1738
J. Phys. Chem. 1992,96, 1738-1742
Fluorescence Quantum Yield of Cresyl Violet in Methanol and Water as a Function of Concentration Stefan J. Isak and Edward M. Eyring* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112 (Received: June 17, 1991; In Final Form: October 7, 1991)
Photothermal spectroscopies and fluorimetry have been used to determine absolute and relative fluorescence quantum yields for cresyl violet perchlorate in methanolic and aqueous solutions as a function of concentration. The concentration dependence of the fluorescencequantum yield is found to be significant and cannot be ignored as in past studies. The values of the fluorescence quantum yield are found to be affected by inner filter effects. In aqueous solution, the fluorescence quantum yield is also affected by quenching attributable to water structure and dye dimerization.
Introduction Cresyl violet perchlorate (CV, Figure 1) has been proposed as a standard for relative fluorescence quantum yield determinations.Iv2 It is an ideal candidate as a fluorescence standard since it is photochemically table,^-^ it has an insignificant triplet-state population,6 and its fluorescence quantum yield appears to be independent of the nature of the solvent, except in water. CV has an accepted literature value for its fluorescence quantum yield, $F, of 0.54 f 0.03 based on a 1979 study by Magde et a1.I In this study1 the value of 4Fwas determined using conventional calorimetry as well as thermal lens spectrometry. The value for 4Fwas reported to be independent of concentration. Since that time, however, several studies have appeared describing the concentration dependence of 4F for a variety of dyes including cresyl and rhodamine 6G,7J0another accepted fluorescence standard. The present study resulted from an attempt to utilize CV as a standard for a series of relative fluorescence quantum yield determinations on a particular group of dyes. We obtained a considerably higher value of -0.65 for $F of CV in dilute methanolic solutions. While not agreeing with most literature values for $F, this value is close to an early reported5 value of 0.70. Our value was determined absolutely utilizing a photothermal spectroscopic technique. This unexpected observation prompted us to characterize the absolute 4Fof CV in methanol over a wide concentration range, from to M. Absolute and relative values of 4Ffor CV in water were also determined. Photothermal beam deflection spectroscopy (PBDS) and photothermal beam deflection photoacoustic spectroscopy (PBDPAS)," as well as fluorescence measurements, were used in our determinations of 4F. While PBDS has been used in a wide variety of studies, PBDPAS has seen relatively little use. We are aware of only one study, conducted by Terazima and Azumi,I2 that has utilized (1) Magde, D.; Brannon, J. H.; Cremers, T. L.; Olmsted, J., 111 J . Phys. Chem. 1979,83, 696. ( 2 ) Reference Compounds for Fluorescence Measurements; IUPAC Pho-
tochemistry Commission: Blackwell Scientific Publications: Boston, 1986. (3) Olmsted. J.. 111 J . Phvs. Chem. 1979. 83. 2581. (4) Drexhage, K.H. In D i e Lasers; Schafer, F. P., Ed.; Springer-Verlag: New York. 1977: D 172. (SjDrexhage, K. H. Laser Focus 1973, 35. (6) Kreller, D. I.; Kamat, P. V. J . Phys. Chem. 1991, 95, 4406. (7) Sakai, Y.; Kawahigashi, M.; Minami, T.; Inoue, T.; Hirayama, S. J . Lumin. 1989, 42, 317. (8) Zhang. G.; Chen, W. Wuli Huaxue Xuebao 1990, 6 , 163. (9) Guilan, Z.; Wenju, C. Acta Phys.-Chim. Sin. 1990, 6, 169. (10) Sathy, P.; Philip, R.; Nampoor, V. P. N.; Vallabhan, C. P. G. Pramana 1990, 34, 585. ( I 1) In PBDS the observed signal is a result of changes in the refractive index of the solvent due to the heat generated by nonradiative processes along the path of the probe beam. In PBDPAS the observed signal is a result of the acoustic wave produced due to the heat generated by nonradiative processes traversing the path of the probe beam. ~
~
0022-3654/92/2096-1738$03.00/0
PBDPAS in the determination of the quantum yield for triplet formation and the triplet lifetime of quinoxaline in benzene. In general, the photoacoustic signal is detected by either a microphone or piezoelectric detector. We have found optical detection of the photoacoustic signal to be just as satisfactory in terms of the signals observed12as well as more sensitivei3and more convenient in terms of the ease with which we can change back and forth between PBDS and PBDPAS measurements. Optical detection allows for the simultaneous detection of both signals,14 if so desired. Currently, we are able to accomplish this using a photomultiplier detector as opposed to two independent photodiode detectors as previously reported.14 The theoretical basis of the optically detected photoacoustic signal has been discussed in detail by both Sullivan and Tam15 and Terazima and Azumi.I2 Experimental Section The experimental setup is shown in Figure 2. The excitation wavelength was 610 nm obtained from the pulsed output of a Lambda Physik FL2002 dye laser using rhodamine 640 perchlorate as the lasing dye. The dye laser was pumped with the frequency-doubled A = 532 nm output from a Quanta Ray DCR-2 Nd:YAG laser. The excitation dye laser pulse energy was varied between 0.1 and 1 J/pulse, and the pulse repetition frequency was 10 Hz. The probe beam was supplied by either a 3-mW diode laser operating at 780 nm or a 5-mW HeNe laser, depending on the absorbance of cresyl violet at the probe wavelength. Neutral density filters were used to adjust the excitation and probe beam energies. Observed signal intensities were checked for linearity with respect to energy for all concentrations. The 610-nm output of the dye laser was spatially filtered for the PBDPAS measurements. A photomultiplier tube (Hamamatsu R928) wired for nanosecond responsei6 was used to detect the probe beam signal. Waveforms were signal-averaged a minimum of lo00 times using a LeCroy 9400 oscilloscope and were stored on disk using an IBM PC compatible computer. The sample cells were made of quartz with a I-cm optical path length. Fluorescence measurements were carried out on a Perkin-Elmer MPF-66 fluorescence spectrophotometer. Emission spectra were corrected using a totally reflecting surface supplied by the manufacturer. A Perkin-Elmer Lambda 9 spectrophotometer was used for absorbance measurements. Refractive indices were taken to (12) Terazima, M.; Azumi, T. Chem. Phys. Lett. 1991, 176, 79.
( I 3) Komorowski, S.J.; Eyring, E. M. In Photoacoustic and Photothermal Phenomena: Springer Series in Optical Sciences; Vol. 58. Hess, P., Pelzl, J., Eds.; Springer-Verlag: New York, 1988; p 484. (14) Komorowski, S.J.; Isak, S.J.; Eyring, E. M. In Photoacoustic and Photothermal Phenomena: Springer Series in Optical Sciences; Hess, P., Pelzl, J., Eds.; Springer-Verlag: New York, 1988; Vol. 58, p 103. (15) Sullivan, B.; Tam, A. C. J . Acoust. SOC.Am. 1984, 75, 437. (16) Harris, J. M.; Lytle, F. E.; McCain, T. C. Anal. Chem. 1976, 48, 2095.
0 1992 American Chemical Society
The Journal of Physical Chemistry, Vo1. 96, No. 4, 1992 1739
Cresyl Violet in Methanol and Water
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Figure 2. Experimental setup: PMT, photomultiplier tube; R., razor edge; P, prism; SC, sample cell; BS, beam splitter; D, diaphragm; IF, interference filter.
be 1.3288 and 1.3326 for methanol and water, respectively.” All spectral measurements were made at an ambient temperature of 24.0 f 0.2 “C. Rhodamine 640 perchlorate and cresyl violet perchlorate were obtained from Exciton and used as received. Potassium iodide was obtained from Mallinckrodt, recrystallized from water, and dried and stored under vacuum in a desiccator. Spectrophotometric grade methanol was obtained from EM Science and used as received. Ultrapure water was obtained from a Corning MP-190 water purification system connected to a MP-1 water still. A typical PBDPAS signal is shown in Figure 3. Absolute fluorescence quantum yields were deduced from the PBDPAS measurements according to the following e q u a t i ~ n ’ , ~ - ’ ~ - ~ ~
4F =
(: 5) 1-
where XF is the mean fluorescence wavelength determined from corrected emission spectra, A,, the excitation wavelength, Hothe nonradiative emission intensity of the sample, and Hrcrthe nonradiative emission intensity of a nonluminescent reference. Samples of cresyl violet quenched with potassium iodide were used as the nonluminescent reference. This choice was made for several reasons. First, in both methanolic and aqueous solutions addition of KI resulted in essentially complete quenching of the fluorescence as determined from fluorescence measurements. Aqueous solutions of cresyl violet were quenched at a potassium iodide concentration of 1 M. Methanolic solutions saturated with potassium iodide were used to quench the fluorescence of cresyl violet in these solutions. Second, the addition of potassium iodide had little or no effect on the absorption spectra of cresyl violet (17) CRC Handbook of Chemistry and Physics; Weast, R. C., Ed.;CRC Press: Boca Raton, FL, 1979. (18) Callis, J. 9. In Standardization in Spectrophotometry and Luminescence Measurements: NBS Special Publication 466; Mielenz, K. D., Velapoldi, R. A., Mavrodineanu, R., Eds.; U S . Government Printing Office: Washington, DC, 1977. (19) Lahmann, W.; Ludewig, H. J. Chem. Phys. Lett. 1977, 45, 177. (20) Starobogatov, I. 0. Opt. Spectrosc. (Engl. Transl.) 1977, 42, 172. (21) Cahen, D.; Garty, H.; Becker, R. S. J . Phys. Chem. 1980,84, 3384. (22) Lesiecki, M. L.; Drake, J. M. Appl. Opr. 1982, 21, 5 5 7 . (23) Zhang, G.; Li, 2.;Yan, J. Chin. Phys. Lett. 1986, 3, 9.
in methanolic or aqueous solutions. In aqueous solutions, however, there was a sudden decrease in absorbance along with precipitation of the sample a t concentrations above 10” M. The reason for this will be discussed below. Finally, by choosing cresyl violet itself as the reference compound, any anomalous absorption, quenching, or reabsorption effect would be canceled out. Absolute values of & for CV in methanol were also obtained by a combination of PBDS and fluorescence measurements using a method developed by Sabol and RockleyZ4requiring low-level luminescence quenching. Again, quenching of the CV fluorescence was accomplished using potassium iodide. The absolute fluorescence quantum yield is described by the following equation
where mF is the slope of the line obtained by plotting the relative fluorescence intensity as FIFO,where F is the fluorescence intensity with quencher added, and Fo without, versus quencher concentration. mH is the slope of the line obtained by plotting the relative nonradiative emission intensities as H/Ho, where H is the nonradiative emission intensity with quencher added, and Howithout, versus quencher concentration. Relative fluorescence quantum yields of CV in water were determined relative to CV in methanol from fluorescence measurements according to the following r e l a t i o n ~ h i p ~ ~ . ~ ~
where 4F,reris the fluorescence quantum yield of a reference compound, A the absorbance, nD the index of refraction of the solvent, and a the area under the fluorescence peak. This expression is only valid, however, assuming that the relationships between absorbance and concentration and the area under the fluorescence peak and concentration are linear. At concentrations of CV above 10” M the relationship between the area under the fluorescence peak and concentration is no longer linear (see Figure 6). The reason for this will be discussed below. To compensate for this nonlinearity, solutions of matching absorbances of CV in methanol and water were used to determine $F from eq 3. Results and Discussion All of our results are combined and presented in Figure 4. Error bars represent the standard deviation of the data points.
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(24) Sabol, J . E.; Rockley, M. G. J . Photochem. Photobiol.. A 1987,40, 245. (25) Guilbault, G.G. Practical Fluorescence; Marcel Dekker: New York, 1973; pp 11-14. (26) Bridges, J. W. In Standards in Fluorescence Spectrometry: Miller, J. N., Ed.; Chapman and Hall: New York, 1981; p 75.
1740 The Journal
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of Physical Chemistry, Vol. 96, No. 4, 1992
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concentration [ M I Figure 4. Absolute and relative values of bFfor CV in methanolic and aqueous solutions as a function of concentration: 0 , absolute bF in methanol determined by PBDPAS; 0,relative bFin methanol adapted from Sakai et al.;’ 0 , absolute bFin methanol determined by method of Sabol and R ~ k l e y ;m, * ~absolute QF in water determined by PBDPAS; V, relative bF in water determination by fluorescence measurements.
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concentration [ M I Figure 7. Absorbance at 610 nm for cresyl violet in methanolic and aqueous solutions with and without potassium iodide quencher. wavelength (mfw) of cresyl violet as a function of concentration in methanolic and aqueous solution with and without the presence of potassium iodide. It can be seen that at a concentration of approximately 10” M the mfw begins to increase rapidly with concentration. This is a consequence of the inner filter e f f e ~ t . ~ ~ ” ~ This observation is confirmed in Figure 6 where the area under the fluorescence peak, for measurements with CV in methanol and water, is plotted as a function of concentration. From Figure 6 it is evident that the relationship between area and concentration is no longer linear at concentrations of CV above approximately 10” M. The mfw of cresyl violet has been listed in Table I along with the 4Fvalues. The data presented in Figures 5 and 6 allow us to interpret the results of others based on their reported values for the concentration studied and the mfw reported. It is also possible to consider whether inner filter effects were taken into account and whether they are significant depending on the method by which $F was determined. Such comparisons can be made if similar optical path lengths of 1 cm were used.
(27) Bevington, P. R.Data Reducrion and Error Analysis for the Physical Sciences; McGraw-Hill: New York, 1969;Chapter 4.
(28)Parker, C.A. Photoluminescence of Solutions; American Elsevier: New York, 1968;pp 220-34. (29)Winefordner, J. D.; Schulman, S.G . ;OHaver, T. C. Luminescence Spectrometry in Analytical Chemistry; Wiley & Sons: New York, 1972;pp si -I 03. (30)Demas, J. N.; Crosby, G. A. J . Phys. Chem. 1971, 75,991. (31) Birks, J. B. J . Res. Nail. Bur. Srand., Secr. A 1976, 80, 389. (32)Lloyd, J. B. F. Standards in Fluorescence Spectrometry; Miller, J. N., Ed.; Chapman and Hall: New York, 1981;p 27. ( 33) Guilbault, G. G . Practical Fluorescence; Marcel Dekker: New York, 1973;pp 17-19. (34)Arbeloa, I. L. J . Chem. Soc., Faruday Trans. 2 1981, 77, 1735.
The Journal of Physical Chemistry, Vol. 96, No. 4, 1992 1741
Cresyl Violet in Methanol and Water
TABLE I: Fluorescence Quantum Yields & of Cresyl Violet (CV) Presented as a Function of CV Concentration, Solvent, and Mean Fluorescence Wavelengtb (mfw) [CV],M solvent A,, nm mfw, nm bF ref 10"- 10-5 10-5 10-5 8.51 x 10-5 7.08 x 10-7 2 x 10" 1 x 10-5 10-5 40-4
9 9 9 9 2
x x x x x
10-5 10-5 10-5 10-5 104-2 x 10-3
5.88 x 9.42 x 2.94 X 5.88 X 9.42 X 2.08 x 3.53 x 5.44 x 8.32 x 1.09 x 2.08 X 2.94 x
8.00 x 1.01 x 2.01 x 7.66 x 5.82 x 1.13 X 3.34 x 6.28 X 8.48 X 1.47 x 3.68 x 6.31 x 9.00 x
10-7 10-7 lod 10" 10" 10-5 10-5 10-5 10-5 10-4 lo4 10-4 10-7 10" 10" 10-7 10-7 lo4 10" lod lo4 10-5 10-5 10-5 10-5
methanol methanol ethanol methanol ethylene glycol ethylene glycol glycerol methanol ethanol methanol methanol methanol ethanol ethanol n-propyl alcohol methanol methanol methanol methanol methanol methanol methanol methanol methanol methanol methanol methanol water water water water water water water water water water water water water
'Adapted from Sakai et al. assuming a value for
632.8 5461578 5461578
638 623 623
480-640 441.6 60 1 632.8 590 600 690 600
638 624 638 654 644 644 647 647 623
610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610 610
621 621 623 624 626 629 63 1 634 637 639 640 64 1 623 623 623 623 623 623 623 624 625 627 630 63 1 633
bF of 0.54 at a concentration of 8.51
Absorbance as a function of concentration for CV is shown in Figure 7. Absorbance maxima as well as the absorbance at 610 nm were found to obey Beer's law up to a concentration between 4X and 5 X M. While the absorbance appears to be independent of the presence of KI in methanol, this is not the case in aqueous solution. At a concentration of approximately 10" M, there is a sudden decrease in the absorbance of CV in the quenched aqueous solutions. At CV concentrations above M, the formation of a precipitate is noted. These observations are consistent with a salt effect33-37of potassium iodide enhancing dye aggregation. Addition of a salt to a dye containing solution tends to favor the formation of aggregates. Depending on the nature of the aggregate, it may precipitate from solution. The fact that dyes are more sensitive to aggregation, with or without added electrolyte, in aqueous solution is well e ~ t a b l i s h e d At .~~~ least two studies of the aggregation of oxazine dyes have ap~ e a r e d . ~ Evidence ',~~ of dimer formation is also obtained from
0.54 f 0.03 0.55 f 0.02 0.51 f 0.02 0.70 0.54 0.68 0.54 f 0.04 0.56 f 0.03 0.59 f 0.03 0.57 0.54 f 0.04 0.54 f 0.04 0.60 f 0.04 0.57 f 0.03 0.56 0.49 f 0.06 0.67 f 0.02 0.66 f 0.02 0.65 f 0.02 0.65 f 0.02 0.63 f 0.04 0.64 f 0.02 0.61 f 0.03 0.56 f 0.02 0.58 f 0.02 0.56 f 0.04 0.54 f 0.03 0.51 f 0.02 0.47 f 0.05 0.44 f 0.03 0.43 f 0.04 0.40 f 0.02 0.40 f 0.02 0.39 f 0.02 0.36 f 0.02 0.34 f 0.02 0.33 f 0.02 0.30 f 0.03 0.26 f 0.04 0.17 f 0.04 0.13 f 0.04
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the absorbance spectra of CV in water. In concentrated solutions (>lo-$ M) a dimer peak is observed with a maximum at 556 nm. The absorbance maximum of the monomer occurs at 585 nm. These observations are consistent with the formation of a stacked or sandwich dimer.35*36,38-42-46 A simple comparison of the ratio of absorbances at these two wavelengths indicates an observable dimer absorbance at a CV concentration of -3 X lod M. This concentration is in agreement with that for which dimerization was observed to occur for a similar oxazine dye.47 It is now possible to interpret the 4Fvalues of CV in aqueous solution as a function of concentration presented in Figure 4. The relative values for 4Fin the aqueous solutions are both lower and apparently more concentration dependent than in the corresponding methanolic solutions. Three factors are responsible for the 4Fvalues obtained. First, the value of 4Fin aqueous solution can be expected to be less than that in methanol due to the ~~~
(35) Valdes-Aquiler, 0.; Necker, D. C. Acc. Chem. Res. 1989, 22, 171. (36) Burdett, B. C. In Aggregation Processes in Solution; Wyn-Jones, E., Gormally, J., Eds.; Elsevier: New York, 1983; p 241. (37) Craven, B. R.; Griffith, J. C.; Kennedy, J. G. Aust. J . Chem. 1975, 28, 1971. (38) Coates, E. J. SOC.Dyers Colour. 1969, 85, 355. (39) Herz, A. H. Adu. Colloid Interface Sci. 1971, 8, 237. (40) Mason, S. F. J. SOC.Dyers Colour. 1968, 84, 604. (41) Morozova, Yu. P.; Zhigalora, E. B. Russ. J . Phys. Chem. (Engl. Transl.) 1982, 56, 1526. (42) Herkstroeter, W. G.; Martic, P. A,; Farid, S . J . Am. Chem. Soc. 1990, 112, 3583.
(43) Bergmann, K.; OKonski, C. T. J. Phys. Chem. 1963, 67, 2169. (44) Arbeloa, I. L. J. Chem. Soc., Faraday Trans. 2 1981, 77, 1725. (45) Chambers, R. W.; Kajiwara, T.; Kearns, D. R. J . Phys. Chem. 1974,
78, 380. (46) (47) (48) (49)
Selwyn, J. E.; Steinfeld, J. I. J. Phys. Chem. 1972, 76, 762. Gvishi, R.; Reisfeld, R. Chem. Phys. Lett. 1989, 156, 181. Sens, R.; Drexhage, K. H. J. Lumin. 1981, 24-25, 709. Petukhov, V. A.; Popov, M. B.; Krymova, A. I. Sou. J. Quantum
Electron. 1986, 16, 503. (50) Nizamov, N.; Umarov, K. U.; Dzhumadinov, R. Kh.; Atakhcdzhaev, A. K. Opf.Spectrosc. (Engl. Transl.) 1983, 54, 600. (51) Wang, T.; Zhang, B.; Pan, J.; Gu, P.; Xu,Q.; Sun, Q. Chin. Sci. Bull. 1989, 34, 1756.
Isak and Eying
1742 The Journal of Physical Chemistry, Vol. 96, No. 4, 1992
quenching effect of water on fluorescence. Second, as in the case of the methanolic solutions, the inner filter effect can be expected to result in a decrease of 4Fwith increasing concentration. These two effects would result in a curve for the aqueous 4F values paralleling those of the methanolic 4Fvalues. This appears to be true in the more dilute aqueous solutions, but at concentrations above 10” M, there is a more rapid decrease in the value of This rapid decrease is due to considerable formation of dimers in the aqueous solutions. The relative values of 4F in aqueous solution are in good agreement with both a 4Fvalue of 0.24 f 5% at a concentration of (2.0 i 0.5) X M for a similar oxazine dye4’ and the absolute values of C#J~ determined by PBDPAS, at least over the limited range for which values of the latter could be measured. The absolute 4Fvalues were limited to a narrow concentration range for two reasons: The lowest concentration was set by the limit of detection for our measurements. This limitation was regulated by the maximum energy of the excitation pulse and the high thermal conductivity of water. The highest concentration was limited by the aggregating effects of the quencher used. It is unlikely that the use of a different quencher would have a significantly different effect on aggregation because of the salt effect. 4Fand mfw values as a function of concentration for the aqueous solutions of CV are given in Table I. Since ref 1 and this study each used two independent methods to measure dF as checks against experimental artifacts, we look elsewhere for the source of the discrepancy. A more likely explanation may be physical differences between the samples of CV studied. These might include quenching impurities or secondary products formed in the synthesis of CV, such as the corresponding oxazone. A sample of the CV studied by Magde et al. was generously provided by Magde so we could determine whether differences between the samples exist. Fluorescence and absorbance measurements were made on the two samples over a wide concentration range. Fluorescence excitation was carried out at both 610 and 632 nm. These wavelengths correspond to the excitation wavelengths utilized in the photothermal measurements made by us and by Magde et al., respectively. The absorbance and fluorescence spectra were found to be essentially identical, except that in concentrated solutions (> 5 X M) the absorbance of the Magde et al. sample was lower by about 5% at 632 nm, while the corresponding fluorescence spectra remained identical. This implies that the $ J ~of the Magde et al. CV sample should be approximately 5% greater than that of our CV sample. These observations rule out the likelihood of the presence of any impurities in the Magde et al. sample directly quenching the fluorescence of CV. However, the presence of other impurities is still a possibility. A difference in purity of the two CV samples of not more than a percent or so would be difficult to observe if the fluorescence was not directly quenched. A small amount of aggregate-promoting salt could prove to be a problem in concentrated nonaqueous solutions. Upon dilution the aggregating effect would be eliminated due to a dilution of the salt below the critical concentration required for dimer formation. The sensitivity of CV dimer formation to the presence of salt in aqueous solution was discussed above. Such salt-induced dimer formation would be further enhanced if the nonaqueous solvents utilized by Magde et al. contained traces of water. The net effect of dimer formation ~ CV. would, of course, result in a diminished r # ~for Dimer formation would result in the appearance of a second absorbance peak blue-shifted by approximately 30 nm with respect to the monomer peak based on the observations of CV dimer
-
-
formation in aqueous solution. As the dimer peak developed, the monomer peak would decrease in intensity. Such a change would be pronounced at 632 nm, which lies in the extreme red tail of absorbance for CV. This reasoning is consistent with our observations in concentrated solutions of the Magde et al. sample. Excitation wavelength dependence of the 4Fof CV may also be another possibility. As already pointed out, 632 nm lies in the extreme red tail of absorbance for CV. Much, possibly all, of the discrepancy may be traceable to Magde et al. not having worked in the “low concentration limit”, below which $F for CV is independent of concentration. Based on our results, for CV in methanol this is 1 X 10” M. Nowhere in the study by Magde et al. are measurements in such a low concentration range reported. Consider the mfw = 638 nm corresponding to the c $ ~value of 0.54 f 0.03 reported by Magde et al. According to our data (see Table I), a mfw = 638 nm corresponds to a concentration of -9.6 X M and a 4F 0.57 f 0.03. These values of 4Fare the same within experimental error. It appears quite likely that Magde et al. were not working with adequately dilute solutions to be in the “low concentration limit”. The fact that Magde et al. did not observe a concentration dependence of $F may be attributed to the fact that the concentration range of CV studied was not large enough to detect the trend. If the first and last few points of our data for 4Fin Figure 4 are removed, the change in $F with concentration becomes less apparent. Magde et al. report that 4F(CV) was determined over the range 6.8 X 10” to 6.8 X lo4 M. No variation [in &] was detected, although random scatter increased at the extremes.” While the degree of scattering is not indicated, it no doubt contributed to a blurring of the concentration dependence of 4p This would have resulted in the determination of an average value for 4Fwhere the average concentration studied by Magde et al. is -6.8 X M. A comparison with our data (see Table I) at a concentration of 6.8 X M yields a mfw = 636 nm and a 4F N 0.57 f 0.02, again essentially the same value as that of Magde et al. within experimental error. Thus, it appears that the differences in 4F for CV observed between Magde et al. and the present paper are primarily a reflection of inner filter effects and the concentration dependence of dimer formation, possibly enhanced by water contamination or salt effects. The lesson for those using CV as a fluorescence standard is the need to work at low concentrations and to be increasingly fastidious about solute and solvent purity as concentrations increase.
-
Conclusion Both absolute and relative fluorescence quantum yields for cresyl violet in methanolic and aqueous solutions as a function of concentration have been determined. It was found that the change in 4F with concentration is significant and cannot be ignored, especially when considering CV as a fluorescence reference standard. In aqueous solution the decreased values of 4F are attributable to the quenching effects of water and dimer formation. Acknowledgment. This research was funded by the Department of Energy, Office of Basic Energy Sciences. The experiments were initiated under a Biomedical Research Support Grant (USPHS No. RR07092). The authors thank J. D. Spikes for the use of his spectrophotometer and fluorimeter and D. Madge for generously providing a sample of his CV and for his helpful comments. Registry No. Rhodamine 640 perchlorate, 72102-91-1; cresyl violet perchlorate, 52659-20-8.
