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The photoacoustic determination of fluorescence yields of dye solutions
Volume 54, number 3
CHEMICAL
THE PHOTOACOUSTIC
DETERMINATION
PHYSICS
OF FLUORESCENCE
Mark G. ROCKLEY
and Kristy M. WAUGH
Chemistry Department,
Oklahoma State University. Stillwater.
Received
1977
14 November
15 March 1978
LPXTERS
YiELDS OF DYE SOLUTIONS
Oklahoma 74074, USA
A new method for the measurement of the primary fluorescence quantum yield of dye solutions is presented. The method is conceptually and experimentally simple, relying on the .S, -- S1 transition probabdity as an internal standard for comparison with the Si radlationless transltlon probability, both probablhties being measured by photoacoustlc spectroscopy.
1. Introduction
It is valuable throughout the field of photophysics to have a reliable measure of the fluorescence quantum yield, this yield being the fraction of those initially excited molecules which return directly to the ground state by a radiative transition. It will be shown by this work that fluorescence yields (+f) of dye solutions (indeed, solutions of any chromophore) can be measured from analysis of the liquid phase photoacoustic spectra VIWhen an aromatic chromophore in solution such as a dye, is excited by light, the transition can be to the lowest energy excited singlet state (S;,with energy relative to the ground state E(S,)) or to higher energy excited singlet states (S,, with energies E(S,)), depending on the wavelength of the incident light. It is well known that, with a few exceptions such as azuIene, the higher states, S,, radiationlessly decay to the lowest excited state, S 1, with an efficiency close to 100%. That is, for every absorbed photon of energy E(S,), that fraction of the energy corresponding to [I!?@,) -WV1 I%%) WI‘11immediately appear as heat coming from the sample. However, S, decay may involve radiative and nonradiative components. (For purposes of this particular experiment, phosphorescence emission yields will be ignored. However, the general method outlined here can be extended to phosphorescent systems by making use of the phase resolution of the photoacoustic spectra_) Indeed, a fraction, (1 - ipf), of all the energy absorbed by St + So transitions, will appear as heat, where Gf is the fluorescence quantum yield.
Photoacoustic spectroscopy (PAS) can be used to measure these heat signals. The essentrals of liquid phase PAS have been described elsewhere [ 1J _ It is sufficient for now to say that the PAS spectrometer will give a signal proporttonal to the total heat emitted by the sample, whether excited into the S, or the S, state. If the proportionality constant is k, then the PAS signal from excitation into S, and St , respectively, can be described by tne two equations: IpAs(Sn)=kiE(Sn)-E(S1)+E(S1)(l
-af)]
(1)
and IpA’
= k[E&)(l
- &)I _
(2)
Since E(S,) and E(S,) are known quantities, whrle IpAs(S,) and I p*s(S,) are measured quantities, the convenient situsrion exists of having two equations with two unknowns to solve. In particular, this enabIes the easy determination of the fluorescence quantum yield, @t, of the molecule, relying only upon the measurement of the corrected photoacoustic signal inrensitres for excitation into the S, and St states of *he molecule.
2. Experimental The sample used to test the expertmental method was recrystaliized crystal violet, [(CH,),N-C6H4] 3C+Cl-, which was tested by TLC on silica with a range of acetone, ethanol, chloroform solvent systems and found to have no visibly detectable impurities. The dye was dissolved in distilled HZ0 at a concentration of 2.02 597
CHEMICAL PHYSICS LETTERS
Volume 54, number 3
mg/ml, giving a solution wrth an optical density 6001 cm. The photoacoustic sample chamber had a total volume of 1S ml and was connected to a CRl9629601 $” diameter foil electret microphone. The signal from the microphone was amplified by an Ithaca 143L preamplifier and processed by an Ithaca 39 1A lock-in amplifier. The internal frequency signal of the lock-in was used to modulate the current supply to the 1 kW xenon arc lamp, hence providing the modulation necessary to observe the photoacoustic signal. The power supply for the arc-lamp was purchased from Photochemical Research Associates in Canada but had tc be modified to ensure relrable lamp operation in the rnodulation mode. While the lamp could be modu!ated anywhere within the 100 Hz to 10 kHz frequency range, this experiment was run at a frequency of 185 Hz. This frequency provided the minimum intemrodulation with the line power frequencies which were superimposed on the power supply output. The PAS liquid phase spectra shown in fig. 1 were obtained by signal averaging. The Sr +-So spectra were more intense than the S, + So spectra both because of the high sample absorptivity and the higher brightness of the xenon lamp in this spectral region. Therefore, three PAS spectra were averaged for the St -+ So spectrum while six spectra had to be averaged to obtam the S,, + St-, PAS spectrum with comparable signal to noise
50
e&o
550
!xcJ 4!xJ -VMbELENGW
400 (NM)
3!3J
598
levels. Because of the poor signa to noise values for the experiment reported here (approximately 5 to 1 for unaveraged spectra in the 300 nm region) the uitimate reliability of the experimental results is low. This is a drawback primarily due to the very low spectral brightness of the arc-lamp monochromator combination (< 1 mW/nm) rather than the experimental method. However, as wil1 be shown, the data are sufficiently reliable to demonstrate the utility of the method, especially when high brightness sources such as lasers are available. For these experiments, a bandpass of 6.4 nm was necessary to obtain a sufficientIy strong signal. The xenon lamp~monochromator combination power spectrum (a PAS spectrum of carbon black) is included in fig. 1 for reference, although the crystal violet spectra shown are not corrected. The wavelength calibration of the monochromator was thus made by referring to the peaks in the power spectrum as well as by the use of a hehum-neon laser. Therefore, the PAS spectra presented are correct with respect to the wavelength-power distribution_ Incidentally, second-order transmission by the monochromator (600 nm setting passing 300 nm light) was checked with a cutoff fiter. It was found to be 5% and so ignored because of the high noise IeveIs of the results reported. The monochromator was a McKee-Pederson 0.5 meter monochromator with a 300 nm blaze grating and a 16 A/mm reciprocal dispersion. The absorption spectrum presented in fig. 1 was obtained with 0.5 rtm resolution on a Beckman model 2.5 absorption spectrometer, calibrated with a holmium oxide filter for wavelength accuracy. Absorption linearity is not a problem since the experiment requires measurement of the PAS signal intensities at two different wavelengths (one for the Sr + So band, the other for the S, f- So band) where the absorbance is the same. To obtain the absorption spectrum of the crystal vrolet solution used here, a drop of the solution was squeezed between two optical flats, forming a layer 0.01 mm thick. The resulting thin “cell” was then sealed and the absorption spectrum measured.
300
TIN. 1. The xenon lamp poner spectrum is represented by the solrd line; the liquid phase PAS spectrum and the absorption spectrum scales are slrahtly displaced for ckrrty of presentatfon.
15 March 1978
3. Results and conclusions The concentration sampIe had an optica
of the solutron was such that the density greater than one in the
CHEMICAL
Volume 54, number 3
PHYSICS LETTERS
thermally active layer [ 1] , but was below the saturation limit. Furthermore. the results descrrbed below are calculated at two wavelengths (617 nm and 306 nm) where the sample has equal optical densities. Therefore, the same fraction of incident photons will be absorbed at each wavelength and the absorbed photons will have an equivalent distribution throughout the thermally active layer. From fig. 1, the following information can be obtained: at 617 nm and 306 nm, the absorbances are the same; the sample PAS intensities are 33 units at 617 run and 29 units at 306 nm; and the power spectrum has intensities of 160 units at 617 nm and 142 units at 306 nm. If eq. (1) is divided by eq. (2), using the wavelengths 617 nm for E(S,) and 306 nm for E(S,,) then the following result is obtained: IPAS(SJ/IPAS(S,)
= (1.02 + 1 - @r)/(l
- (Pf).
(3)
However, the PAS signal intensities need to be corrected so that both correspond to an eqlcal number of phofoons striking the sample. This correction can be made by multiplying IpAs(S,,) by the ratio of the power spectrum intensities at the two wavelengths and by the reciprocal of the ratio of the photon energies. Therefore, the corrected IPAS(S,*) has a value of 66 units. The ratio in eq. (3) is then equal to 2.0. Errors due to noise in the averaged spectra actually make this ratio equal to a value of 2.0 -i- 0.10 giving a fluorescence yield of 0.02 f 0.10. It seems inappropriate to have an error possibility of i 0.10 on a measured yield of 0.02. However, this error is due primarily to the high noise levels associated with measuring PAS liquid phase spectra with low brightness sources. Nevertheless, let it be taken that the fluorescence yield is between 0.00 and 0.10, and that the nonradiative processes have a quantum yield of 0.98 F 0.10. Magde and Windsor [2] measured the singlet state lifetime of crystal violet in H,O, finding a value of 10-l t s. If one assumes that crystal violet (emax = lo5 Q M-l), hke most xanthene derivatives with such large
extinction
coefficients, has an mtrinsic lifetime of 5 X
lo-’ s, then the fluorescence yield could be inferred to be 0.01. This IS in excellent qualitative agreement with the yield of 0.02 reported in this work. It is therefore demonstrated that liquid phase photoacoustic spectroscopy can be used as a powerful tool for measuring primary fluorescence quantum yields. Although the measurement error for these experiments
15 March 1978
is
large, this is due only to the lack of spectral brightness of the exciting source, and does not reflect upon the
wide utihty of this new method. No external standards or calibrat.ons other than a source power spectrum are required. Furthermore, the method may be applied to gas and soPd phase systems, provided high brightness sources at the appropriate wavelengths are available. Starobogatov and co-workers [3,4] have hinted at such methods for determining fluorescence yields. However, they have resorted to using a nonfluorescent “standard” as a means of comparing expected versus obtained PAS signal intensity. This brings back the problem of instrument calibration etc. The method described in this work relies upon the unit probability of S,, S, internal conversion for the provrsion of an intensity cahbrant, in this way providing a much less cumbersome measuring tool. The validity of this technique rests on the unit quantum efficiency for the S,, S, internal conversion. This is a generally accurate assumption for dyes and most aromatics. with the notable exceptions, thioketones, azulenes, etc. Furthermore, it is necessary that the sample optical density be below the saturation limit as explained by McClelland et al. [ I] . (This was true for the experiment reported here -the optical density was 600 cm-’ at 185 Hz.) It is expected that the method will find much use in the study of high optical density dye solutions, especially when lasers are used as exciting sources.
Acknowledgement
The authors gratefully acknowledge partial support of this research by the Research Corporation and by the donors of the Petroleum Research Fund, administered by the ACS. The authors are indebted to Professor G. Waller for the loan of a Tektronix 475 oscilloscope_
References [ 11 JS. McClelland and R.N. Knaeley,
Appl. Phys Letters 28 (1976) 467. 121 D. Magde and M.\V.\Vmdsor,Chem. Phys. Letters 24 (1974) 144. [3] 1.0. .%arobogatov, Opt. Spectry. 42 (1977) 172. [4] T.K. Razumova and I 0. Starobogatov, Opt. Spectrq. 42 (1977) 274.
599
CHEMICAL
THE PHOTOACOUSTIC
DETERMINATION
PHYSICS
OF FLUORESCENCE
Mark G. ROCKLEY
and Kristy M. WAUGH
Chemistry Department,
Oklahoma State University. Stillwater.
Received
1977
14 November
15 March 1978
LPXTERS
YiELDS OF DYE SOLUTIONS
Oklahoma 74074, USA
A new method for the measurement of the primary fluorescence quantum yield of dye solutions is presented. The method is conceptually and experimentally simple, relying on the .S, -- S1 transition probabdity as an internal standard for comparison with the Si radlationless transltlon probability, both probablhties being measured by photoacoustlc spectroscopy.
1. Introduction
It is valuable throughout the field of photophysics to have a reliable measure of the fluorescence quantum yield, this yield being the fraction of those initially excited molecules which return directly to the ground state by a radiative transition. It will be shown by this work that fluorescence yields (+f) of dye solutions (indeed, solutions of any chromophore) can be measured from analysis of the liquid phase photoacoustic spectra VIWhen an aromatic chromophore in solution such as a dye, is excited by light, the transition can be to the lowest energy excited singlet state (S;,with energy relative to the ground state E(S,)) or to higher energy excited singlet states (S,, with energies E(S,)), depending on the wavelength of the incident light. It is well known that, with a few exceptions such as azuIene, the higher states, S,, radiationlessly decay to the lowest excited state, S 1, with an efficiency close to 100%. That is, for every absorbed photon of energy E(S,), that fraction of the energy corresponding to [I!?@,) -WV1 I%%) WI‘11immediately appear as heat coming from the sample. However, S, decay may involve radiative and nonradiative components. (For purposes of this particular experiment, phosphorescence emission yields will be ignored. However, the general method outlined here can be extended to phosphorescent systems by making use of the phase resolution of the photoacoustic spectra_) Indeed, a fraction, (1 - ipf), of all the energy absorbed by St + So transitions, will appear as heat, where Gf is the fluorescence quantum yield.
Photoacoustic spectroscopy (PAS) can be used to measure these heat signals. The essentrals of liquid phase PAS have been described elsewhere [ 1J _ It is sufficient for now to say that the PAS spectrometer will give a signal proporttonal to the total heat emitted by the sample, whether excited into the S, or the S, state. If the proportionality constant is k, then the PAS signal from excitation into S, and St , respectively, can be described by tne two equations: IpAs(Sn)=kiE(Sn)-E(S1)+E(S1)(l
-af)]
(1)
and IpA’
= k[E&)(l
- &)I _
(2)
Since E(S,) and E(S,) are known quantities, whrle IpAs(S,) and I p*s(S,) are measured quantities, the convenient situsrion exists of having two equations with two unknowns to solve. In particular, this enabIes the easy determination of the fluorescence quantum yield, @t, of the molecule, relying only upon the measurement of the corrected photoacoustic signal inrensitres for excitation into the S, and St states of *he molecule.
2. Experimental The sample used to test the expertmental method was recrystaliized crystal violet, [(CH,),N-C6H4] 3C+Cl-, which was tested by TLC on silica with a range of acetone, ethanol, chloroform solvent systems and found to have no visibly detectable impurities. The dye was dissolved in distilled HZ0 at a concentration of 2.02 597
CHEMICAL PHYSICS LETTERS
Volume 54, number 3
mg/ml, giving a solution wrth an optical density 6001 cm. The photoacoustic sample chamber had a total volume of 1S ml and was connected to a CRl9629601 $” diameter foil electret microphone. The signal from the microphone was amplified by an Ithaca 143L preamplifier and processed by an Ithaca 39 1A lock-in amplifier. The internal frequency signal of the lock-in was used to modulate the current supply to the 1 kW xenon arc lamp, hence providing the modulation necessary to observe the photoacoustic signal. The power supply for the arc-lamp was purchased from Photochemical Research Associates in Canada but had tc be modified to ensure relrable lamp operation in the rnodulation mode. While the lamp could be modu!ated anywhere within the 100 Hz to 10 kHz frequency range, this experiment was run at a frequency of 185 Hz. This frequency provided the minimum intemrodulation with the line power frequencies which were superimposed on the power supply output. The PAS liquid phase spectra shown in fig. 1 were obtained by signal averaging. The Sr +-So spectra were more intense than the S, + So spectra both because of the high sample absorptivity and the higher brightness of the xenon lamp in this spectral region. Therefore, three PAS spectra were averaged for the St -+ So spectrum while six spectra had to be averaged to obtam the S,, + St-, PAS spectrum with comparable signal to noise
50
e&o
550
!xcJ 4!xJ -VMbELENGW
400 (NM)
3!3J
598
levels. Because of the poor signa to noise values for the experiment reported here (approximately 5 to 1 for unaveraged spectra in the 300 nm region) the uitimate reliability of the experimental results is low. This is a drawback primarily due to the very low spectral brightness of the arc-lamp monochromator combination (< 1 mW/nm) rather than the experimental method. However, as wil1 be shown, the data are sufficiently reliable to demonstrate the utility of the method, especially when high brightness sources such as lasers are available. For these experiments, a bandpass of 6.4 nm was necessary to obtain a sufficientIy strong signal. The xenon lamp~monochromator combination power spectrum (a PAS spectrum of carbon black) is included in fig. 1 for reference, although the crystal violet spectra shown are not corrected. The wavelength calibration of the monochromator was thus made by referring to the peaks in the power spectrum as well as by the use of a hehum-neon laser. Therefore, the PAS spectra presented are correct with respect to the wavelength-power distribution_ Incidentally, second-order transmission by the monochromator (600 nm setting passing 300 nm light) was checked with a cutoff fiter. It was found to be 5% and so ignored because of the high noise IeveIs of the results reported. The monochromator was a McKee-Pederson 0.5 meter monochromator with a 300 nm blaze grating and a 16 A/mm reciprocal dispersion. The absorption spectrum presented in fig. 1 was obtained with 0.5 rtm resolution on a Beckman model 2.5 absorption spectrometer, calibrated with a holmium oxide filter for wavelength accuracy. Absorption linearity is not a problem since the experiment requires measurement of the PAS signal intensities at two different wavelengths (one for the Sr + So band, the other for the S, f- So band) where the absorbance is the same. To obtain the absorption spectrum of the crystal vrolet solution used here, a drop of the solution was squeezed between two optical flats, forming a layer 0.01 mm thick. The resulting thin “cell” was then sealed and the absorption spectrum measured.
300
TIN. 1. The xenon lamp poner spectrum is represented by the solrd line; the liquid phase PAS spectrum and the absorption spectrum scales are slrahtly displaced for ckrrty of presentatfon.
15 March 1978
3. Results and conclusions The concentration sampIe had an optica
of the solutron was such that the density greater than one in the
CHEMICAL
Volume 54, number 3
PHYSICS LETTERS
thermally active layer [ 1] , but was below the saturation limit. Furthermore. the results descrrbed below are calculated at two wavelengths (617 nm and 306 nm) where the sample has equal optical densities. Therefore, the same fraction of incident photons will be absorbed at each wavelength and the absorbed photons will have an equivalent distribution throughout the thermally active layer. From fig. 1, the following information can be obtained: at 617 nm and 306 nm, the absorbances are the same; the sample PAS intensities are 33 units at 617 run and 29 units at 306 nm; and the power spectrum has intensities of 160 units at 617 nm and 142 units at 306 nm. If eq. (1) is divided by eq. (2), using the wavelengths 617 nm for E(S,) and 306 nm for E(S,,) then the following result is obtained: IPAS(SJ/IPAS(S,)
= (1.02 + 1 - @r)/(l
- (Pf).
(3)
However, the PAS signal intensities need to be corrected so that both correspond to an eqlcal number of phofoons striking the sample. This correction can be made by multiplying IpAs(S,,) by the ratio of the power spectrum intensities at the two wavelengths and by the reciprocal of the ratio of the photon energies. Therefore, the corrected IPAS(S,*) has a value of 66 units. The ratio in eq. (3) is then equal to 2.0. Errors due to noise in the averaged spectra actually make this ratio equal to a value of 2.0 -i- 0.10 giving a fluorescence yield of 0.02 f 0.10. It seems inappropriate to have an error possibility of i 0.10 on a measured yield of 0.02. However, this error is due primarily to the high noise levels associated with measuring PAS liquid phase spectra with low brightness sources. Nevertheless, let it be taken that the fluorescence yield is between 0.00 and 0.10, and that the nonradiative processes have a quantum yield of 0.98 F 0.10. Magde and Windsor [2] measured the singlet state lifetime of crystal violet in H,O, finding a value of 10-l t s. If one assumes that crystal violet (emax = lo5 Q M-l), hke most xanthene derivatives with such large
extinction
coefficients, has an mtrinsic lifetime of 5 X
lo-’ s, then the fluorescence yield could be inferred to be 0.01. This IS in excellent qualitative agreement with the yield of 0.02 reported in this work. It is therefore demonstrated that liquid phase photoacoustic spectroscopy can be used as a powerful tool for measuring primary fluorescence quantum yields. Although the measurement error for these experiments
15 March 1978
is
large, this is due only to the lack of spectral brightness of the exciting source, and does not reflect upon the
wide utihty of this new method. No external standards or calibrat.ons other than a source power spectrum are required. Furthermore, the method may be applied to gas and soPd phase systems, provided high brightness sources at the appropriate wavelengths are available. Starobogatov and co-workers [3,4] have hinted at such methods for determining fluorescence yields. However, they have resorted to using a nonfluorescent “standard” as a means of comparing expected versus obtained PAS signal intensity. This brings back the problem of instrument calibration etc. The method described in this work relies upon the unit probability of S,, S, internal conversion for the provrsion of an intensity cahbrant, in this way providing a much less cumbersome measuring tool. The validity of this technique rests on the unit quantum efficiency for the S,, S, internal conversion. This is a generally accurate assumption for dyes and most aromatics. with the notable exceptions, thioketones, azulenes, etc. Furthermore, it is necessary that the sample optical density be below the saturation limit as explained by McClelland et al. [ I] . (This was true for the experiment reported here -the optical density was 600 cm-’ at 185 Hz.) It is expected that the method will find much use in the study of high optical density dye solutions, especially when lasers are used as exciting sources.
Acknowledgement
The authors gratefully acknowledge partial support of this research by the Research Corporation and by the donors of the Petroleum Research Fund, administered by the ACS. The authors are indebted to Professor G. Waller for the loan of a Tektronix 475 oscilloscope_
References [ 11 JS. McClelland and R.N. Knaeley,
Appl. Phys Letters 28 (1976) 467. 121 D. Magde and M.\V.\Vmdsor,Chem. Phys. Letters 24 (1974) 144. [3] 1.0. .%arobogatov, Opt. Spectry. 42 (1977) 172. [4] T.K. Razumova and I 0. Starobogatov, Opt. Spectrq. 42 (1977) 274.
599