Excited State pK Values from Fluorescence Measurements would be correspondingly more exothermic. Taking into account the intensity of the product absorption bands, we conclude that reaction 3 is the dominant process in this system. As before, we take S to react with 0 2 or with 0 3 . No evidence was found for the reaction of atomic sulfur with OCS even undjer the favorable conditions obtaining when OCS is used as matrix host for 03.In these experiments, some ef the light was absorbed by OCS, but this introduced no new reaction products. Other work9 has shown that there is very little net photodecomposition of OCS, either neat or dispersed, in a rigid inert medium a t low temperature using either 228.8- or 253.7-nm light. The tracer experiments show the isotopic course of reaction 4 to be *0651 -i- Q(’D) ---cC*O C SO and show that the oxygen in the SO2 originates in the 03(Oi2) component of the reaction system. This is expected on the basis of the major path assumed for the production of SO2 s c 0 2 +-SO, and on the basis OF the isotopic course assigned to the minor reaction path (4). The clean isotopic stoichiometry shows that the OC!3-O(1D) encounter complex does not last long enough to bring about scrambling of the oxygen atoms. Several factors may contribute to the observed differences between the reaction of O(1D) with 6 0 2 , on the one
Excited ~
t
toll
hand, and with CS2 and OCS, on the other. First, the reactions of O(1D) with CS2 and/or OCS are very exothermic, requiring a large number of collisions to thermalize the adduct. A second consideration resLs on the substitution of the heavier sulfur atom for oxygen. Spin-orbit interaction may considerably reduce the forbiddenness of a transition to a dissociative triplet state. DeMore and Raper estimate that in the case of O(1D) reacting with N2, only one in about 75 collision complexes are deactivated to produce an N20 molecule.10 Also, a study of the formation of CO2 by the reaction of O(lD) in liquid CO has led to the conclusion that predissociation of the initially formed collision complex is,the dominant process.ll Since we have observed only products which are attributable to dissociative paths from the reaction of O(1D) with either CS2 or OCS, and since sulfur is likely to enhance spin conversion, it is believed that for the collision complex with O(lD) deactivation of the encounter complex to an oxygen adduct is precluded by dissociation to the trip) SO(3Z) and SPP). let sulfur products S Z ( ~ Zor
Acknowledgment. Financial support for this research, both for Grant No. GP 5322 and for Jones’ Traineeship, 1967-1970, by the National Science Foundation, is gratefully acknowledged. (9) W . H. Breckenridge. Ph.D. Thesis, Stanford University, 1968. (10) W . B. DeMore and 0. F. Raper,J. Chem. Phys., 37,2048 (1962). (11) 0. F. Raperand W. B. DeMore, J . Chem. Phys., 40, 1053 (1964).
Values ~ t from ~ Fluorescence Measurements
t8cIharna Lasser and Jehuda Feitelson* Deoartment of Physical Chemistry, The Hebrew University, Jerusalem, Israel (Received August 17, 1972)
Aromatic acids and bases have often different pK values in the excited and in the ground states. Atteimpts have been made to determine these excited state pK* values from the fluorescence-pH dependence of the acidic or the basic form of the molecule. Here we show that such data generally do not yield the excited state pK, and what often is measured is the rate of the back reaction in AH* F? A - * Hf. This is shown for riboflavin monophosphate (FMN) by measuring the fluorescence-pH dependence in the presence of various concentrations of Br- ions. Br- is a known quencher for the FMN fluorescence and consequently it shortens the FMN* lifetime. Different “pK*” values, as measured by the FMN fluorescence, are obtained for different B r - concentrations. This is due to the fact that a t shorter lifetimes higher H+ concentrations are required for the same degree of FMN fluorescence quenching. A similar effect is described for a series of indole derivatives.
+
It is well recognised now that acids and bases in their excited states are characterized by dissociation constants which could differ by several orders of magnitude from the corresponding ground-state values.1 The few experimental methods available for the determination of the pK* values of aromatic molecules in their lower excited singlet state are based mainly on absorption and emission spectra of these molecules,
One method,l.Z based on thermodynamic considerations and known as the Forster cycle, attributes the difference between the absorption (or emission) peaks of the acidic form and its conjugate base to the difference in pK values for the ground and the excited states. This method in(1) T. Forster, 2. Eiektrochem., 54, 42 (19S0); 54, 531 (1950) (2) A . Weller, Z. Nektrochem., 56, 662 (1952).
The Journal of Physical Chemistry, Vol. 77, No. 8, 7973
Nechama Lasser and Jehuda Feitelson
101
volves an approximation; it is assumed that the entropies associated with the ground and excited state dissociations are equal. An alternative kinetic approach has been developed by Weller,3 which is hased on the pH dependence of the fluorescence of the acid and its conjugate base, respectively. This method is applicable to acids (or bases) in which the prototropic equilibrium is a t least partially established within the lifetime of the excited system. Following Weller’s work a number of studies on aromatic systems based on fluorescence measurements have been published. In some of these4-’ pK* values were determined by fluorimetric titration of one member of a conjugate acid-base pair the other member being nonfluorescent. The fluorescence intensity as a function of pH yielded in these castes a sigmoid curve, whose inflection point was considered to correspond to the excited state P K. The aim of the present study is to show what really is being measured when the fluorescence as a function of pH is recorded for one member of the conjugate acid-base pair, and that these data do not necessarily yield a pK value of the excited state.
Experimental Section Materials and SolzAtions. Tryptophan, tryptamine, 3-/3hydroxyethylindole, and tryptophanamide were purissimim grade, and ~-aminoindole-3-propanol and FMN (riboflavine-5’-monophcisphate sodium salt) were purum grade Fluka (Switzerland) products. N-Acetyltryptophan methyl ester and AI-aicetyltryptophanamide were obtained from Miles-Yeda (Rehovot) and N-acetyltryptophan from Matheson Coleman aind Bell. All other solutes were analytical grade reagents. Triply distilled water was used throughout. Fresh solutions were prepared before each experiment, so as to minimize photolytic degradation of the substances (which was especially important in the case of FMN), Absorption spectra were measured on a Cary 14 spectrophotometer. The fluorescence intensity was measured at right angles to the incident light in an apparatus consisting of a Xe arc, two Bausch and Lomb 500-mm focal length monochromators, an EM1 6256 S photomultiplier, and an EIL electrometer. The apparatus has been described previously.8 The solutions were of optical density less than 0.1. Spectrosil cells, I-can optical path, were used. FMPJ containing solutionsqwere excited a t X 375 nm and indole derivatives at >\ 280 nm. Since the shape of the fluorescence spectra of both FMN and of the indole derivatives do not change with pH, the peak emission intensity was taken as a measure for the relative quantum yield. Lifetime measurements were carried out with the aid of an apparatus basically similar to that described by Berlmans (Figure 1). Tht? equipment consisted of a nanosecond light pulser (‘TRW Instrument) with a deuterium lamp of about 4-nsec flash half-width, a Jarrel-Ash Model 82-410 monochromator, a Philips DUVP photomultiplier, a sampling oscilloscope (Tektronix 535 A with 1S1 sampling unit), a Computer of Average Transients (TMC, CAT 400c), and a 1%-Y recorder (Electro Instruments line.). The fluorescence intensity as a function of time was measured at right angle to the incident light in 1-cm optical path quartz cells. The optical densities of the solutions in these experirnenls were between 0.2 and 0.3. Corning The Jourrial of Physical Chemistry, Vol. 77,No. 8, 1973
-
1 I I II
I I
I
Figure 1. Scheme of
CAT
I
lifetime measuring apparatus.
filters (3-73 for FMN and 0-54 for indole derivative solutions) were used to cut off the scattered light from the exciting source. The sampling oscilloscope was triggered by an IP28 photomultiplier which received part of the light flash from the lamp via a quartz fiber light guide. The lifetime was calculated with the aid of the “convolution integral”
R(t’) = f Y ( t ) P ( t ’ -- t ) dt
(1)
in which R(t’) is the intensity of fluorescence a t a given time t’, Y ( t )is the intensity of the exciting light at time t, and P(t’ - t ) is the response function of the experimental system. Since, in all the systems measured, we could expect an exponential decay (according to a first-order decrease in the number of excited molecules)
P(t‘
-t) =
e-(t’-t)/r
(2)
Y ( t ) e t l rd t
(3)
and therefore
R(t’) = e+’/‘
The values of R(t’) and Y ( t ) as a function of time, which were measured in the apparatus, were fed into a 6400 CDC computer to determine the value of T which would give the minimum deviation between the experimental and the calculated curves. Because of the relatively long lifetime of the deuterium lamp, the above procedure results in an increase in the relative error of our measurements as the measured lifetime decreases.
Results (a) Quenching Effect of Br- on FMN Fluorescence. Halogen ions are known to quench the fluorescence of FMN in the order I- > Br- > Cl- = F-.10 Since it is (3) A. Weller, Progr React Kmet., 1, 189 (1961), and references therein (4) R. E. Ballard and J W Edwards, Specfrochm Acta, 20, 1275 (1964) (5) S. Schuiman and Q Fernando, Tetrahedron, 24, 1777 (1968) (6) S. G Schulrnan, J Pharm Sc/ , 60,628 (1971) (7) E Vander Donckt, Progr. React. Kmet., 5, 273 (1970). (8) J. Feitelson, J Pbys. Chem., 68, 391 (1964) (9) I . B Berlman, “Handbook of Fluorescence Spectra of Aromatic Molecules,” Academic Press, New York, N Y , 1965. (10) H. Theorell and A P Nygaard, Ark Kern!, 7, 205 (1954)
1013
Excited State pK Values from Fluorescence Measurements TABLE I: Lifetime of FMN as a Function of T , nsec
[H+], M
4.5 f 0.1= 4.1 f 0.2
10-3
3.1 f 0.2 2.5 f 0.3 1.5
I
i I!
HL Ion Concentration
7 , nsec
[H+], M
1.4f 0.3 5x
61.8
lo-’
0.5 M) ((@/&) - I)/[H+] = k~ is constant over the whole pH range. These constants far a variety of indole derivatives are presented in Table T’U. This table also shows the relative quantum yields with respect to /3-hydroxyethylindole (which we used as a standard) and the fluorescence lifetimes of these compounds. From the Stern-Volmer constants, we can calculate the H+ concentration at which the fluorescence intensity decreases to 50% of its value in neutral solutions, JH+]so%. These pHbo%values or apparent pR* values are also shown in Table I11 for the different indole derivatives. iscussion
In acid-base dissociation equilibria 4 HA t. EI,O ---L- H30+ + Ak l
the rate of the “baclr reaction” is usually diffusion controlled, with a bimolecular rate constant k - 1 in the 1010 M-1 sec -1 range. It follows that the dissociation constant of HA, is primarily determined by the rate constant of the forward reaction, k l . If the dissociation takes place in the excited singlet state the following scheme applies h”
HzO -1- 4H* G A-* =tH3Q+ k ,*
11
(5)
hf’ ”d’
where kf and k d are the rate constants for fluorescence and for the other deactivation steps which compete for the excited molecule, because of the various reactions taking place during the singlet lifetime of 10-9-10-8 sec. The above equilibrium i s never fully established. Nevertheless, if both moieties AH* and A-* are fluorescent the dissociation constant can be computed as shown by Weller3 from the pH dependencies of the two fluorescence quantum yields. We shall now enquire whether it is possible to The Journal of Physical Chemistry, Voi. 77, No. 8,1973
PH
fluorescence intensity as a function of pH various Br-. ion concentrations: [Br-] = 0.0 M (a),0.01 M 0.03 M (c),0.05 M (d). Figure 4. FMN
obtain the K* value in those cases where only one of the two forms, HA* or A-*, is fluorescent which means that the other form usually has a lifetime of 7 C 5 x 10-11 sec. The Acidic Form HA* o n l y is Fluorescent. This means that kd” >> k f ” and the lifetime of A-* i s so short that the association reactions A-* H30+ cannot take place. The forward dissociation reaction AH* HzO is pH independent and therefore the pH dependence of the AH* fluorescence will be determined by the ground-state AH concentration and will therefore follow the ground-state titration curve. The excited state pK* will only determine the absolute fluorescence quantum yield of AH* in that the lower the pK* the faster does the dissociation take place and the shorter the lifetime of AH* will be. The Basic Form A - * Only is Fluorescent, The molecular systems described in this study all belong to this class of compounds. Here the concentration of A- ions which are excited at a given pH is determined by the degree of dissociation of the HA molecules in the ground state, If the ground-state pK is sufficiently low, cited in the presence of a comparatively high H+ ion concentration. The fluorescence of A-* will then be at least &0+ partly quenched due to the back reaction A-* (eq 5 ) . In such a case, therefore, the fluorescence yield of A-* at low pH will be smaller than in the high pH region and the fluorescence us. pH curve will be displaced toward higher pH values when compared to the groundstate titration curve. This shift in the fluorescence-pH curve is in fact a measure of the rate of protonation k-l[H+] (eq 5) and of the lifetime of A-*, but it is entirely independent of the dissociation of (the very short lived) HA*. If the ground-state pK is higher, say p K > 2 or 3, the available H+ ion concentration might become too small for protonation to occur during the lifetime of the excited A-*. The fluorescence will then follow the ground-state titration curve. We see that in all the cases discussed the fluorescence of either the acidic or the basic form of a molecule alone is insufficient to determine pK*. In the above two cases where excited state pK* values are apparently obtained we can show that the data do not really represent the dissociation constant. The pH dependence of the fluorescence of FMN shows an inflection point from which Schulmane has concluded that pK* = pK + 1.7. In the Results section we have shown that
+
+
-
+
Excited State pK Values from Fluarescence Measurements TABLE 111:
1015
Quenching01 Indole Derivative Fluorescence by H+ Ions
Indole derivative
Relativea quantum yield
Measured lifetime
*
-
I I
_ _ _ l _ _ l _ _ _ _ _ _ l -
SternVoimer constant k, M - I
kHm/k PKapp
@HEI*/@
kHErC/k
$HEI/@
1 3-P-P~ydroxyethyl-
I
7.7
0.2
110
indole 2 N-Ac~~yl~ryptophan-
0.37
2.7 f 0.2
28
1 .J7
2.68
3.9%
1.47
3 N-Acelyltryptophan
0.17
