11493
J. Phys. Chem. 1993,97, 11493-1 1496
Excited-State Acid-Base Kinetics and Equilibria in Norharman Sonja Draxler and Max E. Lippitsch' Karl-Franzens- Universitht Graz, Institut f i r Experimentalphysik, A-801 0 Graz, Austria Received: June 25, 1993; In Final Form: August 19, 1993' Absorption and CW and time-resolved fluorescence measurements were performed to investigate the excitedstate acid-base kinetics and equilibria of norharman in aqueous solutions of different pH. Four different excited-state species, namely cation, neutral molecule, anion, and zwitterion, can be distinguished. Rate equations were applied to explain the observed decay curves and to calculate the pH-dependent kinetic parameters for the transitions between the species.
Introduction Norharman or @-carboline(9H-pyrido[3,441indole) belongs to the group of alkaloids. These compounds have gained much interest in pharmacological sciences as central nervous system stimulants, hallucinogens, and paralysants of cardiac muscle.I4 In recent years norharman has been proposed as a fluorescence standard6t7because the fluorescence decay of its cation can be fittdvery well with a single exponential. Norharman is composed of a r-deficient pyridinic ring fused to a r-excessive indole ring. Thereforetwo functional sites for acid-base chemistry are present, and consequently two pK, values exist in the ground state and the first excited state. In addition the existence of an excitedstate zwitterion has been Hence the dependence of absorption and fluorescence behavior of this molecule on the kind of solvent and on the pH value in aqueous solutions is quite interesting. Though many papers have been published on that the mechanisms underlying the excited-state acid-base chemistry of norharman are not yet fully explained and no quantitative study on its kinetics has been reported. In order to obtain further information about the acid-base equilibria in the excited state, we performed absorption and both CW and time-resolved fluorescence mdasurements in aqueous solutions of different pH between 2 and 14. A rate equation model was applied to simulate the measurements. Using this model it was possible to deduce numerical values for the kinetic parameters as well as for the excited-state equilibria of norharman.
ExperimentalSection Norharman was purchased from Aldrich-Chemie GmbH (Steinheim, Germany), methanol p.a. from Merck (Darmstadt, Germany) and used without further purification. For preparing the buffer solutions,highly purified water (Bamstead NANOpur; specific resistivity 18 M Q cm) was used. pH was adjusted by adding NaOH or H2S04 to phosphate buffer. Solutions of norharman (10-5 M) in buffer with 1% methanol were used. All measurements were performed at a temperature of 22 OC. Absorption spectra were measured on a Hewlett-Packard 8451A diode array spectrophotometerusing quartzcuvettes with 1-mm path length. The CW fluorescence measurements were performed using a SPEX Fluorolog I1 fluorimeter. The spectra were corrected with respect to excitation by using a quantum counter (Rhodamine B in ethylene glycol) and with respect to emission by reference to an NBS standard light source. Time-resolved fluorescence measurements were performed using a N2 laser as light source, a streak camera (Hamamatsu C 1370) or a fast photomultiplier as detector, and an optical multichannel analyzer or a digital signal analyzer for signal acquisition. The time resolution of the whole system was below 300 ps with the streak camera and about 1 ns with the *Abstract published in Advance ACS Abstracts. October 15, 1993. 0022-3654/93/2097-11493304.00/0
H
Ci + ,*+
"
Ma?\
\
,
zx,
PK,al* N *
1
A' -
A' -
C+ N AFigure 1. Acid-base equilibria of norharman in the ground and excited state. photomultiplier. A sum of exponentials was fitted to the decay data using a commercial mathematical program. Results Norharman contains a weakly acidic indole N H group and a basic pyridine ring nitrogen. Therefore three different species can exist in aqueous solutions in the ground state: a cation, a neutral molecule, and an anion. Measurements of the dependence of absorption on pH give a pK,l= 6.5 for the transformation fromcation to neutral molecule and a pK,2 = 14.5 for the transformation of neutral molecule to anion. For the lowest excited singlet state, SI, the pKa* values can be calculated applying the F6rster-Weller equation
where PB and ~ ' B H are the respective wavenumbers of the 0-0 transitions of acid and base molecules and Tis the temperature in Kelvin. Following that equation we get pK,2* = 7.7 for the transformation of the neutral molecule to the anion and pKal* = 12.2 for that from cation to the neutral form. Thus in the excited state the sequenceof pK,values is reversed. This behavior had been predicted before from molecular orbital calculations19 and suggeststhe possibility of a zwitterionicspeciesin the excited state. It should be noted that the pKal and pK,1* values found in this work are in good agreement with values given by Wolfbeis et al.8 but differ from those given by Sakurovs and Ghiggin~.~ Figure 1 summarizes the ground- and excited-state acid-base chemistry of norharman. The fluorescencespectra at different pH are presented in Figure 2, and the results of the time-resolved measurements, in Figure 3. For acidic solutions (pH < 5 ) only the cation emission is observed &,,,= 450 nm). The decay is single-exponential, with 0 1993 American Chemical Society
11494 The Journal of Physical Chemistry, Vol. 97, No. 44, 1993
Draxler and Lippitsch
f
'b 400
500
600
wovelength (nm)
Figure 2. Fluorescence spectra of norharman at pH 3.3 (a), 8.9 (b), and 14.0 (c). The band at 380 nm, most pronounced in spectrum b, is due to the neutral species. The cation and anion emit around 450 nm. and the 520.nm band originates from the zwitterion. 0
20
90
-
Time (ns) Figure 4. Fluorescencedecay of norharman at &,= 380 nm (pH 6.0) showing two exponential components.
20 r\
bl
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only two of the four excited-state species. The neutral molecule can be monitored at wavelengths below 380 nm, while the zwitterion emits above 500 nm. The cation and the anion, however, havenearly identical emission spectra.*JOJ1 Hence their emissions cannot be separated spectrally.
+
x
10
10
U
0
0 0
I
I
5
10
PH
Figure 3. pH dependence of the fluorescence decay time in norharman at > 400 nm. Note that below pH 11 a single-exponential fit is adequate, while at higher pH two components are observed.
a decay time of 22 ns, in agreement with earlier work.' Above pH 5.5 an inconspicuous shoulder appears at 520 nm and a small additional band arises at 380 nm. The shoulder can be attributed to the zwitterion while the UV emission is due to the neutral molecule.' Despite the fact that the spectrum in the visible (b,,, > 400 nm) now originates from two different species, the decay is singleexponential within the experimental accuracy and the decay time is independent of wavelength. Between pH 5 and 8 the decay time decreases continuously with increasing pH. Between pH 8 and 11 it has a nearly constant value of about 8 ns. Above pH 11 a double-exponentialdecay can be observed. The longer component increases slightly from 8 to about 10 ns, and a short component of about 1 ns appears. In the near-UV region (bm< 400 nm) where we have only the fluorescenceof the neutral molecule, the decay was reported earlier to be single-e~ponential.~As is obvious from Figure 4, this is not the case. All goodness-of-fit parameters showed unambiguously the necessity of two exponentials. The shorter component had a pH-independent decay time of about 250 ps (which was at the limit of resolution and hence may be only approximate). The second componentwas as long as or slightly longer than the decay time in the visible region. This long component is definitely not, as one may suspect, due to a cross talk from the cation fluorescence. It was also observed when using a narrow-band interference filter centered at X = 350 nm at pH values above 5 , whereas no signal wasobservableat thatwavelength with lower pH, when thecation fluorescence is the only emission. The results of the time-resolved fluorescence measurements yield directly the temporal evolution of the concentrations of
Discussion To find a quantitative explanation for the dependence of the decay times on pH as shown in Figure 3, all four excited-state species, namely cation (c), neutral molecule (n), anion (a), and zwitterion (z), have to be taken into consideration. All those species are kinetically coupled to each other, as shown in the scheme of Figure 5 . The asterisks indicate the lowest excited states of the molecules. Arrows from one species to another show possible transformations between them. For the most general case, 4 intramolecular(ki,radiativeand nonradiativedeactivation) and 12 intermolecular (kg, reactive transformation from species i into speciesj) rate coefficients are possible. To model the kinetics of the excited-state processes presented in the scheme of Figure 5 , we need the following set of differential equations:
-dn* - k,c* + kana* + k,z* - k,g* - k,n* - k d * - k,n* dt _ dc* -- kncn*+ k d * + kaca* - k,c* - k,c* - kczc* - kac* dt -da* - k,n* + k,z* + kac* - kaa* -kana* - k,a* - kaca* dt -dz* - k,n* + k,c* + ka,a* - kg* - k,z* dt
- k d * - k,z*
(2) where n*, c*, a*, and z* represent the concentrations of the respective excited-state species. A similar treatment was used by Schulman and Rosenberg20 for methylumbelliferone,where the problem is basically the same (a molecule with two acid-base functional groups) but the evaluation is somewhat simpler, however, since the cation does not play a significant role. A rate equation treatment of norharman has been undertaken before,I6 but only two of the possible twelve reaction paths have been taken into account. Some of the coefficients in eq 2 can be determined directly by measuring the fluorescence decay for the isolated species. This
The Journal of Physical Chemistry, Vol. 97, No. 44, 1993 11495
Excited-State Kinetics in Norharman
a
+ -
2’ kza
a
QJcly+-
9z
Figure 5. Excited-state acid-base reactions and corresponding kinetic parameters.
I
L
u1 Q
\
0 300
350
400
-m ” H ---n/H
