Measurement of thermal energy recovery using thermal lens spectrometry in fluorescence quenching experiments

SpectrochimicaActa, Vol. 50A, No. 5, pp, 953-959, 1994 Copyright ~) 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0584-8539/94 $6,00 + 0.00

Pergamon

Measurement of thermal energy recovery using thermal lens spectrometry in fluorescence quenching experiments J . GEORGES* a n d J. M . MERMET Laboratoire des Sciences Analytiques, URA CNRS 435, Brit. 308, Universit6 Claude Bernard-Lyon 1, 69622 Villeurbanne C6dex, France

(Received 14 June 1993; in final form 26 July 1993; accepted 28 July 1993) Abstract--This paper describes some limitations of the pulsed-laser thermal lens method in measuring the heat produced by non-radiative relaxation of excited fluorescent molecules. Simultaneous measurements of the fluorescence intensity and thermal lens signal have been carried out for fluorescein dianion in water and perylene in ethanol. It is shown that complete fluorescence quenching of fluorescein and perylene by potassium iodide and nitromethane, respectively, does not result in full recovery of the thermal energy. The results depend on either the operating mode of the excitation laser or the solvent composition.

INTRODUCTION

THERMAL LENS spectrometry is a convenient photothermal method used for the direct observation of non-radiative relaxation processes following absorption of radiation [1]. The various applications of the thermal lens method in this field have been recently reviewed [2]. Especially, thermal lens spectrometry, as well as photoacoustic spectrometry, has been applied to the determination of fluorescence quantum yields and to fluorescence quenching studies [3-11]. It is generally accepted that these methods offer significant advantages over photometric measurements because they can be used without any standard. Since fluorescence and thermal deactivation are competitive processes, and for most solutes the absorbed energy can be dissipated mainly as thermal energy and radiant energy, it should be possible to quantify the extent of fluorescence quenching by monitoring the change in thermal lens signal. The simultaneous and complementary use of fluorescence and thermal lens spectrometries is based on the principle of energy conservation. The fraction of absorbed energy not released as light is dissipated as thermal energy and should contribute to the formation of the photothermal signal. During fluorescence quenching experiments, the observed thermal signal should increase with the quencher concentration until complete fluorescence quenching is obtained. Practically, the conversion efficiency of the absorbed energy into heat is measured by comparing the thermal signal of the fluorescent sample with that of a reference absorber, in the same solvent and with the same experimental arrangement [2]. The method is valid only if one verifies that all the thermal energy contributes to the formation of the photothermal signal. In other words, the ratio of the photothermal signal of an unknown sample to that of a reference absorber should depend only on the amount of thermal energy that is actually released. Previous studies have shown that the thermal lens signal can depend on the solute and the solvent [12-14], on the excitation wavelength [14] and on whether the excitation laser is continuous or pulsed [15]. The aim of the present work was to correlate the thermal lens signal with the fluorescence intensity in two kinds of fluorescence quenching experiments. Simultaneous fluorescence and thermal lens measurements have been carried out for the fluorescein dianion quenched by potassium iodide and for perylene quenched by nitromethane. Deviations from the expected thermal lens signal have been observed depending on the excitation mode (cw or pulsed) and the solvent composition, respectively.

* Author to whom correspondence should be addressed. 953

954

J. GEORGESand J. M. MERMET EXPERIMENTAL

Instrumentation Both thermal lens experimental setups were based on a double-beam p u m p - p r o b e configuration. Fluorescence and thermal lens experiments with pulsed excitation were made with the same laser-based apparatus [16, 17]. The pulsed laser was a nitrogen-pumped dye laser. The experiments were carried out either around 435 nm with 2 x 10 -3 M coumarin 440 in ethanol or at 490 nm with 6 x 10 -3 M coumarin 503 in ethanol. The sample cell was a 1 cm four-window fluorescence cell. After the cell, the excitation beam was blocked by a bandpass interference filter. The thermal lens effect was probed colinearly with a H e - N e laser and the intensity variation was measured through a I mm pinhole with a PIN silicon photodiode. The fluorescence, collected at right angles to the laser beams, was focused on the entrance of a monochromator and then detected by a photomultiplier (R928) operating at 900 V. Both signals were monitored with an oscilloscope and fed into a boxcar averager. In all our experiments, the thermal lens signal was sampled with a delay of 400 ixs from the laser pulse in order to measure the whole signal including any slow component that could arise, within this delay, from possible slow decay processes. The thermal lens apparatus with cw excitation was the same as previously described [18]. The cw laser was an air-cooled argon-ion laser working in the single line mode with an output power of 20 mW at 488 nm, and modulated at 40 Hz. The thermal lens signal was detected with a photodiode and monitored with a lock-in amplifier. Steady-state fluorescence measurements were obtained on a Jobin-Yvon JY3 spectrofluorimeter with a 1 cm quartz cell equipped with 10 nm slits for both excitation and emission. Absorbances were measured with a conventional spectrophotometer (Beckman, Model 25). The working wavelength of the dye laser was measured with a monochromator (Jobin-Yvon, H-20 VIS) which has been previously calibrated with the 632.8-nm line of the H e - N e laser. Also, absorbance measurements and thermal lens experiments were carried out at the top of the absorption band, so that a small wavelength discrepancy between both measurements was not critical.

Reagents The studied compounds were fluorescein dianion in water (pH = 10) and perylene in ethanol. Fluorescence quenching was obtained with potassium iodide for fluorescein in water and nitromethane for perylene in ethanol. All reagents were the purest grade available and were used as received. A t the excitation wavelength used for perylene (435nm), nitromethane (Janssen Chimica) had an absorbance of about 0.003 (1 cm cell versus water). Low sample concentrations (3 × 10-7-4 × 10 -6 M, i.e. about 5 x 10-3-5 × 10 -2 in absorbance) were used in order to satisfy the linearity of the thermal lens response and to reduce concentration quenching of fluorescence, but the lowest concentration was set by the limit of detection of our measurements. Previous studies with cresyl violet in methanol have shown that concentration quenching no longer occurs below 10-6M [11]. When necessary, perylene samples were deoxygenated directly in the measurement cell with pure helium according to a procedure described elsewhere [19].

Procedure Fluorescein dianion in water was excited at a wavelength corresponding to the maximum of the absorption band, i.e. 490 nm with the pulsed dye laser and 488 nm with the cw argon-ion laser. Complete fluorescence quenching was obtained by the addition of 1.35 M potassium iodide [5]. Perylene was excited at the top of the main absorption band (Fig. 1). The exact wavelength, depending on the composition of the solvent, was selected by tuning the dye laser around the peak value in order to obtain the greatest thermal lens signal and to match the excitation wavelength in the thermal lens experiment with that used to measure the absorbance of the sample. The absorhance of dilute samples was estimated from the dilution factor of stock solutions. Practically, absolute fluorescence quantum yields have been deduced from thermal lens measurements according to the following equation [2]:

• r=~ 1 ASr],

(1)

Thermal energy recovery

955

0.8

20

0.7 o.8

15

c-

0.5

O

0 c ¢g e~

e@

0.4

1 0 =~ o

0.3

0 1 i1

0.2

0.1

0 370

0 400

430 460 490 Wavelength (nm)

520

Fig. 1. Absorption and fluorescence spectra of perylene in ethanol and nitromethane. (1) and (2) Absorption spectra of perylene in ethanol and nitromethane, respectively. (3) Absorption spectrum of neat nitromethane against water. (4) and (5) Fluorescence spectra of perylene in ethanol and nitromethane, respectively (right ordinate with different scales); excitation wavelength = 410 nm.

where 2; is the mean fluorescence wavelength determined from the emission spectrum, 2, the absorption wavelength, S the thermal lens signal of the sample with absorbance A, and S" the thermal lens signal of the reference absorber with absorbance At. Conversely, the same equation may be used to calculate the theoretical thermal lens signal, Qth, expected from the value of ~f, and expressed as the ratio of thermal energy to energy absorbed: Q,h = 1 --

(2)

with Qth = 1 for a non-fluorescent compound. In polar solvents, the quencher nitromethane does not drastically change the polarity of the medium and, for low quencher concentration, the quenching effect is shown to follow the Stern-Volmer relationship [20, 21]: -~t= 1 + Kq[Q]

(3)

where ~ and ~f are the fluorescence quantum yields in the absence and in the presence of the quenching agent, respectively, Kq is the Stern-Volmer quenching constant defined as the quenching rate constant (kq) multiplied by the intrinsic lifetime of the fluorophore, and [Q] is the concentration of the quencher. If the fluorescence spectrum of the fluorophore is not altered by the quencher, then the fluorescence intensity F is directly proportional to the fluorescence quantum yield: O~

¢f

F° F"

(4)

Keeping the excitation power and the fluorophore concentration constant, the quenching efficiency was followed by the decrease in fluorescence intensity at a fixed wavelength. Emission of perylene was selected at 470 nm, i.e. outside the absorption range of both the fluorophore and the quencher.

956

J. GEORGES and J. M. MERMET RESULTS AND DISCUSSION

Fluorescence quenching of fluorescein dianion in water Before investigating the quenching effect of potassium iodide on fluorescein, one has to consider the effect of the quencher on the properties of the solvent. PHILLIPSet al. [22] have shown that the addition of salts in a sample solution has a real effect on the thermal lens signal of the sample. The signal enhancement of a sample upon addition of salts in water is caused mainly by a change in the thermoopticai parameters of the solution. The experimental verification was made by using a non-luminescent reference absorber, that is an aqueous solution of cobalt nitrate. Our results (Table 1) show an average signal enhancement of 45 and 50% with cw and pulsed excitation, respectively, when 1.35 M KI is added in water. The signals for cobalt nitrate with and without potassium iodide were compared with solutions of matching absorbances, after substraction of the background signal due to the residual absorbance of KI in water at the excitation wavelength. Taking this effect into account, the quenching action of KI on fluorescein was then considered. The thermal lens signal was shown to increase linearly with concentration for cobalt nitrate (reference absorber), fluorescein and quenched fluorescein, An increase in the thermal lens signal of fluorescein dianion in water was observed upon addition of KI, indicating an increase in the conversion efficiency of the absorbed energy into thermal energy (Table 1). Moreover, in the case of cw excitation, the total signal for quenched fluorescein is even greater than that of the reference absorber. Corrections for the effect of KI on the thermooptical properties of water led to Qth values equal to 0.99 and 0.23 for cw and pulsed excitation, respectively. The value obtained with cw excitation is close to the expected one and indicates that all the thermal energy actually contributes to the thermal lens signal. On the contrary, with pulsed excitation, the experimental value represents only 23% of the theoretical one, corroborating previous studies with pulsedlaser excitation [12-15]. The influence of the operating mode of the excitation laser on the recovery of thermal energy has been previously discussed with respect to the time scales of sample irradiation and thermal lens formation [15].

Fluorescence quenching of perylene In ethanol, perylene exhibits a wide absorption spectrum in the visible range with three distinct bands centered around 435, 410 and 385 nm. The fluorescence spectrum presents an intense maximum at 440 nm and another of average intensity at 470 nm (Fig. 1). Nitromethane has been shown to quench the fluorescence of polynuclear aromatic hydrocarbons, such as perylene, containing only six-membered rings [23]. In nitromethane as solvent, the absorption spectrum of perylene is not changed. The fluorescence intensity is much lower, but the emission spectrum is not drastically different. The intensity ratio between peaks 1 and 2 is equal to 1.27 against 1.46 in ethanol, indicating a more important quenching effect on the main fluorescent band. Table 1. Relative values of the thermal lens signal, expressed as Qth, for cobalt nitrate and lluorescein in water with and without potassium iodide. Cobalt nitrate in pure water is taken as the reference absorber, except in (a) where the values are corrected for the effect of KI on the thermooptical properties of water. In the absence of KI, the theoretical value of Qth is 0.10 [13] cw excitation Pulsedexcitation Co(NO3)2

water water+ 1.35M KI

1 1.45

1 1.5

Fluorescein

water water + 1.35 M KI

0.12 1.44 0.99(a)

0.11 0.35 0.23(a)

Thermal energy recovery

957

Therefore, the quenching effect, measured as the decrease in fluorescence intensity at 470 nm, is rather under estimated. The absorption range of neat nitromethane is also represented in Fig. 1. Because the cw argon-ion laser has no emission line available in the absorption range of perylene, the experiments were carried out only with pulsed-laser excitation. The fluorescence intensity at 470 nm was measured simultaneously with the thermal lens signal. For each perylene concentration and solvent composition, the thermal lens signal was compared with that of ferrocene, taken as the reference absorber and prepared in the same medium in order to take into account changes in the thermooptical properties of the solvent upon addition of nitromethane.

Oxygen and concentration quenching. Figure 2 shows the dependence of the fluorescence intensity as a function of perylene concentration, along with the variation of Qth. The fluorescence intensity does not vary linearly in the concentration range used to satisfy the requirements of thermal lens measurements. The shape of the curve is consistent with that reported previously for cresyl violet in methanol and water [11]. Although excimer formation is likely only at relatively high concentrations, it can be one of the processes, with inner-filter effects, by which a solute can quench its own fluorescence. Whatever the origin of the phenomenon, it can be expected to result in an increase of the relative amount of heat released with increasing concentration. Instead, the Qth value remains constant over the whole concentration range studied, with an average value of 0.136. This invariance of Qt~ is consistent with the linear dependence of the thermal signal with concentration (Fig. 3). Taking hI from the emission spectrum, Eqn (2) allows the calculation of Or; one obtains 0.89, which is close to 0.87 given in the literature [24]. Deoxygenation of the solutions resulted in a slight increase of the fluorescence intensity, but no variation of the photothermal signal was observed. Our result means that the experimental thermal lens signal equals the theoretical value when no quenching effect occurs. Then, the decrease in fluorescence intensity caused by concentration and/or oxygen quenching does not result in an increase of the thermal lens signal. 100

/

80

0.8

c

60

0.6 .g:

/

o c o

~ 40 i1

c7 0.4

0.2

20 13

0

C

[]

I

0

1

t3

[]

t

[3

I

2 3 Conoentration (uM)

0

4

Fig. 2. (*) Fluorescence intensity and ([3) thermal lens signal, expressed as Q,h, versus concentration for perylene in ethanol.

J. GEORGES and J. M. MERMET

958

Nitromethane quenching. The addition of nitromethane in the ethanolic solution resulted in a fast decrease of the fluorescence intensity (Fig. 4). Comparatively, the thermal lens signal increases more slowly than that expected. The expected values of Qth were deduced from Eqn (2), taking an estimated value of ¢br for each solvent composition. Since the dependence of the photothermal signal on perylene concentration was also linear in neat nitromethane (Fig. 3), it was assumed that the same relationship was true in every ethanol/nitromethane mixture. Therefore, the values of ~I were obtained from Eqn (3), taking the absolute fluorescence quantum yield in ethanol without any quenching effect (0.87) multiplied by the ratio of the fluorescence intensity in the presence of nitromethane to that in the absence of any quenching effect. The latter was estimated from the slope of the straight line crossing the origin and tangent to the fluorescence curve in Fig. 2. Using this calculation, one obtained the curve in Fig. 4 showing the theoretical variation of Qth upon addition of nitromethane in ethanol. While the experimental value agrees with the theoretical one in the absence of quencher, the curves drastically diverge when the amount of nitromethane is just necessary to quench almost completely the fluorescence of perylene. Then, the thermal lens signal increases with the volume percent of nitromethane while the fluorescence intensity remains nearly constant. The photothermal signal is much greater in neat nitromethane than in ethanol containing a few per cent of nitromethane as quencher. According to DREESKAMP[20], the quenching effect of nitromethane on non-fluoranthenic hydrocarbons occurs most probably via energy transfer from the excited singlet state of the fluorophore to the quencher followed by dissociation of the quencher. If such a deactivation process occurs, a small enthalpy difference between dissociated and undissociated nitromethane could partly explain the discrepancy observed between theoretical and experimental values of Qth. However, variation of this effect upon addition of increasing amounts of nitromethane should be, at the most, in the same order as the fluorescence intensity variation (Fig. 4). Our result means that the recovery of thermal energy using the thermal lens method varies with the solvent composition independently of the variation of the thermooptical properties of the medium. A similar effect, but to a lesser extent, has been previously observed and explained as occurring from differences in solute-solvent interactions and in energy-transfer processes between the excited solute and the solvent. 25

20

c

•~15 c

E ~10 J¢ p-

OK¢ "~'-'''

0

I

I

L

1

2 Con¢ontration (uM)

3

Fig. 3. Dependence of the thermal lens signal on perylene concentration in (*) ethanol and (t-l) nitromethane; same scale for both lines.

Thermal energy recovery

959

3.8 C C

20

3.6

o e-

(3:

(.1 L. O

0.4

14.

10 0.2

01

j

i

i

0

25

50

75

~0 I00

Volume percent of nitromethane

Fig. 4. (*) Fluorescence intensity and (l~) experimental and ([7) theoretical thermal lens signals, expressed as Qth, for perylene in ethanol with increasing amounts of nitromethane.

Although this work does not provide full information about the origin of the incomplete recovery of the thermal signal, it corroborates previous studies and adds new results in this field. Further experiments carried out with other solutes and comparing cw and pulsed excitation are still necessary to establish an obvious interpretation of this phenomenon.

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