 min.
109
shutter, the probe beam intensity decreases, indicating the formation of a diverging lens. However, after a very short time ( B 1 s), the probe beam intensity increases quickly while the beam is seen to converge. Then, the probe beam intensity increases slowly, but remains greater than the initial value before the beginning of irradiation. When the excitation laser is turned off, the probe beam intensity rapidly increases, indicating the relaxation of the diverging thermal lens, and then decays more slowly to its initial value. The signals in Figs. 8 and 9 indicate that the ‘true’ thermal lens signal created by the temperature-induced refractive index gradient, dn/dT, is mixed with another signal of opposite sign and resulting in a convergent behaviour of the probe beam. This anomalous signal was independent of the surfactant concentration and was observed even below the critical micelle concentration. However, as shown in Fig. 9, the convergent behaviour of the probe beam depended strongly on the time during which the sample was allowed to rest in the cell before the beginning of the experiment. This observation, along with the time constant of the anomalous phenomenon, suggests the formation of another refractive index gradient which is the reverse to that produced by the temperature gradient and would be governed by mass diffusion. Such an inversion of the refractive index gradient has been observed by Roach and Snook in the study of uranyl ions in the presence of acetic acid [29]. A possible mechanism for this phenomenon was based on energy transfer between the uranyl ion and acetic acid and subsequent photodecarboxylation of acetic acid. The process would be endothermic and would remove heat from the beam centre and hence weaken or even reverse the thermal lens. The anomalous thermal lens signal exhibited in our quenching experiments probably explains the disagreement observed between fluorescence and photothermal data. The chemical or physical origin of this effect has not been elucidated and cannot be solved easily with some further experiments. The time constant of the abnormal signal is quite greater than the time constant of the thermal lens effect. Moreover, preliminary studies have shown that the observed effect depends on a lot of parameters
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
110
including the solvent, the concentration of the reagents, convection effects into the solution and the excitation mode (cw, modulated, or pulsed). A detailed study would probably require the use of other techniques in addition to thermal lens spectrometry.
References [1] K.H. Drexhage, Structure and properties of laser dyes, in: F.P. Scha¨fer (Ed.), Dye Lasers, Topics in Applied Physics, Springer, Berlin, 1973, chapter 4. [2] A. Penzkofer, Y. Lu, Chem. Phys. 103 (1986) 399. [3] M. Fischer, J. Georges, Chem. Phys. Lett. 260 (1996) 115. [4] M.M. Wong, Z.A. Schelly, J. Phys. Chem. 78 (1974) 1891. [5] M. Fischer, J. Georges, Spectrochim. Acta Part A, 53 (1997) 1419. [6] J.H. Brannon, D. Magde, J. Phys. Chem. 82 (1978) 705. [7] D. Magde, J.H. Brannon, T.L. Cremers, J. Olmsted, J. Phys. Chem. 83 (1979) 696. [8] J. Georges, Spectrochim. Acta Rev. 15 (1993) 39. [9] M. Fischer, J. Georges, Anal. Chim. Acta 334 (1996) 337. [10] M.J. Adams, J.G. Highfield, G.F. Kirkbright, Anal. Chem. 49 (1977) 1850. [11] R.S. Quimby, W.M. Yen, Opt. Lett. 3 (1978) 181. [12] J. Shen, R.D. Snook, Chem. Phys. Lett. 155 (1989) 583.
.
[13] J.E. Sabol, M.G. Rockley, J. Photochem. Photobiol. 40 (1987) 245. [14] J. Georges, Spectrochim. Acta 51A (1995) 985. [15] M. Fischer, J. Georges, Anal. Chim. Acta 322 (1996) 117. [16] M.R. Eftink, in: J.R. Lakowicz (Ed.), Topics in Fluorescence Spectroscopy: Principles, Vol. 2, Plenum, New York, 1991. [17] A.K. Babko, A.P. Kostyshina, Ukr. Khim. Zh. 35 (1969) 544. [18] G.A. Ketsle, L.V. Levshin, V.V. Bryukhanov, Zh. Prikl. Spektrosk. 24 (1976) 809. [19] M. Franko, C.D. Tran, J. Phys. Chem. 95 (1991) 6688. [20] H.D. Burrows, S.J. Formosinho, M.F.J.R. Paiva, J. Photochem. 12 (1980) 285. [21] R.R. Hautala, N.E. Schore, N.J. Turro, J. Am. Chem. Soc. 95 (1973) 5508. [22] M.R. Eftink, C.A. Ghiron, Anal. Biochem. 114 (1981) 199. [23] E. Blatt, R.C. Chatelier, W.H. Sawyer, Biophys. J. 50 (1986) 349. [24] M.E. Daraio, P.F. Aramendia, E. San Roman, Chem. Phys. Lett. 250 (1996) 203. [25] E. Blatt, K.P. Ghiggino, J. Chem. Soc. Faraday Trans. 177 (1981) 2551. [26] F. Grieser, J. Phys. Chem. 85 (1981) 928. [27] K.-J. Choi, L.A. Turkevich, R. Loza, J. Phys. Chem. 92 (1988) 2248. [28] J. Georges, N. Arnaud, L. Parise, Appl. Spectrosc. 50 (1996) 1505. [29] R.J. Roach, R.D. Snook, Anal. Chim. Acta 262 (1992) 231.
Limitations arising in the study of the fluorescence quenching of rhodamine 6G by iodides using cw-laser thermal lens spectrometry Marc Fischer, Joseph Georges * Laboratoire des Sciences Analytiques, UMR 5619, Uni6ersite´ Claude Bernard-Lyon 1, Baˆtiment 308, 43 Boule6arde du 11No6embre 1918, 69622 Villeurbanne Cedex, France Received 31 May 1997; accepted 15 July 1997
Abstract Fluorescence quenching of rhodamine 6G by iodides has been carried out in ethanol and aqueous micellar solutions using cw-laser thermal lens spectrometry. Comparison of the photothermal results with those obtained from conventional fluorescence has shown some limitations of the photothermal method. Depending on the medium and the quencher concentration, the quenching level calculated from the photothermal data is higher than that derived from fluorescence. In some cases, the thermal lens signal is even higher than that expected for a nonfluorescent compound. A detailed study of the thermal lens behaviour has shown a converging effect on the probe beam arising simultaneously with the normal diverging effect of the thermal lens. The time scale of this abnormal signal is longer than that generated by thermal relaxation of the excited states and could originate from an endothermic (photo)chemical reaction subsequent to the quenching reaction. © 1998 Elsevier Science B.V. Keywords: Rhodamine 6G; Quenching by iodides; Thermal lens spectrometry; Anomalous signal
1. Introduction Rhodamine 6G (R 6G) is commonly used as a fluorescent standard and laser dye. This is because it is photochemically stable, has a high fluorescence quantum yield and has an insignificant triplet-state population [1]. In ethanol, R 6G dissolves completely into monomers and its fluorescence quantum yield is independent of the * Corresponding author. Tel.: + 33 78949510; fax: + 33 472431078; e-mail: [email protected]
concentration [2,3]. In contrast, the dye tends to aggregate in aqueous solutions because of hydrophobic interactions between the alkyl substituents, which results in an significant decrease in the fluorescence efficiency of the dye in concentrated solutions [4,5]. Quenching can also originate from an external quencher and the knowledge of the efficiency of any quenching process is of theoretical and practical interest. Two kinds of techniques, based on the complementary nature of excited-state relaxation through radiative and nonradiative processes, can
1386-1425/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 6 - 1 4 2 5 ( 9 7 ) 0 0 2 0 5 - 9
102
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
be used. Usually, fluorescence quantum yields are determined by relative measurements with respect to a standard for which the absolute fluorescence quantum yield must be known. Similarly, fluorescence quenching experiments are carried out by recording the decrease in fluorescence intensity as the concentration of the quencher is increased. The second kind of technique is based on the measurement of the heat yield of the sample, i.e. the ratio of the power converted to heat over the power absorbed. The heat yield of the fluorescent compound can be obtained by comparing the photothermal signal of the fluorescent sample with that of a reference absorber under the same experimental conditions [6 – 8]. The reference absorber is a compound of known absorbance and heat yield. Then, the fluorescence quantum yield is determined from the knowledge of the heat yield for the fluorescent compound. The method is absolute only if some care is taken in order to avoid errors which may arise when the sample signal is compared with the reference signal [9]. Another method, based on fluorescence quenching, has been proposed to eliminate the need of a reference absorber, by using fluorescence quenching [10–13]. The photothermal signal of the fluorescent sample is compared with that obtained with the same compound, the fluorescence of which has been quenched with a quencher. The method is expected to be more accurate provided that the absorption features of the sample and the thermo-optical properties of the medium are not changed upon addition of the quencher. This study results from an attempt to determine the fluorescence quantum yield of R 6G using thermal lens spectrometry and complete fluorescence quenching. The decrease in fluorescence efficiency upon addition of the quencher should result in a concomitant increase in the thermal power released by the sample. When the fluorescence quantum yield is high, the photothermal signal is expected to vary more rapidly than fluorescence does [5,14]. However, preliminary results have shown that complete quenching of R 6G by iodide could lead to erroneous values of the fluorescence quantum yield. The aim of the present work was to investigate the quenching of R 6G as a function of the quencher concentration
using thermal lens spectrometry. The thermal lens experiments were carried out in various media including ethanol, water and aqueous micellar solutions and the results were compared with those obtained from conventional fluorescence measurements.
2. Experimental
2.1. Instrumentation The dual-beam photothermal experimental setup consisted of an air-cooled argon–ion laser as the excitation laser and a helium–neon laser as the probe laser. The excitation laser was operated at 488 nm with the power adjusted to :4 mW at the sample cell in order to prevent optical saturation. Also, a low excitation power allowed the formation of weak thermal lenses, ensuring a linear response of the thermal lens signal with respect to the amount of heat released. The excitation beam was modulated at 15 Hz by an optical chopper with symmetrical or dissymmetrical slot blades depending on the solvent [15]. Both beams were focused independently by two lenses (f= 80 mm) in order to operate in a mode-mismatched configuration. The thermal lens signal was detected through a 1 mm pinhole with a PIN silicon photodiode. The photodiode output, converted to a voltage, was fed into a lock-in amplifier. For the time-resolved experiments, the signal from the photodiode was processed with a numerical memory (Metrix, VK12-2). Steady-state fluorescence measurements were obtained with a spectrofluorometer (Jobin-Yvon, JY3) working with a spectral bandpass of 2 nm for both excitation and emission. A 1.5 mm cell was used in order to minimize inner-filter effects. Absorbances were measured with a conventional spectrophotometer using 0.1 and 1 cm pathlength cells depending on the concentration.
2.2. Reagents Solutions of R 6G were prepared from the rhodamine chloride (Exciton) in ethanol, distilled water or micellar solutions. Micellar solutions
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
were prepared by dissolving either sodium dodecylsulfate (SDS), cetyl N,N,N-trimethylammonium bromide (CTABr) or polyoxyethylene 23 lauryl ether (Brij 35) in distilled water. Surfactant concentrations are indicated in weight percent (w/v). The pH of aqueous and micellar solutions were set at :4.8 with 10 − 2 M acetate buffer in order to prevent any pH variation which would result in a change of the absorption spectra. The reference absorbers used for the determination of the fluorescence quantum yield of R 6G were m-cresol purple in its acidic form (HCl, 10 − 3 M) and the basic form of phenol red in ethanol and in the aqueous solution, respectively [9]. All experiments were performed at room temperature (22 – 23°C) except those in CTABr micelles for which the solutions were slightly heated in order to work above the Krafft solubility boundary.
2.3. Procedure The basic relationship which describes the effect of a quencher Q on the steady-state fluorescence of a sample is given by the Stern-Volmer equation [16]
verted into F0/F from the knowledge of F0. Absolute fluorescence quantum yields were previously calculated from photothermal experiments according to the following equation [6–8]:
Ff = 1−
A rS l( f , AS r la
(1)
where F0 and F are the fluorescence quantum yields in the absence and in the presence of the quencher, respectively, Ksv (l mol − 1) is the SternVolmer quenching constant, defined as the quenching rate constant, kq (l mol − 1 s − 1), multiplied by the intrinsic lifetime of the fluorophore, and [Q] is the quencher concentration. If the fluorescence spectrum of the fluorophore is not altered by the quencher, then the fluorescence intensity is directly proportional to F. In order to compare fluorescence and thermal lens experiments, both kinds of measurements were expressed as F0/F vs. the quencher concentration. The fluorescence intensity (F) was measured at 600 nm, i.e. outside the absorption spectrum of the fluorophore, in order to avoid reabsorption effects, and the ratio F0/F was con-
(2)
where l( f is the mean fluorescence wavelength determined from the emission spectrum, la is the absorption wavelength, S is the thermal lens signal of the sample with absorbance A, and S r the thermal lens signal of the reference absorber with absorbance A r. The F0 values are 0.95, 0.89, 0.91, 0.90 and 0.81 in ethanol, water, 2% SDS, 5% Brij 35 and 2% CTABr, respectively [5]. The photothermal data were also expressed as F0/F according to the following calculation. The photothermal signal is proportional to the amount of heat released, Pth. In the absence of quencher, Pth(0) can be defined as: Pth(0) = (1− 10 − A) Pexc (1−F0x),
F0 = 1+Ks6 [Q], F
103
(3)
where Pexc is the excitation power and x= n¯ f / na accounts for the heat generated by the Stokes shift. In the presence of quenching, the same relation applies, defining a series of values for Pth and F. The thermal lens signals S0 and S, measured in the absence and in the presence of quencher, respectively, are therefore related by the following equation:
S0 1−F0x . = 1−Fx S
(4)
From the experimental values of S0/S and with the knowledge of F0, one can calculate F as a function of the quencher concentration. Conversely, the photothermal data can be expressed as a heat yield, Qth, defined as the ratio of thermal energy to energy absorbed:
104
Qth =
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Pth =1 − Fx. (1−10 − A) Pexc
(5)
When quenching of R 6G is complete, or more generally for a non-fluorescent compound, Qth = 1. The quenching experiments were performed as follows. A series of samples were prepared from the same diluted rhodamine solution. In each of them, graduated amounts of the quencher (NaI) were added, either by weighing small amounts of the salt or by adding small aliquots of a concentrated solution in order to minimise dilution effects. The addition of NaI had no effect on the absorption spectrum of the dye in ethanol and in the Brij 35, SDS or CTABr micellar solutions. However, in the Brij 35 micellar solution, absorbance at 488 nm was found to increase slightly upon the addition of NaI. This is because, in this solution, iodide has some tendency to form iodine which absorbs light at the excitation wavelength. On the contrary, in water, the absorption spectrum of R 6G was significantly altered upon the addition of NaI. This effect originates from the formation of nonluminescing complexes of the monoprotonated form of the dye with ionic halides, which results in a decrease of the absorbance at 488 nm and in static quenching of the dye solution [17,18]. Therefore, before each set of fluorescence and photothermal experiments, absorbances were measured in order to account for these variations in the experimental data. In water, the correction factor was applied with respect to the actual amount of energy absorbed by the solution. In the Brij 35 micellar solution, the correction factor also included the attenuation of the incident light due to the presence of small amounts of iodine absorbing at the working wavelength. In addition, the photothermal data were corrected for the amount of thermal energy released by iodine according to the relationship previously defined for a fluorescent compound in the presence of a concomitant [5]. Electrolytes have also been found to change the thermo-optical properties of solutions, leading to an enhancement or a decrease of the sensitivity of the thermal lens method depending on the solvent
[15,19]. For every solution studied, this effect has been measured and a correction factor was applied to the experimental photothermal data.
3. Results and discussion
3.1. Fluorescence data For a homogeneously fluorescing solution, the Stern-Volmer relation predicts a linear plot of F0/F vs. [Q]. The dynamic quenching process is diffusion controlled and requires collisional interactions between the excited fluorophore and the quencher [16]. When the Stern-Volmer relation is strictly obeyed, plotting F0/F should give a straight line with an intercept at unity and a slope equal to KSV which is a measure of the fluorophore’s sensitivity to the quencher. As shown in Figs. 1–5, quenching of R 6G by iodides depends greatly on the medium. In water, the Stern-Volmer plot is linear while deviations from the Stern-Volmer relation are observed in the other solutions studied when the iodide concentration increases. However, the first part of all the plots is linear and goes through unity, allowing the determination of KSV (Table 1). The sensi-
Fig. 1. Plots of F0/F for the quenching of R 6G by I − in ethanol using ( ) fluroescence and () photothermal data.
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Fig. 2. Plots of F0/F for the quenching of R 6G by I − in water using ( ) fluroescence and () photothermal data.
105
Fig. 4. Plots of F0/F for the quenching of R 6G by I − in water + 2% CTABr using ( ) fluroescence and () photothermal data.
tivity of R 6G to iodides is about twice as great in ethanol than in water. In the micellar solutions, it is observed that micelles have a different effect on the quenching efficiency of iodides depending on the surfactant charge. Iodides are more effective in micelles of opposite charge (CTABr) than in micelles of the same charge (SDS). In cationic CTABr micelles, the Stern-Volmer constant is about three times greater than in water, while in anionic SDS micelles the KSV value is only 0.7
M − 1, a factor of : 60-fold lower than in water. In a previous study, we have shown that the monoprotonated form of R 6G associates more or less with any kind of micelles thanks to the combination of hydrophobic and electrostatic interactions [5]. The increase in the extent of quenching of R 6G when the quencher is associated to the cationic micelles (CTABr) and, on the contrary, the high protection afforded by SDS, due to
Fig. 3. Plots of F0/F for the quenching of R 6G by I − in water+ 5% Brij 35 using ( ) fluroescence and () photothermal data.
Fig. 5. Plots of F0/F for the quenching of R 6G by I − in water + 2% SDS using ( ) fluroescence and () photothermal data.
106
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Table 1 Stern-Volmer constants for the quenching of rhodamine 6G by iodides in ethanol and in aqueous solutions Solvent
KSV (l mol−1)
EtOH
Water
5% Brij 35
2% CTABr
2% SDS
108
44
46
131
0.7
electrical repulsions between iodides and anionic micelles, reflect the importance of ions in the micellar electrical double layer [20]. In CTABr micelles, there is a competition between Br − and I − for counterion sites upon the addition of iodides. This results in a high local concentration of iodide ions which are much more efficient quenchers than bromides and in a ‘catalysis’ of the quenching reaction [21]. In the nonionic micellar solution (Brij 35), the Stern-Volmer constant determined at low quencher concentrations is of the same order as that determined in water, indicating that nonionic micelles have little effect on the quenching reaction. However, in all solutions except water, upward or downward curvings of the F0/F plots are rapidly observed, indicating a loss of the SternVolmer linearity. Similar plots have been found for many homogeneous liquid-phase systems [22]. Upward curving is generally accounted for by the presence of an additional quenching mechanism, usually referred to as static quenching. In addition to the possible formation of a nonfluorescent ground-state complex between the chromophore and the quencher, static quenching refers also to a very fast quenching reaction between a chromophore and a quencher, in close proximity, at the instant that the former becomes excited. This process can be observed in viscous solvents where the fluorophore, located within a spherical volume surrounding the quencher, is quenched instantaneously without collisional interactions. In contrast, downward curving of the Stern-Volmer plot can arise when the quenching efficiency (g) is less than 1. In this case, every encounter between the quencher and the excited fluorophore does not result in deactivation of the excited state and the quenching constant is a function of [Q].
In compartmentalised or microheterogeneous systems, such as micellar solutions, dynamic and/ or static quenching mechanisms have been proposed to explain the shape of the Stern-Volmer plots. The characteristics of these plots are also affected by the nature of the distribution of fluorophore and quencher species between the aqueous phase and micelles [22–25]. Especially, three kinds of associations have been considered for the quencher: partition, binding and the combination of both partition and binding [23]. In the case of partition, the distribution of the quencher is determined only by a partition equilibrium and the ratio of the quencher concentrations between the aqueous phase and micelles is constant. In the case of binding, where an ionic quencher can be bound by micelles with head-groups of opposite charge, the process implies that the interaction is saturable to a limited number of binding sites. As suggested by Blatt et al. [23], binding of I − to cationic CTABr micelles can enhance static quenching. In SDS solutions, upward curving of the Stern-Volmer plot can originate from the high quencher concentration necessary to decrease significantly the fluorescence. Such an increase of the quenching rate constant upon the addition of salts has been observed for nitrite quenching of Tb3 + bound to SDS micelles [26]. Upon the addition of Na + , the surface potential of SDS micelles is reduced and the repulsion between the anionic head-groups is partially shielded by the counterions. This change in surfactant–surfactant interaction makes the aggregation number and the micelle size increase [27]. Such changes in micelle size and structure would alter the interactions between the fluorophore and micelles as well as the distribution of iodide around the micelle surface.
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Therefore, the Stern-Volmer plots obtained in micellar solutions result from the combination of different kinds of quencher association processes and quenching mechanisms. In addition, it is likely that R 6G is also distributed and quenching can occur both in micelles and in the aqueous phase [20]. Understanding the processes involved in each medium studied would require computer simulations of the Stern-Volmer plots and is outside the scope of this paper.
3.2. Photothermal data Quenching experiments have also been carried out using thermal lens spectrometry. As suggested by previous works [5,14], recording the variation of thermal energy associated to a variation of fluorescence can be more sensitive, especially when the fluorescence quantum yield is high. Fig. 6 compares the relative variations of fluorescence, F, and of the photothermal signal, S, for quenching of R 6G by iodides in ethanol. As expected, S/S0 and F/F0 vary in the opposite direction, but the photothermal signal increases more rapidly than fluorescence decreases. In order to compare both kinds of experiments, the photothermal data were arranged as described in Section 2 and the results are combined in Figs.
Fig. 6. Quenching of R 6G by iodide in ethanol: relative () thermal lens signal and ( ) fluroescence intensity vs. quencher concentration.
107
Table 2 Values of the heat yield, Qth, for the quenching of rhodamine 6G by iodides in ethanol and in the Brij 35 micellar solution NaI (M)
Ff
Qth (fluo)
Qth (tls)
Ethanol 0 0.01 0.04 0.105
0.94 0.53 0.26 0.14
0.185 0.54 0.77 0.88
0.185 0.59 0.99 1.27
Water+5% Brij 35 0 0.18 0.32 0.61 0.98
0.90 0.13 0.08 0.05 0.04
0.23 0.89 0.93 0.96 0.97
0.23 0.93 0.99 1.08 1.10
(fluo), Values expected from fluorescence measurements; (tls), values derived from the thermal lens data
1–5 with those obtained by fluorescence. It can be seen that, in every medium studied except in water, the photothermal results are consistent with those obtained by fluorescence only over a limited range of quencher concentrations. Otherwise, the F0/F ratios obtained by thermal lens spectrometry deviate positively with respect to the fluorescence plots. These results are very surprising because they would mean that the amount of thermal energy produced by the quenching reaction is greater than the associate loss of radiative energy. The discrepancy between both experiments can be illustrated by comparing theoretical and experimental values of the heat yield. Calculations were made for high quenching levels in ethanol and the Brij 35 (Table 2). As previously noticed, Qth should maximise at unity for a non-fluorescent compound. However, it can be seen that the experimental values are greater than the expected ones and can be greater than unity, even when fluorescence quenching is not complete. Not only are these deviations detrimental when the photothermal method is used to investigate fluorescence quenching reactions, but also they are an important drawback for the determination of absolute fluorescence quantum yields using the completely quenched fluorophore as internal reference [10–12].
108
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Some experiments previously made with the same fluorophore but using pulsed-laser excitation did not show such abnormal results [28]. This suggested to us, that the difference could originate from the operating mode of the excitation laser and the time scale for the thermal lens to form. In cw-laser experiments, excitation occurs over a rather long time, typically in the order of ms or s, depending on whether the signal is sampled at the steady-state or before. In pulsed-laser experiments, sample irradiation occurs within a very short time ( : 10 ns) and the lens forms almost instantaneously (:ms). Therefore, the pulsedlaser method would be less sensitive to the presence of slow chemical and/or physical processes interfering with the formation of the thermal lens. Further evidence of such processes occurring in cw-laser thermal lens spectrometry came from the observation of abnormal signals when doing timeresolved experiments. These experiments were carried out with R 6G quenched by iodide in SDS micellar solutions, where the effects observed are the most typical. Three kinds of recordings were made using either a chopper or a mechanical shutter: the time dependence of the demodulated signal at the output of the lock-in amplifier, the time-profile of the ac signal during the first chopper cycles after the beginning of irradiation, and the build-up and relaxation of the thermal lens to steady-state. When the excitation laser is modulated with an optical chopper, the ac signal produced by the periodical build-up and relaxation of the thermal lens is converted into a dc signal through a lock-in amplifier. Since the formation of the thermal lens is of the order of the thermal time constant tc ( : ms), the signal risetime is limited by the time constant of either the lock-in amplifier or the recorder. Generally, the time required to get a steady-state value of the signal is some seconds after the beginning of irradiation (Fig. 7(a)). However, in most cases, when rhodamine is quenched by the addition of significant amounts of iodides, the time-dependent dc signal can be divided into two components that correspond to different time constants (Fig. 7(b)): a fast rising part, which is the typical signal induced by the thermal lens effect and a slower component with a
time constant of the order of minutes. Even though the signal was sampled at the top of the fast rising part, thus ignoring the slow component, the experimental values were generally too great. However, the demodulated signal does not account for the sign of the thermal lens formed and the time profile of the signal during the relaxation and the formation of the thermal lens. Fig. 8 shows the time dependence of the probe beam intensity during the first chopping cycles from the beginning of irradiation. When the chopper opens at the first cycle, the sample is irradiated and the probe beam intensity decreases as the thermal lens forms. At the end of the first ‘on’ period, when the excitation beam is blocked, the probe beam intensity not only increases due to the relaxation of the thermal lens, but also becomes greater than its initial value before the beginning of irradiation. Then, the probe beam intensity at the end of each successive relaxation period gradually increases until a steady-state. The ac component of the signal behaves as usually observed for the formation of a diverging lens, but its amplitude increases as the relative probe beam intensity increases. The slow rising component in Fig. 7 could originate from this effect and could explain
Fig. 7. Time-dependence of the demodulated signal for 2.5 × 10 − 6 M R 6G in 2% SDS micellar solution (a) in the absence of NaI and (b) in the presence of 0.22 M NaI; chopping frequency =15 Hz.
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
Fig. 8. Time-profile of the ac signal during the first chopping cycles for R 6G quenched by iodides in 2% SDS micellar solution; [R 6G]=10 − 5 M; [NaI]=0.25 M; solution rested for some minutes before irradiation; chopping frequency= 15 Hz.
the errors in the measurement of the ‘true’ thermal lens signal. The third recording shows the build-up of the signal over a longer irradiation time (:10 s) and its subsequent relaxation (Fig. 9). When the excitation laser is turned on, using a mechanical
Fig. 9. Time-resolved build-up and relaxation of the thermal lens signal for 2.5 ×10 − 6 M R 6G quenched by 0.22 M NaI in 2% SDS micellar solution (a) immediately after refilling the sample cell and (b) after the solution was let to rest for 10 min.
109
shutter, the probe beam intensity decreases, indicating the formation of a diverging lens. However, after a very short time ( B 1 s), the probe beam intensity increases quickly while the beam is seen to converge. Then, the probe beam intensity increases slowly, but remains greater than the initial value before the beginning of irradiation. When the excitation laser is turned off, the probe beam intensity rapidly increases, indicating the relaxation of the diverging thermal lens, and then decays more slowly to its initial value. The signals in Figs. 8 and 9 indicate that the ‘true’ thermal lens signal created by the temperature-induced refractive index gradient, dn/dT, is mixed with another signal of opposite sign and resulting in a convergent behaviour of the probe beam. This anomalous signal was independent of the surfactant concentration and was observed even below the critical micelle concentration. However, as shown in Fig. 9, the convergent behaviour of the probe beam depended strongly on the time during which the sample was allowed to rest in the cell before the beginning of the experiment. This observation, along with the time constant of the anomalous phenomenon, suggests the formation of another refractive index gradient which is the reverse to that produced by the temperature gradient and would be governed by mass diffusion. Such an inversion of the refractive index gradient has been observed by Roach and Snook in the study of uranyl ions in the presence of acetic acid [29]. A possible mechanism for this phenomenon was based on energy transfer between the uranyl ion and acetic acid and subsequent photodecarboxylation of acetic acid. The process would be endothermic and would remove heat from the beam centre and hence weaken or even reverse the thermal lens. The anomalous thermal lens signal exhibited in our quenching experiments probably explains the disagreement observed between fluorescence and photothermal data. The chemical or physical origin of this effect has not been elucidated and cannot be solved easily with some further experiments. The time constant of the abnormal signal is quite greater than the time constant of the thermal lens effect. Moreover, preliminary studies have shown that the observed effect depends on a lot of parameters
M. Fischer, J. Georges / Spectrochimica Acta Part A 54 (1998) 101–110
110
including the solvent, the concentration of the reagents, convection effects into the solution and the excitation mode (cw, modulated, or pulsed). A detailed study would probably require the use of other techniques in addition to thermal lens spectrometry.
References [1] K.H. Drexhage, Structure and properties of laser dyes, in: F.P. Scha¨fer (Ed.), Dye Lasers, Topics in Applied Physics, Springer, Berlin, 1973, chapter 4. [2] A. Penzkofer, Y. Lu, Chem. Phys. 103 (1986) 399. [3] M. Fischer, J. Georges, Chem. Phys. Lett. 260 (1996) 115. [4] M.M. Wong, Z.A. Schelly, J. Phys. Chem. 78 (1974) 1891. [5] M. Fischer, J. Georges, Spectrochim. Acta Part A, 53 (1997) 1419. [6] J.H. Brannon, D. Magde, J. Phys. Chem. 82 (1978) 705. [7] D. Magde, J.H. Brannon, T.L. Cremers, J. Olmsted, J. Phys. Chem. 83 (1979) 696. [8] J. Georges, Spectrochim. Acta Rev. 15 (1993) 39. [9] M. Fischer, J. Georges, Anal. Chim. Acta 334 (1996) 337. [10] M.J. Adams, J.G. Highfield, G.F. Kirkbright, Anal. Chem. 49 (1977) 1850. [11] R.S. Quimby, W.M. Yen, Opt. Lett. 3 (1978) 181. [12] J. Shen, R.D. Snook, Chem. Phys. Lett. 155 (1989) 583.
.
[13] J.E. Sabol, M.G. Rockley, J. Photochem. Photobiol. 40 (1987) 245. [14] J. Georges, Spectrochim. Acta 51A (1995) 985. [15] M. Fischer, J. Georges, Anal. Chim. Acta 322 (1996) 117. [16] M.R. Eftink, in: J.R. Lakowicz (Ed.), Topics in Fluorescence Spectroscopy: Principles, Vol. 2, Plenum, New York, 1991. [17] A.K. Babko, A.P. Kostyshina, Ukr. Khim. Zh. 35 (1969) 544. [18] G.A. Ketsle, L.V. Levshin, V.V. Bryukhanov, Zh. Prikl. Spektrosk. 24 (1976) 809. [19] M. Franko, C.D. Tran, J. Phys. Chem. 95 (1991) 6688. [20] H.D. Burrows, S.J. Formosinho, M.F.J.R. Paiva, J. Photochem. 12 (1980) 285. [21] R.R. Hautala, N.E. Schore, N.J. Turro, J. Am. Chem. Soc. 95 (1973) 5508. [22] M.R. Eftink, C.A. Ghiron, Anal. Biochem. 114 (1981) 199. [23] E. Blatt, R.C. Chatelier, W.H. Sawyer, Biophys. J. 50 (1986) 349. [24] M.E. Daraio, P.F. Aramendia, E. San Roman, Chem. Phys. Lett. 250 (1996) 203. [25] E. Blatt, K.P. Ghiggino, J. Chem. Soc. Faraday Trans. 177 (1981) 2551. [26] F. Grieser, J. Phys. Chem. 85 (1981) 928. [27] K.-J. Choi, L.A. Turkevich, R. Loza, J. Phys. Chem. 92 (1988) 2248. [28] J. Georges, N. Arnaud, L. Parise, Appl. Spectrosc. 50 (1996) 1505. [29] R.J. Roach, R.D. Snook, Anal. Chim. Acta 262 (1992) 231.