Phoruchntisny ondPhurubiology. 197U. Vol. I I, pp. 247-293. Pergamon Press. Printed in Great Britain SPECTRAL CHARACTERISTICS OF THE EXCITED STATES OF THE PRODUCT OF THE CHEMILUMINESCENCE OF LUMINOL* JOHN LEEt and H. H. SELIGER New England Institute. Ridgefield, Conn. 06877. U.S.A.: and McCollum-Pratt Institute and Department of Biology. The Johns Hopkins University. Baltimore. Md. 2 I2 18. U.S.A. (Received 30 December 1968: in revisedform 26June 1969) Abstract - In dimethylsulfoxide the emission spectrum of luminol chemiluminescence is red- shifted by 300 cm-' from the photoexcited fluorescence of the product 3-aminophthalate dianion. while in aqueous solvent the two spectra are identical. The spectral properties of the product dianion have been measured in aqueous solvent and in a number of aprotic solvents. both at room temperature and at 77°K. The ground states and the excited states from which emissions are observed are characterized. Two alternatives are presented to explain the aprotic emission spectra. INTRODUCTION THERE is general agreement that the oxidation of luminol(3-aminophthalhydrazide. I, Fig. I) is of the class of direct chemiluminescence where the product molecule is formed directly in the excited state[ I]. White and co-workers[2] have found the mechanism of Fig. I to be the main chemical reaction path for the luminol auto-oxidation in dimethylsulfoxide (DMSO) made basic by the addition of 30% of N NaOH. In dry DMSO made basic with potassium tertiary butoxide (r-BuOK) the chemiluminescent emission spectrum has been reported to be the same as the fluorescence of either the reaction product or of 3-aminophthalic acid under the same dry basic conditions[3.4]. Analogues of luminol are also stated to produce chemiluminescent spectra similar to the fluorescence of the corresponding substituted carboxylic acid [ 5-71. although discrepancies have also been reported [ 81. In aqueous solution the chemiluminescent emission spectrum (P = 23250 cm-', A, = 425 nm) and the fluorescence of the 3-aminophthalate ion are the same. The fluorescence of luminol itself is only very slightly different and a lack of precise spectra led to errors in interpretation of the reaction mechanism in earlier studies[9]. Seliger [ 101 showed the spectral differences and also that luminol is not fluorescent under the basic conditions used for the chemiluminescent reaction. He found that when the solution is more basic than pH 10 the fluorescence yield (QF) of 111 showed a pH dependence similar to the luminol chemiluminescent quantum yield (Q,.)$. *Supported in part by the U.S. Atomic Energy Commission, Division of Biology and Medicine, Contracts AT (30-1) 3401 and AT (30-1) 2802 and by the Connecticut Research Commission. Contribution Number (583) of the McCollum-Pratt Institute. tPresenf uddress: Department of Biochemistry, University of Georgia. Athens, Georgia 30601. USA. $The chemiluminescent quantum yield is defined with reference to a specific reactant as the total number of photons emitted divided by the number of molecules of the reactant consumed by the chemical reaction. 247 P.P.Vol IINo.4-C
Transcript
Phoruchntisny ondPhurubiology. 197U. Vol. I I , pp. 247-293. Pergamon Press. Printed in Great Britain
SPECTRAL CHARACTERISTICS OF THE EXCITED STATES OF THE PRODUCT OF THE CHEMILUMINESCENCE OF
LUMINOL*
JOHN LEEt and H. H. SELIGER New England Institute. Ridgefield, Conn. 06877. U.S.A.: and McCollum-Pratt Institute and
Department of Biology. The Johns Hopkins University. Baltimore. Md. 2 I2 18. U.S.A.
(Received 30 December 1968: in revisedform 26June 1969)
Abstract - In dimethylsulfoxide the emission spectrum of luminol chemiluminescence is red- shifted by 300 cm-' from the photoexcited fluorescence of the product 3-aminophthalate dianion. while in aqueous solvent the two spectra are identical. The spectral properties of the product dianion have been measured in aqueous solvent and in a number of aprotic solvents. both at room temperature and at 77°K. The ground states and the excited states from which emissions are observed are characterized. Two alternatives are presented to explain the aprotic emission spectra.
INTRODUCTION THERE is general agreement that the oxidation of luminol(3-aminophthalhydrazide. I , Fig. I ) is of the class of direct chemiluminescence where the product molecule is formed directly in the excited state[ I ] .
White and co-workers[2] have found the mechanism of Fig. I to be the main chemical reaction path for the luminol auto-oxidation in dimethylsulfoxide (DMSO) made basic by the addition of 30% of N NaOH. I n dry DMSO made basic with potassium tertiary butoxide (r-BuOK) the chemiluminescent emission spectrum has been reported to be the same as the fluorescence of either the reaction product or of 3-aminophthalic acid under the same dry basic conditions[3.4]. Analogues of luminol are also stated to produce chemiluminescent spectra similar to the fluorescence of the corresponding substituted carboxylic acid [ 5-71. although discrepancies have also been reported [ 81.
I n aqueous solution the chemiluminescent emission spectrum ( P = 23250 cm-', A, = 425 nm) and the fluorescence of the 3-aminophthalate ion are the same. The fluorescence of luminol itself is only very slightly different and a lack of precise spectra led to errors in interpretation of the reaction mechanism in earlier studies[9]. Seliger [ 101 showed the spectral differences and also that luminol is not fluorescent under the basic conditions used for the chemiluminescent reaction. He found that when the solution is more basic than pH 10 the fluorescence yield ( Q F ) of 111 showed a pH dependence similar to the luminol chemiluminescent quantum yield (Q,.)$.
*Supported in part by the U.S. Atomic Energy Commission, Division of Biology and Medicine, Contracts AT (30-1) 3401 and AT (30-1) 2802 and by the Connecticut Research Commission. Contribution Number (583) of the McCollum-Pratt Institute.
tPresenf uddress: Department of Biochemistry, University of Georgia. Athens, Georgia 30601. U S A . $The chemiluminescent quantum yield is defined with reference to a specific reactant as the total number
of photons emitted divided by the number of molecules of the reactant consumed by the chemical reaction. 247
P.P.Vol I INo .4 -C
248 J . LEE and H. H. SELIGER
OHe
m Fig. I . Mechanism suggested by White and co-workers (reference [2]) for the chemiluminescent
oxidation of luminol ( I ) in DMSO.
I t is the purpose of this paper to describe quantitatively the spectral properties of the product molecule, the 3-aminophthalate anion (I I I ) , and to identify the emitting species under aqueous or aprotic conditions.
MATERIALS A N D METHODS Materials
The 3-aminophthalic acid hydrochloride was obtained as 'white-label' grade from Eastman Organic Chemicals, Rochester, New York and recrystallized until white from a 1 : I mixture of acetone and 0.1 N HCI. Nitration of phthalic acid yielded a mixture of 3-nitro and 4-nitro phthalic acid which were separated by crystallization. The 4-nitro derivative (m.p. 2 1 1-2 14°C) was reduced to 4-aminophthalic acid by catalytic hydrogenation and recrystallized from dilute HCI (m.p. 161 -1 64°C). Spectro- grade DMSO was from Crown Zellerbach Corporation, Camus, Washington; dimethyl- formamide (DMF), acetonitrile (AN) and propylene glycol (PrG) were fluorescence grade from the Hartman-Ledden Co., Philadelphia, Penn.; potassium tertiary butoxide (t-BuOK) was obtained as the alcohol-free powder and was a gift of the Mine Safety Appliance Research Corporation, Callary , Pennsylvania.
Quinine hydrogen sulfate (Reagent grade) was from British Drug House, Poole. England, and was recrystallized several times (0.01 N H,S04). H ydroquinone and 9 : 10-diphenylanthracene (DPA) were also recrystallized before use. All other chemicals were used without further purification.
Instrumentation Absorption and photoluminescent spectra were measured using a Cary 14 Spectro-
photometer. The three-port illuminator attachment enabled the emission spectra to be measured using the Cary 14 optical system'for detection. In this device an excitation beam is chopped at 10 cycleslsec alternately on a sample and a reference phototube. The reference phototube was generally allowed to view the excitation beam via a quantum counter of rhodamine B in PrG (30 mglml). The chopper could be synchron- ized either in or out of phase with the internal chopper of the Cary 14, allowing either prompt or delayed luminescence to be measured. A Bausch and Lomb f/3.5 grating monochromator type 33086 having a grating blazed at 250 nm was used for excitation.
Spectral characteristics 249
The light source was usually a 200 W a.c. 'superpressure' short arc quartz Hg lamp although a 150 W d.c. Xe lamp was occasionally substituted. The excitation beam was passed through a glass filter C.S. 7-54 (Corning Glass Works, Corning, New York) to further attenuate scattered radiation of wavelength above 400 nm. For the phosphores- cence spectral measurements the sample was dissolved in an appropriate glass and contained in a 'Spectrosil' quartz tube and dewar vessel. The dewar containing liquid nitrogen was placed in the position normally occupied by the fluorescent sample so that the phosphorescence was viewed at right-angles to the excitation beam. The aqueous glass was a 1 : 1 mixture of 0.1 M K&03 and propylene glycol, PrG. The aprotic glass was a 7:3 volume mixture of DMF and AN which when rapidly quenched to 77°K produced a rigid solution fairly free from cracks.
Lifetimes of phosphorescence were measured on a separate apparatus which used the excitation and detection optics of an Aminco-Bowman Spectrofluorometer (American Instrument Co., Silver Spring, Md.). The computer program and other details of this analysis are described elsewhere[ I I J . Quantum yield meusurements
The apparatus was calibrated for measurement of absolute quantum yields of fluorescence by comparison with standard substances. The QF of an oxygen-free solution of DPA in cyclohexane excited at 366 nm was taken as unity[ 121. Degassing was carried out by the freeze-thaw technique under vacuum ( Torr). On the basis of DPA. QF for quinine (lops M) in air-saturated solution (0.1 N H2S04) was found to be 0.46 and did not change after repeated recrystallization. Solvent refractive index corrections were made in all cases[4].
The spectral sensitivity of the apparatus was calibrated by reference to the 366 nm- excited fluorescent emission of quinine ( M) in H,SO, (0.1 N). The spectrum tabulated in Table I represents the average of the published results of Lippert et ul. [ 131, Melhuisht 141, Borresen[ IS] and Eastman[ 161. Individual results are generally in satisfactory agreement (k 5 per cent) for wavenumbers above 18000 cm-I but the scatter is as much as 2 20 per cent below this.
Table I . Averaged results of published values of the absolute fluorescence emission, E(D) of quinine sulfate ( M )
The E(P) are in units of photons per wavenumber interval normalized to I00 at the maximum(references[l3-16]).
250 J . LEE and H. H. SELIGER
RESULTS
Spectra of chemiluminescence and fluorescence Figures 2 and 3 compare for aqueous and DMSO solutions respectively the emis-
sion spectra of chemiluminescence from the oxidation of luminol with that of the fluorescence of the 3-aminophthalic acid salt under the same conditions of solvent and base. In water (Fig. 2) the chemiluminescent and fluorescent emission spectra are identical (V, = FF = 23250 cm-I). In DMSO (Fig. 3) the chemiluminescence (Fc = I9900 cm-l) is red shifted from the fluorescence (vF = 20200 cm-I). The fluorescence excitation spectra are identical to the main absorption spectra also shown in the figures. The weak absorption at 27,000cmr' for 111 in DMSO (Fig. 3) is due to an impurity which develops during the chemiluminescent oxidation. I t is included in the figure to
A , nm
550 4 5 0 4 0 0 3 5 0 300 275 I 1 I I I I I I I
- I b 1 3
15 20 25 30 35 40
D , Id an-'
Fig. 2. Fluorescence and absorption spectra (full lines) of 3-aminophthalic acid ( M ) in aqueous solution (pH 11.6; 0.1 M K,CO,). Chemiluminescent emission spectrum (dashed line)
from luminol ( I W,i M ) oxidized by H,O,/hemin under the same aqueous conditions.
A , nm
550 450 400 450 500 215 1 1 1 I I I I
15 20 25 30
i; , Id an-'
Fig. 3. Fluorescence and absorption spectra (full lines) of 3-aminophthalic acid (lo-' M ) in DMSO made basic by the addition of 1-BuOH (4%) saturated with f-BuOK (= 10-*M). Chemiluminescent emission spectrum (dashed line) from luminol ( M) oxidized by oxygen
under the same conditions.
Spectral characteristics 25 1
show its relative contribution. Freshly recrystallized samples of 111 do not exhibit this shoulder.
Solvent effects Table 2 shows the absorption and fluorescent properties of the 3-aminophthalate
ion in aqueous solution and in aprotic solvents in order of decreasing dielectric constant. Solvent absorption did not allow accurate measurements in acetonitrile and sulfolane but absorption and excitation spectra appeared to correspond in all solvents. The data of Table 2 were independent of 3-aminophthalate concentration (< 2 X M) and of base except as described in the next section. In all aprotic solvents two fluorescent peaks are observed and the ratio of the peak intensity of the one at higher energy to that of lower energy is tabulated. In DMSO the addition of proton donors such as H,O or t-BuOH enhances the higher energy fluorescence peak. In spite of careful drying it is possible that the higher ratios found for the less polar solvents arise from the presence of traces of water.
Table 2. Absorption and fluorescence characteristics of the 3-aminophthalate dianion ( I 1 I ) in water (pH 1 I .5,0.I M K,CO,) and aprotic solvents (4% /-BuOH saturated with r-BuOK)
Absorption Fluorescence Solvent
dM-lcm-I) c,(cm-l) ijF(cm-l) ijF(cm-') QF(%) Ratio ~~
Water 2250 33000 23250 30 DMSO 2450 31900 (24000) 20200 14 0.1 DMF 2500 32200 (24600) 20100k100 5 * 1 1.5 AN - - (25500k300) 2oooO~300 2 Sulfolane - - (25300 & 300) 2oooO k 300 4 TH F 2500 32400 (25300) 2oooO&300 4 k l 4
Spectra are measured in photons per wavenumber interval. Errors are k50cm-1 in spectral maximum (D,,. o F ) except where indicated and k 10% in fluorescence quantum yield Q F : e is the molar extinction coefficient and the last column is the ratio is of the intensity of the higher wavenumber fluorescence peak height to that of the lower.
p H effects As a function of pH of aqueous solutions of 3-aminophthalic acid, there appear
different ionic species as evidenced from the absorption characteristics summarized in Table 3. The fluorescent emission peaks corresponding to these ionic species and their respective QF values are also given. Figure 4 shows the variation with pH of QF of the 23.250 cm-I fluorescence.
Table 3. Spectral characteristics and ground state pK's of 3-arninophthalic acid salts in water
ij,,(cm-l) e(M-'cm-') pK Fp(cm-') QF(%)
36400 1000 1.5 19800 1 30300 1400 22100 4
33000 1900 - -
33000 2250 23250 30
3-4
5
252
30-
2 0 -
# d.
10
J . LEE and H. H. SELIGER
-
I I 1 I I I I 1 I I
Low temperature luminescence Table 4 gives the characteristics of the emission of I11 at liquid nitrogen temperature
in two types of solvents. The peaks of the excitation spectra are unchanged from room temperature but the half-height band width is narrowed by a factor of 0.5 to about 5500 cm-l. The phosphorescence in both cases exhibits complex decay kinetics, analyzed as the sum of two or three first order processes. The delayed emission spectra were integrated over the first 0.1 sec following a 0.1 sec illumination period. We assumed that identical emission spectra were associated with each decay process. The temperature dependence of these lifetimes was not examined. Over the first 0.1 sec following the illumination period the intensity of the delayed emission was independent of excita- tion intensity and of 3-aminophthalate concentration. The low-temperature vF for the PrG : 0.1 M K,CO, glassy solution is quite blue-shifted relative to vF in water at room temperature. This suggests that considerable hydrogen bonding or other solvation re- arrangements follow excitation in fluid solution but are inhibited under the glassy solvent conditions. I n contrast, in the DMF: AN the low energy fluorescence (vF = 20,000 cm-I) and the ratio of high to low energy peaks are the same for both glassy and fluid solvent at room temperature. The high energy peak is only slightly blue shifted. No delayed emission peaks were found below 20,000 cm-l.
Table 4. Low temperature luminescence of 3-aminophthalate and aprotic glassy solutions (77°K)
T~ is the mean lifetime for phosphorescence decay, Qp the phosphorescence quantum yield; errors are AvF f 50 cm-I, AvP +- 100 cm-I, AQF+ 10 per cent, AQD2 20 per cent.
Spectral characteristics 253
Quenching The addition of proton donors to aprotic solvents reduces the QF of the 3-amino-
phthalate ion and at the same time increases the proportion of the higher-energy fluores- cent component (24.000cm-' in DMSO). Figure 5 shows the overall reduction in QF produced by increasing concentrations of r-BuOH in DMSO. Similar effects are observed for addition of H20. Initial base concentrations had no effect on pF or QF
of 3-aminophthalate. Oxygen, KI, 3-amino-2-naphthoic acid, butylated hydroxytoluene and hydroquinone
show the normal behavior of fluorescence quenching, reducing QF without affecting vF. This quenching follows the Stern-Volmer relation
where the initial fluorescence yield (QF)o is reduced to (QFlC by the presence of concen- tration C of quencher and K is the Stern-Volmer constant (Table 5 ) . Excitation was at the absorption maximum in each case and the results are corrected for self-absorption.
In order to illustrate the trend of spectral changes due to inter- and intra-molecular hydrogen bonding a series of analogous compounds was studied. Table 6 summarizes
5
0 1.0 2.0 3.0 Camantrotion hrl.-BuOH , M
Fig. 5 . Quenching of the 3-aminophthalate fluorescent yield (el.) in DMSO by addition of t-BuOH.
Table 5 . Stern-Volmer quenching constants (K) for the fluorescence of 3-aminophthalate anion in aqueous (0.1 MK,CO,) and DMSO (4%
1-BuOKlt-BuOH) solutions
Quencher Solvent K(M-')
0 2 DMSO 60 0 2 K O 80 BHT DMSO 130 Hydroquinone H 2 0 4OOo
254 J . LEE and H. H. SELIGER
Table 6. Fluorescence characteristics of substituted phthalic and benzoic acids in aqueous (0.1 M K,CO,, pH 11.5) solution and in DMSO (4% 1-BuOH saturated
the fluorescence and absorption characteristics of substituted phthalate and benzoate dianions in DMSO and in basic aqueous solution.
DISCUSSION The interpretation of the absorption, photoluminescence and chemiluminescence
spectra of 3-aminophthalic acid and its salts is complicated by the number of acid-base transitions together with the possibilities for intra- and inter-molecular hydrogen bond- ing. By analogy with benzoic and phthalic acid the absorption spectra of Table 2 can be explained by the following equilibria.
APH,+ * APHP e APH- e AP=
AP' corresponds to I 1 I (Fig. I ). Well-defined isobestic points enable the pK's of I .5 and 5.0 to be easily established. In the intermediate pH range more than two absorbing species are present and so the pK of the neutral to mono-anion species is less well defined.
Changes in pK going from ground to first excited singlet states are now well docu- mented[ 17, 181. Forster has suggested that the loss of fluorescence of a-naphthylamine in aqueous solution around pH 13 is due to loss of a proton from the amine group to form the non-fluorescent anion [ 19,201. Figure 4 shows that a fluorescence decrease also occurs for 3-aminophthalate in this pH region and may be attributed to the same proton removal from the amine, since the N:N-dimethylated derivative shows no change in QF with increasing basicity[5, 20, 211. The drop in 3-aminophthalate QF
below pH 6 can be attributed to the presence of the non-fluorescent mono-anion, the pK for its formation lying at 5.0 (Table 3).
The rest of the discussion will concern only the species AP= and its excited states. If the hydrogen bonding effects of water are avoided by using dry aprotic solvents
Spectral characteristics 255
(Table 2) the absorption maximum vo is blue shifted by decreasing solvent polarity. This shift suggests a mr* character for this lowest energy absorption[22]. A strong blue shift of 1 100 cm-l going from DMSO to water, however, suggests the dominating influence of intermolecular hydrogen bonding[23]. In the ground state therefore the dianion exists in the form
(AP= ...... H,O)
,_...I@'" 0
Table 6 further documents this suggestion. The aminophthalate and aminobenzoate Fa are all blue shifted on going from DMSO to aqueous solvent. On the other hand the dimethylated derivatives are red shifted for two of the three cases showing regular behavior with increase in solvent polarity. The blue shift of the m-dimethylamino of course shows that the situation is not quite so simple.
There is some evidence that in the excited state the hydrogen bond of the type HNH ... OH2 may be weakened and that of the type HNH ... O=C strengthened [23-251. This latter effect is aided by the known changes in opposite directions in acid- base strength of the amine and carboxyl groups in the excited state. I t can be expected therefore that there will be hydrogen bond re-arrangements following excitation. A basic aqueous environment probably prevents a complete proton shift from the amine of AP= to the carboxyl as is found. for the orthohydroxybenzoic and naphthoic acids [19,25,26]. In DMSO, however, this could occur since it is known to allow marked changes of pK of acid-base groups in the ground state[27,28].
Proton transfer can explain the large Stokes shifts observed for AP' both in water (33,000 + 23,250 cm-I) and in DMSO (3 1,900 + 24,000; 20,000 cm-l). These have been observed for other molecules, notably firefly luciferin in aqueous solution [29] (30,500 4 22,250; 18,50Ocm-') and the ortho substituted carboxylic acids[23,26, 30,3 I].
Table 6 provides further evidence particularly for the DMSO solvent where inter- molecular hydrogen bonding is minimized.
Relative to 3-aminophthalate we would expect both 4-aminophthalate and 5-amino- isophthalate to exhibit sterically hindered intra-molecular proton transfer. Thus while 3-aminophthalate exhibits a major fluorescence at 20,200 cm-' with only a 9 per cent contribution from the non-proton-transferred higher-energy level at 24,000 cm-I (Table 2), the latter two emit only from their higher energy levels. This is again obvious from inspection of o- and m-amino-benzoate respectively. Thus we would t . :pect large Stokes shifts ( I 1,000 crn-') where excited state proton transfer occurs and more normal (4000-8000 cm-') Stokes shifts for the other cases.
The 20,200 cm-1 fluorescence of 3-aminophthalate in DMSO is therefore proposed to originate from the excited state species
256 J. LEE and H. H. SELIGER
and in the aqueous system the 23,250 crn-* fluorescence originates from
0 ,.H' 'H
'bP=).: .... H,O
The energy level diagram of Fig. 6 is constructed for aprotic and aqueous systems using the data of Table 4. The ground states of the emitting species are not known but would be expected to be about 1000 cm-' above the species in equilibrium. Since this is a spectroscopic energy level diagram we have arbitrarily set all ground states at zero. The 25,600cm-' and 20,00Ocm-' levels observed at low temperature in aprotic solvent (Table 4) are essentially the same as those observed for DMF and AN at room temperature (Table 2). In the aqueous glassy solution however inter-molecular re- arrangements will be hindered and emission would be expected from the '(AP= ... H,O)* excited state (corresponding to the ground state) rather than from the I(AP=)* ... H,O level, the latter accessible at room temperature. Since the triplet levels are long- lived they are assumed to be completely relaxed at the low temperature and therefore will not change energy on going to room temperature.
- c
h
Fig. 6. Spectroscopic energy level diagram for the 3-aminophthalate dianion in aprotic and aqueous systems at 77°K and at mom temperature (Tables 2 and 4). The levels correspond to FF, not to the 0-0 transition, since the absorption spectra of some of the species are not
known.
Spectral characteristics 257
1 t has recently been suggested [32] that intramolecular proton transfer provides an efficient route for the S' -+ So internal conversion. For 3-aminophthalate in DMSO the proton-transferred state itself is also efficiently fluorescent. The measured QF is thus a measure of the overall yield, e.g. in general, QF = a + Py.
hu A P= + (AP=)*
/ I \
Fluorescence Quenched (-A P-) *
/ \
J I Y / \ 1-7
Fluorescence Quenched
For luminol chemiluminescence and photoluminescence at room temperature a -- 0 and Q F = P y . Thus y , the fluorescence yield of I(-AP-)* may considerably exceed QF, since p can be much less than unity. The quenching by proton donors in aprotic solvents may be explained by the energetically favored reaction with the primary excited species: (AP=)* + HA + (APH-)* -k A- where the mono-anion is not fluores- cent.
The primary species formed by photoexcitation relaxes rapidly to the fluorescent state. The relatively small quenching effect of oxygen on this singlet level is consistent with a singlet lifetime of the order of 10-gsec, since diffusion controlled oxygen quenching has a rate of 2 x 1O1O M-I sec-I [331.
There remains the small but significant difference between the chemiluminescent emission of luminol and the photoexcited emission spectrum of 3-aminophthalate in DMSO (Fig. 3). In aqueous solution the two spectra are identical within our experi- mental precision. The spectral difference of 300 cm-' in DMSO is energetically within the range of hydrogen bonding perturbations or solvent shifts as seen by the difference between I(AP')* (DMSO, VF = 24000 crn-I) and I(AP=)* ... HzO, (FF = 23250 cm-I). Thus it is possible that the immediate environment of the chemically produced '(-AP-)* is not the same as photoexcited '(-AP-)*. Alternatively the chemiluminescence red shift may arise from the same source as the shift in tF seen for excitation in the anti- Stokes region [34]. This is caused by a finite relaxation time for solvent re-arrangement or distribution between symmetry states of the excited molecule which may not be negligible in comparison with the radiative lifetime. The chemical excitation may mimic the anti-Stokes excitation process in that the available free energy is not sufficient to proceed via '(AP')* as is the case with photoluminescence, but I(-AP-)* is populated directly.
Acknowledgements-We thank J . F. Ferguson for technical assistance in part of this work and Dr. W. Yeranos for valuable discussions.
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