the anion, I. As the acidity is increased, there are two possible sites of protonation-either at the dissociated phenolic oxygen, leading to the neutral molecule, 111, o r at the carbonyl oxygen, forming the zwitterionic species, 11. I n very strong acid media, the second protonation will lead to the cation, IV, whether I1 or I11 is the uncharged species from which it is derived. If the stepwise addition of the protons led to a path such as I 111 IV, one would expect to see the fluorescence bands shift from the blue of the anion, I, to shorter wavelengths o n protonation of the phenolate oxygen, and finally to longer wavelengths again, o n protonation of the carbonyl group to give IV. These are the changes observed in the absorption spectra as a function of acidity indicating that I11 is the predominant uncharged species in the ground state. The fluorescence spectral shifts, however, change from longer to shorter wavelengths as the acidity increases in concentrated sulfuric acid. Consideration of the pathway I -., I1 + IV indicates that this route in the excited state, will produce the required fluorescence spectral changes. That the blue-green fluorescence of 4-methyl-7-hydroxycoumarin does not originate from the excited neutral species (111), is supported by the acidity dependence of the emission spectra of the 7-methoxy derivative and that of coumarin itself. Neither of the latter compounds contains a dissociable proton and thus neither can form a n anion or zwitterion. In alkaline, neutral, and dilute acid solutions, the fluorescences of coumarin and the 7-methoxy derivative are unchanged, very weak, and occur at -400 nm and 386 nm, respectively. These emissions, especially that of the 7-methoxy derivative, arise from an electronic configuration very close to that of the excited neutral species. I n concentrated acid, both molecules are protonated, presumably at the carbonyl group, and the emission spectra as well as the absorption spectra shift to longer wavelengths (Table I) in accordance with the predicted emission behavior for protonation at a caibonyl group. I n chloroform, whose low dielectric strength and weak hydrogen bonding properties might not stabilize the excited zwitterion, the 7-hydroxy derivative fluoresces at 380 nm, presumably from the true neutral species. That the bluegreen fluorescence of the 7-hydroxy derivative does not originate from a dimer is supported by the lack of dependence of the emission wavelength and the linearity of the fluorescence signal, at p H 1, with varying 4-methyl-7-hydroxycoumarin concentration in the range 1 x 10-3M-l x 10-7M. Thus the zwitterion (11) appears to be the predominant uncharged species, derived from 7-hydroxy-4-methylcoumarin in the lowest excited singlet state, indicating that in the latter electronic state the carbonyl oxygen is more basic than the phenoxy anion. I n the range p H 2 t o Ho -4, the formation of the excited zwitterion must proceed entirely by tautomerization of the excited neutral species, since the latter is the sole absorbing species in this range. This requires a n extremely rapid twoproton transfer, to the solvent by the hydroxy group and from the solvent t o the carbonyl group. Presumably this process is mediated by hydrogen bonding with the solvent in the FranckCondon excited state of the neutral molecule and indicates that even moderately concentrated sulfuric acid-water solutions have substantial proton-accepting ability. A rather interesting application of this study lies in the interpretation of the fluorescence changes observed when
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
essentially nonfluorescent esters of umbelliferone and its derivatives are enzymatically hydrolyzed to yield fluorescent species derived from umbelliferone (4-8). I n the ester form, umbelliferone is covalently bound through the 7-hydroxy group to a substrate and is constrained to a n electronic configuration analogous to that of the neutral species or methoxy derivative. Thus the ester is weakly or not at all fluorescent, depending upon the nature of the interactions of the substrate with the umbelliferone moiety. Enzymic hydrolysis liberates the free umbelliferone moiety which rapidly equilibrates in the excited state, subsequent to excitation, to the excited anion in neutral solutions, resulting in intense blue fluorescence, o r to the excited zwitterion in acidic solutions, giving rise to blue green fluorescenced If the neutral species derived from umbelliferone did fluoresce intensely, studies of enzyme kinetics by this method might be seriously complicated by overlap of the fluorescence of the bound moiety with that of the free fluorescing species. The data of Table I1 show that the pK, values obtained by fluorimetry and absorptiometry in concentrated sulfuric acid differ. This would be expected if both excited state and ground state prototropic reactions are occurring in this acidity region. However, quantitative estimation of the dissociation constants for the ground state (and possibly excited state) protonations of the carbonyl groups of coumarin and the 7-methoxy derivative, and for the excited state protonation of the phenolic group of the 7-hydroxy derivative are complicated by the fact that the coumarins undergo some type of reaction in sulfuric acid solutions as evidenced by changes in the absorption spectra with time. The absorption spectra as a function of acidity did not show isosbestic points indicating that more than a simple transformation between acid and conjugate base was occurring. Thus, the pK, values obtained in strongly acidic sulfuric acid media should only be taken as approximate even though the measurements were made as quickly as possible. RECEIVED for review October 12, 1971. Accepted December 13, 1971. Taken in part from a dissertation submitted by 6 . J. Yakatan to the University of Florida, in partial fulfillment of the Doctor of Philosophy degree, December 1971.
Correction Simultaneous Automated Determination of Hydralazine Hydrochloride, Hydrochlorothiazide, and Reserpine in Single Tablet Formulations In this paper by Tibor Urbrinyi and Arthur O’Connell [ANAL.CHEM.,44, 565 (1972)], the authors would like to add the following acknowledgment. “The authors thank Henry Stober for his advice in using for separation the ion exchange resin.”
