pH Effects on Fluorescence of Umbelliferone David W. Fink and Walter R. Koehler Lever Bros. Research and Development, 45 River Road, Edgewater, N. J. 07020
The intense blue and blue-green fluorescence bands of umbelliferone (7-hydroxycoumarin) in aqueous solution are studied as a function of pH and excitation wavelength. Based upon the correlation of absorption and excitation spectra at several pH levels, ground and excited state acid-base species are assigned and the fluorescence intensity-pH profiles are explained. The excited state is a much stronger acid than the ground state species. The green fluorescence band which develops with irradiation in alkaline solution is assigned to the cinnamic acid photolysis product of u m belliferone. SOMECOUMARINS are naturally occurring intensely fluorescent physiologically active compounds of general analytical and biological interest. For example, fluorogenic coumarin substrates have been used in enzyme determinations at pH levels adjusted for optimal enzyme activity ( 1 - 2 ) , the fluorescence of 7-hydroxycoumarins (umbelliferones) has been employed in analytical methods for malic acid (3-3, umbelliferone derivatives are found as biologically originated components of air pollution samples (6), and the excited (triplet) states of these compounds are suspected of participating in photobiological energy-transfer processes ( 7 ) . The fluorescence of substituted coumarins is so efficient that these compounds are often used as fluorescent whiteners in detergent products (8,9). A recent report on the total luminescence of coumarins (10) recorded data for absolute ethyl alcohol solutions ; however no data were given for changes in emission spectra with pH, solvent, excitation wavelength, or other conditions. Similarly, another recent study which correlated umbelliferone fluorescence intensity at the reported “pH of maximum fluorescence” (Le., pH 10) with Hammett substituent constants (11) did not consider any other pH’s, nor reasons for the dependence of fluorescence wavelengths and intensities on PH. The numerous applications of umbelliferone fluorescence underscore the need for a description of the fluorescence properties of this molecule in fluid solution. I n particular, a description of the effects of pH, solvent, irradiation, concentration, and excitation wavelength might increase the sensitivity and applicability of fluorometric enzyme and other determinations as well as elucidate other observations of biological and chemical interest. EXPERIMENTAL Apparatus. An Aminco fluorometer was used for all
fluorescence measurements. It was equipped with an Aminco 416-992 Xenon lamp and a 1P21 photomultiplier tube. For (1) G. G. Guilbault and J. Hieserman, ANAL. CHEM., 41, 2006
(1969). (2) G. G. Guilbault, ibid., 40, 459R (1968). (3) E. Lieninger and S . Katz, ibid., 21,1375 (1949). (4) J. P. Hummel, J. Bid. Chem., 180, 1225(1949). (5) C. G. Barr, Plant Physiol., 23, 443 (1948). (6) E. Sawicki and C. Golden, Microchem. J., 14,437 (1969). (7) D. R. Graber, M. W. Grimes, and A. Haug, J. Chem. Phys., 50,1623 (1969). (8) K. R. Lange, Deterg. Spec., 6,19 (1969). (9) H. Haeciserman, U. S . Patent 2,881,186 (1959). (10) H. W. Latz and B. C. Madsen, ANAL.CHEM., 41, 1180 (1969). (11) W. R. Sherman and E. Robins, ibid., 40, 803 (1968). 990
0
all fluorescence measurements, the excitation monochromator slit widths were 5.0, 4.0, and 3.0 mm; the emission monochromator slit widths were 4.0, 3.0, and 2.0 mm for the entrance beam and 3.0 mm for the exit beam to the photomultiplier tube. The sample container was a fused quartz fluorescence cell, 10.5 mm i d . , square X 46 mm high. All emission intensities were read on the Aminco “Photomultiplier Microphotometer” and recorded relative to the fluorescence intensity (450 nm) of a 1.0-ppm quinine sulfate in 0.1N sulfuric acid solution (350 nm excitation). Excitation and emission spectra were recorded on a strip chart recorder uncorrected for frequency dependence of source excitation intensity, photomultiplier output, or monochromators; peak wavelengths reported are probably =k ca. 2 nm, but correlation with absorption data was satisfactory for this investigation. pH was measured on a Leeds and Northrup pH meter standardized against Fisher certified standard buffer solutions. Ultraviolet absorption spectra were recorded on a PerkinElmer Model 202 spectrophotometer, but all quantitative absorption measurements were made with a Shimadzu QV-50 spectrophotometer. Reagents. 7-Hydroxycoumarin was obtained from Aldrich Chemical Co. Purity was checked by thin-layer chromatography: ethyl alcohol solutions of the compound were spotted (1 pl) on 20- X 20-cm silica gel plates; water was the developing solvent. The blue fluorescence of 7-hydroxycoumarin (R, 0.6) was observed on the developed TLC plates under ultraviolet light. No additional spots were detected. Unsubstituted coumarin and o-hydroxycinnamic acid were purchased from the same supplier.
-
RESULTS BLUE EMISSION.7-Hydroxycoumarin exhibits an intense blue or blue-green fluorescence band in the pH range 1.2-1 1.2. The shape of the intense blue fluorescence band (peak emission: 460 nm) illustrated in Figure 1 was pH-independent for all pH’s greater than 2.2. The emission color appears blue-green only at very low pH (less than 2.2) ; the concomitant change in fluorescence spectrum (peak emission: 480 nm) is also illustrated in Figure 1. The blue emission observed above pH 2.2 is more intense than the lower energy band of the more acidic solutions. The blue band (at 460 nm) was observed by excitation at either 330 nm or 370 nm, depending upon pH. The excitation wavelength for solutions of pH less than 6 was 330 nm; 370 nm was used at pH greater than 8. For the intermediate pH values such as 7.3 and 7.6, the excitation wavelengths used were 340 nm and 350 nm, respectively. The fact that the same emission spectrum was recorded under different excitation wavelengths is another indication of the purity of the emitting species (12,13). The pH of an 8.0 X lO-7M aqueous solution of 7-hydroxycoumarin was adjusted by dropwise addition of hydrochloric acid and sodium hydroxide and the fluorescence intensity of the blue band was measured at 460 nm as a function of pH at Fluorescence Spectra.
(12) I. B. Berlman, “Handbook of Fluorescence Spectra of Aromatic Molecules,” Academic Press, New York,N. Y.,1965, p 18. (13) I. B. Berlman, H. 0. Wirth, and 0. J. Steingraber, J. Amer. Chem. Soc., 90,566 (1968).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 9, AUGUST 1970
30
ta I
I
I
l
l
1
1
1
1
1
l
l
l
l
l
I
I
I
I
- B
0.8
0.6
Y
0
z
0.4
2E
-
-
-
$
9
0.2-
L ..~
00
d I
A--! 2
P
I
4
6
I
8
10
12
PH
360
400
450
500
510
WAVELENGTH, nm
Figure 1. Umbelliferone fluorescence spectrum as a function of p H 7-Hydroxycoumarin concentration = 8 X lO-'M; excitation wavelength: 330 nm; spectrum I , pH 2.2; 2, pH 1.8; 3, pH 1 .5; 4, pH 1.2
two excitation wavelengths. The data are presented in Figure 2A. GREEN (500 nm) EMISSION BAND. An intense green fluorescence was observed in aqueous 7-hydroxycoumarin solutions of high pH, as well as in similar solutions of the unsubstituted parent molecule, coumarin. The unsubstituted coumarin does not exhibit the blue or blue-green fluorescence band which would be analogous to umbelliferone. Therefore, to avoid the complications of other emitting states and of ground and excited state phenolic acid-base equilibria, the low energy (green) band was investigated through the unsubstituted coumarin. The intense green fluorescence of coumarin and of umbelliferone is absent when solutions are initially prepared-this band develops with time. Several 1.1 x 10-4M solutions of coumarin were prepared at different sodium hydroxide concentrations and the fluorescence intensity which developed at 500 nm was measured as a function of time. The data for the first 10 minutes for some of these solutions are presented in Figure 3. These samples were under constant irradiation in the fluorometer at the excitation wavelength, 370 nm. Samples of the same solutions were stored in the dark and the fluorescence intensity at 500 nm (370 nm excitation) was measured after 0.5 hr. The measured intensities were 0.07, 2.6,
Figure 2. A . Umbelliferone fluorescence intensity us. p H 7-Hydroxycoumarin concentration = 8.0 X 10-7M; emission wavelength: 460 nm 1. Excitation wavelength: 370 nm; 2. Excitation wavelength: 330 nm
B. Umbelliferone absorbance us. p H 7-Hydroxycoumarin concentration = 6.0 X 10-5M; 1.00-cm cells I . Absorbance at 370 nm 2. Absorbance at 330 nm 7.0, and 12 for hydroxide concentrations of 1 x 10-4N, 5 X 10-4N, 5 x lO-aN, and 1 x 10-2N, respectively. These values are comparable to those values given in Figure 3 after 1 minute of irradiation. The intensity of the green fluorescence of these solutions then increased rapidly once irradiation was begun. A 1.1 X 10-5M coumarin in 0.1N NaOH solution was exposed to daylight for 0.5 hr and the fluorescence spectrum was recorded (370 nm excitation). The green fluorescence band, Figure 4, was superimposable upon the fluorescence spectrum of o-hydroxycinnamic acid (3 X 10-6M in 0.1N NaOH; 370 nm excitation). Excitation Spectrum. The excitation spectrum of the blue fluorescence band was studied by varying the pH of an 8 X lO-7M umbelliferone solution and recording fluorescence intensity at 460 nm us. excitation wavelength and pH. The resulting spectra are presented in Figure 5 . Absorption Spectrum. The ultraviolet absorption spectrum of umbelliferone has been studied in detail. Previous reports demonstrate that the protonated umbelliferone molecule in acid solution (pH less than 6) (14,15) or in methyl (14) G. K. Sutherland, Arch. Biochem. Biophys., 75, 412 (1958). (15) B. N.Mattoo, Trans. Faraday Soc., 52, 1184 (1956).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 9, AUGUST 1970
991
./.
I
0
L 0
1
2
4
3
5
6
7
8
9
10
TIME, min.
Figure 3. Umbelliferone fluorescence intensity at 500 nm V S . time 7-Hydroxycoumarin concentration = 1.1 X 1O-W; excitation wavelength: 370 nm 1. [OH-] = 1 X 10-4N; 2. [OH-] = 5 X 3. [OH-] = 1 X lO-3N: 4. [OH-] = 5 X lO+N; 5. [OH-] = 5 X 10-2N
250
300
350
400
WAVELENGTH, nm
Figure 5. Umbelliferone fluorescence excitation spectrum as a function of pH 7-Hydroxycoumarin concentration = 8 X lO-'M; emission wavelength: 460 nm; spectrum 1, pH 1.9; 2, pH 6.3; 3, pH 7.3; 4, pH 8.0; 5, pH8.6
-
I
alcohol (16, 17) or ethyl alcohol (18-21) exhibits an intense absorption band with, , ,X 324-330 nm, log e 4.1, In basic solution (pH greater than 8) the absorption band which appears at A, 365-370 nm, log e ~ 4 . (11, 2 14, 15, 22) can be assigned to the anion formed from the dissociation of the acidic phenolic proton at the 7-position. Indeed, one study even illustrated an isosbestic point common to the two bands (22). The results of the present study agree with the literature: A, of acid-325 nm,, , ,X of anion-367 nm. The absorbance (1.0-cm cells) of a 6.0 X 10-5M aqueous solution of umbelliferone was measured as a function of pH at 370 nm and at 330 nm. The data are presented in Figure 2B.
\
DISCUSSION
The intense blue fluorescence of umbelliferone at 460 nm is assigned as a transition from the first excited singlet state of the 7-hydroxycoumarin anion. Excitation at 330 nm or at 370 nm yields the same emission spectrum over the pH range 2-11 regardless of which ground state species (protoI
450
500
sm
I 600
650
WAVELENGTH, nm
Figure 4. Fluorescence spectrum of umbelliferone photolysis product Initially: 1.1 X 10-6M coumarin in 0.1N NaOH, spectrum recorded after 0.5 hr daylight irradiation, excitation wavelength: 370 nm (same fluorescence spectrum recorded for o-hydroxycinnamic acid: 3 X 10-GMin0.1 NNaOH; 370 nm excitation) 992
R. S. Shah and S . L. Bafna, Indian J. Chem., 1,400 (1963). D. G . Crosby and R. V . Berthold, Anal. Biochem., 4,349 (1962). C. E. Wheelock, J. h e r . Chem. SOC.,81, 1348 (1959). K. Sen and P. Bagchi, J . Org. Chem., 24, 316 (1959). (20) R . H. Goodwin and B. M. Pollack, Arch. Biochem. Biophys., 49, l(1954). (21) C. R . Jacobson, K. R. Brower, and E. D. Amstutz, J. Org. Chem., 18, 117 (1953). (22) A . Foffani, E. Fornasari, and M. Foffani, Ric. Sei., 27, Suppl. A , Polurogruja, 3, 115 (1957).
(16) (17) (18) (19)
ANALYTICAL CHEMISTRY, VOL. 42, NO. 9, AUGUST 1970
nated or anion) absorbs the excitation energy. These results imply a rapid excited state acid-base equilibrium which takes place during the lifetime of the singlet excited state, 10-8 seconds. The emission spectrum changes only in highly acidic solution (pH less than 2) where it shifts to longer wavelength and appears more green (Figure 1). This lower energy emission can be assigned to the protonated species, yielding an excited state pK,* for umbelliferone which is much different from that of the ground state-Le., pK,* in the range of 1-2. That the excited state of this molecule is a much stronger acid than the ground state is not surprising-similar results have been reported for phenol and other substituted phenols and naphthols ( 2 3 , 24). These data, then, describe the acid dissociation of umbelliferone after excitation by ultraviolet absorption prior to radiative emission in the pH range 2 to 8. There is a striking similarity between the 7-hydroxycoumarin excitation and absorption spectra which allows the assignment of the ground state species which absorb the excitation energy. Both spectra exhibit the same Amax’sand the same pH dependence. Consideration of Figures 5 and 2B explains the results of Figure 2A in the pH range 6-8. The changes in fluorescence intensities are the results of changes in absorbance--i.e., a ground state phenomenon (pK, 8), not an excited state (pKB*)effect, although the fluorescence technique is a measure of the excited state. Further consideration of Figures 5 and 2B confirms that the emission intensity at 460 nm is function of which ground state species is excited. Thus, whether the ground state acid or anion is excited, the shape of the emission spectrum is the same, but the fluorescence is more intense if the excited state acid dissociation is unnecessary. (The molar absorptivities of the acid and the anion are approximately the same at their respective Amax’s.) Conversely, the change in the fluorescence spectrum below pH 2 (Figure 1) explains the measurements of Figure2A-2 below pH 2. In this instance the excitation absorbance does not decrease (Figure 2B-2), confirming that the changed intensity is caused by an excited state phenomenon, the pK.* for the species. Guilbault and Heiserman (I) were able to increase the sensitivity of sulfatase determinations by measuring the fluorescence intensity of the 4-methylumbelliferone sulfate substrate at a pH of 10 because of the effects described above. Goodwin and Kavanagh have reported the fluorescence of coumarin derivatives as a function of pH, but because they used only the 366 nm mercury line for excitation, they erroneously reported that 7-hydroxycoumarin is not fluorescent in acid solution ( 2 5 , 2 6 ) . Figure 2A-1 agrees with the data of Goodwin and Kavanagh ( 2 5 ) and the remainder of the figure demonstrates the importance of excitation wavelength. Other studies of coumarin fluorescence have included similar omissions by studying either only the anion ( 5 , I I ) or only the (ground state) acid (IO,18). Crosby and Berthold did report a 7-hydroxycoumarin fluorescence peak shift to longer wavelength when solvent was changed from pH 10 buffer to 0.1N sulfuric acid (17). They also reported the changes in excitation maximum and the relative emission intensities included in the present study. Two other investigations of substituted
-
(23) H. H. Jaffe and M. Orchin, “Theory and Applications of Ultraviolet Spectroscopy,” Wiley, New York, N. Y . , 1962, p 156. (24) W. Bartok, P. J. Lucchesi, and N. S . Snider, J. Amer. Chem. Soc., 84, 1842 (1962). (25) R. H. Goodwin and F. Kavanagh, Arch. Biochem. Biophys., 27, 152 (1950). (26) Ibid., 36, 442 (1952).
coumarins recorded similar emission peak shifts with acidification (15, 27). However, none of these considered absorbing and emitting species nor did they correlate spectra. The low energy ultraviolet absorption bands of coumarins have been assigned as a* + 7r transitions (28). The large molar absorptivities and the disappearance of these bands with saturation at the 3,4 positions ( 2 0 , 2 9 ) confirm the assignment. In addition, Graber, Grimes, and Haug recently assigned the lowest triplet state of coumarin as (a*,a) and observed the 3a* a 0-0 phosphorescence peak of 7-hydroxycoumarin at 467 nm by irradiation in the long wavelength band (7). The intense blue emissions observed in fluid solution are probably not phosphorescence transitions; there is no reason not to assign these bands as normal la* a fluorescence. Analogous to the absorptions, the fluorescence of coumarins has also been traced to the cr,@-unsaturatedcarbonyl group conjugated with the benzene ring (27). It is valid to compare, approximately, the relative quantum efficiencies of the umbelliferone acid and anion forms from these data under comparable conditions; Le., at the same excitation wavelength (330 nm) so that source intensity is the same, at emission wavelengths in close proximity so that detector sensitivity is similar, and under conditions such that the same amount of excitation energy is absorbed (Figure 2B-2). Figure 1 indicates that the anion has approximately twice the quantum efficiency of the 7-hydroxycoumarin acid. This might be related to resonance stabilization in the anion which is absent in the protonated species. The apparent correlation of fluorescence intensity and the Hammett substituent constant recently reported by Sherman and Robins (II) is then related to increased electron delocalization in the substituted 7-hydroxycoumarin anion. The reason for the correlation with the meta constants for 3-substituted coumarins is still not known, but must be related to excited state (a*)electron redistribution in the anion as they suggest. Several studies have attributed the intense green fluorescence at 500 nm to a transition of coumarin or substituted coumarins (IO,14,27). The results of the present investigation demonstrate that the green band develops with time and that it develops more rapidly under irradiation and at high pH. These data and the spectral identification (Figure 4) prove that the green fluorescence is a property of o-hydroxycinnamic acid, a basic photolysis product of coumarin, which may subsequently undergo cis-trans isomerization in water (30). The 500 nm fluorescence peak of o-hydroxycinnamic acid has been reported previously (31) and splitting of the lactone ring in extreme alkaline solution is expected. Some studies have mentioned that umbelliferone is photolabile at high pH ( 5 , 15, 26) and others have also previously identified the cinnamic acid product ( 1 8 , 3 2 ) . It is reasonable to assume the photolysis of umbelliferone to 2,4-dihydroxycinnamic acid, analogous to the photolysis product of coumarin.
-
-
RECEIVED for review February 10, 1970. Accepted May 26, 1970. (27) Y . Ichimura, YukugakuZusshi, 80,771 (1960). (28) M. E. Perel’son and Yu. N. Sheinker, Teor. Eksp. Khim., 3, 697 (1967); Chem. Abstr., 68,86646d(1968). (29) M. Dezelic, M. Trkovnik, and M. Zovko, Glasnik Hemicara Technol. Bosne Hercegouine, 12, 17 (1963); Chem. Abstr., 63, 17846h (1965). (30) E. F. Ullman, E. Babed, and M. Sung, J. Amer. Chem. Soc., 91, 5792 (1969). (31) “Luminescence Data Sheet,” American Instrument Company, Silver Spring, Md., 1969, p 12. (32) H. Bohme and T. Severin, Arch. Pharm., 290,486 (1957).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 9, AUGUST 1970
993
