Photochemisty and Photobiology, Vol. 61, No. 4, pp. 313-318, 1995 Printed in the United States. All rights reserved

003 1-8655/93 $05.00+0.00 0 1995 American Society for Photobiology

USE OF TRIS (2,2’-BIPYRIDINE) RUTHENIUM(I1) DICATION IN A NOVEL METHOD FOR THE DETERMINATION OF SINGLET

OXYGEN REACTION RATES: APPLICATION TO STUDIES OF GELATIN

PETER DOUGLAS*, PHILIPPA c. EVANS? and KEVIN HENBEST Chemistry Department, University College of Swansea, Singleton Park, Swansea SA2 8PP, UK

(Received 3 June 1994; accepted 1 November 1994)

Abstract- A novel method for the determination of singlet oxygen reaction rate constants is described and applied to studies of cyclohexadiene in methanol and gelatins in H 2 0 and DzO. The technique uses tris (2,2’-bipyridine) ruthenium(I1) dication (R~(b ipy)~~+) as both singlet oxygen sensitizer and in situ oxygen concentration monitor during irradiation of sealed samples. Because of the high efficiency with which the luminescence of R ~ ( b i p y ) ~ ~ + * can be detected, and the fact that emission lifetimes are used, the method offers some advantages over those previously described. The advantages and disadvantages of the method are discussed. A rate constant of 2.1 (+0.3) x lo6 mol-I dm3 s-* has been determined for the reaction of lo2 with cyclohexadiene in methanol. For two different photographic gelatins the sums of reaction and quenching rate constants are 2.0 (f0.4) x lo6 and 3.1 (k2.0) x lo5 mol-I dm3 S-I; for swine skin gelatin this value is 3.9 (f2.4) x los mol-l dm3 s-I. Chemical reaction, rather than physical quenching, is the dominant reaction route for gelatins and lo2.

INTRODUCTION

Singlet oxygen, lo2,$ is an important intermediate in many photooxidations. Our interest in the photodegradation of photographic materials has led us to study the kinetics of the interaction of lo2 with gelatin,j which is commonly used as an emulsion medium in photographic product^.^ Gelatin may also be of more general interest as a model substrate for other protein singlet oxygen interactions. We have developed a novel method for the determination of singlet oxygen rates, which only requires the determination of emission lifetimes of t r is (2,2‘-bipyridine) ruthenium(I1) dication (R~(bipy),~+) as sealed samples are irradiated. Because ofthe high efficiency with which luminescence can be detected, and the fact that emission lifetimes are used, the method offers some advan- tages over the laser kinetic absorption techniques or steady- state emission intensity measurements previously de~cribed.~ In addition the emission of R~(bipy),~+ lies in the visible spectrum and it can be readily monitored using standard laser luminescence techniques without recourse to the specialized detectors required for the direct measurement of the near IR lo2 emission.6

Oxygen quenching of the charge transfer triplet excited state of Ru(bipy)32+ produces lo2 with = 100% efficien~y.’.~

R~(bipy),~+ + hu + Ru(bi~y),~+* (1)

(2) Ru(bipy),2+* + R ~ ( b i p y ) ~ ~ + + hv

Ru(bipy),’+* + 02(38,) - Ru(bipy),’+ + 02(lA,) (3)

Since the concentration of oxygen in air-equilibrated water is 2.7 x mol dm-3,9 and k2 and k3 are 1.64 x lo6 s-’ and 3.1 x lo9 mol-1 dm3 dm3 s-I,Io then the quantum ef- ficiency of lo2 formation is substantial in air-equilibrated water. A similar situation is found for most other solvents and air-equilibrated environments. In addition, the lumi- nemnce decay rate of Ru(bipy)32+ * ( kL) is directly dependent on the oxygen concentration in solution and can be used as an in situ monitor of this,”J2 i.e.

The combination of these characteristics suggested to us that Ru(bipy),’+ might prove useful in the determination of (0, reaction rates; in the presence of oxygen, R~(bipy),~+ will act as a photosensitizer for lo2 production, and if a substrate that reacts with and consumes lo2 is present then the rate of decrease in kL will follow the rate of uptake of oxygen. Thus, Ru(bipy),2+ can act as both a source of lo2 and a monitor of lo2 uptake.

In order to confirm the validity of our approach we have first applied the technique to the study of the well-charac- terized substrates 2-methylfuran and cycl~hexadiene~ in methanol. We have then combined the basic method with the known variation of lo2 lifetime in D 2 0 and H 2 0 5 to determine the lo2 reaction rates of photographic gelatin and swine skin gelatin.

*To whom correspondence should be addressed. ?Current address: Cray Valley, Machen Technology Centre, Water-

$Abbreviations: lo2, singlet oxygen; Ru(bipy)3z+, tris (2,2’-bipyri- loo Works, Machen, Newport, Gwent NP1 8YN, UK.

dine) ruthenium(I1) dication.

MATERIALS AND METHODS

Materials. Samples of photographic gelatin (Kodak G56, and Kodak E852 class 46 TCGII type 4) were a gift from Kodak Ltd. Harrow, UK, Ru(bipy),*+ was obtained as the dichloride salt from

313

314 PETER DOUGLAS et al.

5'53'

Hmelseconds

Figure 1. Variation in the decay rate of Ru(bipy)32+ * with irradiation time in methanol in the presence of 2-methylfuran in a sealed vial. Ru(bipy),z+ = 8 x mol dm-, (fitted curve has no theoretical significance).

mol drn-,; 2-methylfuran = 1 x

Strem Chemicals (Newburyport, MA); D20 was obtained fi-om Sigma Chemical Co. Ltd. (Poole, UK); 1,3-cyclohexadiene, 2-methylfuran and swine skin gelatin from Aldrich Chemical Co. Ltd. (Gillingham, UK); and analytical reagent grade methanol from BDH Chemicals Ltd. (Poole, UK). Water was deionized and double distilled.

Methods. Solutions of Ru(bipy)32+ were made up at 8 x rnol dm - 3 in methanol, gelatidH,O or gelatin/D,O, with substrates as appropriate. Small screw-cap glass sample vials (2 mL) with alu- minized paper seals were completely filled with solution and sealed by tightening the screw cap (tests showed that when the cap was adequately tightened the diffusion of oxygen from the atmosphere into the vial was negligible over a time scale of at least an hour). The path length and concentration of Ru(bipy),2+ used results in an optically dilute sample with uniform absorption throughout the vial. The entire volume of the samples was irradiated for fixed time in- tervals using a 900 W Xe arc lamp equipped with a filter cell con- taining a saturated solution of sodium nitrite to limit excitation to wavelengths 2410 nm and to minimize heating of the sample. The luminescence lifetimes of Ru@ipy),2+* were obtained using a fre- quency-doubled Spectron NdNAG laser (A 532 nm, pulse duration -6 ns) as the excitation source and an Applied Photophysics laser kinetic spectrometer as detector. The luminescence at 610 nm was detected at 90" to the excitation beam using a Hamamatsu R928 photomultiplier, with a red glass cut-off filter (A t 580 nm) to remove scattered light from the excitation pulse. The photomultiplier re- sponse was recorded on a Gould 034072 oscilloscope and trans- ferred to a BBC Masterclass computer for kinetic analysis. In all cases emission decays followed first-order kinetics. The extent of photodegradation of Ru(bipy)3z+ following irradiation was assessed by comparing the emission intensity of air-equilibrated samples be- fore and after irradiation using a Jobin-Yvon JY3D spectrofluorom- eter, in all cases photodegradation of Ru(bipy),Z+ was negligible.

RESULTS

2-Methylfuran and cyclohexadiene in methanol

Figures 1 and 2 show the decrease in kL as R~(bipy)~,+ is irradiated in the presence of 2-methylfuran and methylcy- clohexane. In both cases k, decreases as the irradiation pro- ceeds and approaches a limiting value. When the seal on the sample tube was broken, then, over a period of time, kL returned to the initial value. Spectrofluorometry before and after irradiation showed that irradiation caused no significant degradation of Ru(bipy),2+. We are led to the conclusion that as the irradiation proceeds '0, formed by energy transfer from R~(bipy),~+* reacts with the substrate and is consumed in the process, and it is the reduction in the concentration

tlmelrecondo

Figure 2. Variation in the decay rate of Ru(bipy),,+* with irradiation time in methanol in the presence of cyclohexadiene in a sealed vial. Ru(bipy)32+ = 8 x mol dm-, (fitted curve has no theoretical significance).

mol drn-,; cyclohexadiene = 2 x

of 0, that causes kL to decrease. The reactions of lo2 are shown in Eqs. 5-7, where Eq. 5 represents reactive quenching by substrate (SUB), which consumes oxygen, Eq. 6 is physical quenching in which lo2 is deactivated but not consumed, and Eq. 7 represents deactivation of lo2 by the solvent alone.

loz + SUB -+ products (consumption of 0,) (5)

(6)

lo2 -+ 302 (7)

'02 + SUB-+302 + SUB

Using this kinetic scheme the rate of photooxygenation of the substrate and consumption of oxygen, d[02]/dt, is given by

d[O21/dt = I&Y(~[SUBI /{ (~ , + k6)[SUB] + k7i) (8)

where I is the rate of photon absorption by Ru(bipy),2+, $I is the quantum yield of R~(bipy),~+* formation and y is the fraction of the sensitizer excited state quenched by oxygen. Because kL is linearly dependent on [O,], then

d(kL)ldt = k,d[O,]/dt (9)

and hence the initial slope of Fig. 1 is proportional to d[02]/ dt.

[d(kL)ldt]initial

= Kk3yi(k5[SUB]/{(k5 + k6)[SUB] + k,}) (10) where K is a composite constant incorporating I and 9, which are constant for any given Ru(bipy),z+ concentration and irra- diation arrangement, and the initial y, yl, is given by Eq. 11

7' = k3[02Ii/(k2 + k3[O2li) = (kLi - kLf)/kLf ( 1 1)

where kLi and kLf refer to luminescence lifetimes before ir- radiation and after complete exhaustion of O2 in the sample. For many solvent systems k2 and k,[O,] are available in the literature. In other cases, or when components are present that may act as quenchers of Ru(bipy),2+, yi can be obtained from kL before and after prolonged irradiation, i.e. kL in air- equilibrated solution and in oxygen-depleted solution.

Equation 10 can be rearranged to give

I/(initial rate) = (k, + k6)/(Kk3yik5)

+ k,/(Kk3yik5[SUB]) (12)

Use of Ru(bipy),*+ for lo2 reaction rates 315

I no0 800

000 -* 700 5 GOO

7 600 p 400

300

.o . h

-

- /

200 ,' ,' FURAN

0 . . . . . . - 100 6' " 1 i,n v 0 9 0

0 100 200 300 400 500 600 700 800 (concentratlon ) ' I mol I dm'

Figure 3. (Initial rate)-l against (substrate concentration)-l for the decrease in kL in the presence of different concentrations of cyclohex- adiene (filled circles) and 2-methylfuran (open circles).

where rate refers to rate of change of kL (units of s-~) . Hence a plot of l/(initial rate) against I/[SUB] should be linear with slope k7/(Kk3yik,) and intercept (k, + k6)/(Kk3yik5) and slope/ intercept will give k,/(k, + k6). For furans and dienes k6 <<

and hence slope over intercept will give k,/k,. Figure 3 shows such a plot for data obtained with 2-methylfuran and cyclohexadiene. This type of graphical analysis has been de- scribed previ~us ly~J~-- '~ and can lead to rather large errors in the determination of k, because either the intercept or slope are not well defined by the data. Our cyclohexadiene data arc examples of the first case, while the 2-methylfuran data are examples of the second case. Analysis of the individual plots in Fig. 3 gives slopes and intercepts for cyclohexadiene and 2-methylfuran of 1.40 (fO.O1) x sz mol dm-3 and 46 (+21) x sz, and 0.008 (k0.003) x s2 mol dm-3 and 29.7 (51.1) x sz, respectively. From these values and using a value of 1 x lo5 s-l for k75 gives k, cyclohex- adiene = 3.3 (kl .5) x lo6 mol-I dm3 s-I and k5 2-meth- ylfuran = 3.7 (k 1.4) x lo8 mol-I dm3 s-I, which are within experimental error of those reported by others5 However, because the irradiation arrangement is the same for both substrates we can use the data for 2-methylfuran to give an accurate intercept for use with the slope from the cyclohexa- diene data. Essentially, the initial rate of reaction with loz is so rapid over the range of concentrations used that almost every '02 molecule generated is consumed by the 2-methylfuran; in this sense we are using the 2-methylfuran as an actinometer. Using this approach we obtain a precise value of k, for cyclohexadiene of 2.1 (k0.3) x lo6 mol-' dm3 s- I ; a value that is in agreement with, but which is more precise than, those obtained previo~sly.~

Gelatin studies

Figure 4 (top) shows the decrease in kL following irradiation of R~(bipy),~+ in 5% photographic gelatin/H,O. The initial kL value, kL1, of 2.23 x lo6 s-I is reduced upon irradiation and approaches the value found in degassed aqueous solu- tions. In DzO, k7 is less than one-tenth of that in H 2 0 (see Table 1) and Fig. 4 (bottom) shows kL as a function of ir- radiation time for R ~ ( b i p y ) ~ ~ + in a 5% gelatin/DzO (wt/wt) mixture. The rate of decrease of kL is substantially faster in D20 than HzO, consistent with the longer lifetime of 'OZ in

2.51 , , , . , , , . , , , . , , , . , I , . , , , . ,

2.31 2.41 i 2 x 2.211 *\

- 2.li

181-1 I I ' I ' I ' I , I

0 20 40 60 80 100 120 140 160 180 200 220 240

tlmelrecond

1.87

1.571 !/

I Y

1.37 1 \ r 1*271 1.1 7 \

1.07 . , , , , , . , . z , , , ? , s , t s

0 10 20 30 40 50 80 70 80 90 Tlmeleocondr

Figure 4. Variation in the decay rate of R~(bipy),~+* in: (top) 5% wt/wt gelatin/H,O; (bottom) in 5% wt/wt gelatin/D20 following ir- radiation in a sealed vial. Fitted curves are monoexponential; [(kL - kLf) = e-k].

D20. Table 1 collects rate parameters for Ru(bipy),z+* and lo2 in D20 and H20. If we make the reasonable assumption that k, and k6 are the same in HzO and D20, then the ratio of the initial slopes of Fig. 4 (top) and (bottom) can be used, along with values for k,, to give (k5 + k,)[GEL] via Eq. 13.

slopei(H20) slopei(D20)

- - k3H20yiHzO{(k5 kd[GEL] + k7(D20)} (13) k3DzOyiDzO{(k5 + k,)[GEL] + k,(H,O)}

Table 1. Rate parameters for Ru(bipy),*+ and '02 in D20, H 2 0 and CH30H

k 4 0 4 SKI* 44 3.1 10 k,/106 s-I 1.64$§ 1.02$6 1.246 kL (aerated)/106 s-l 2.688 1.966 4.846 K,/109 mol-I dm3 s-I 3.9 3.5 1.66 [02]i/10-4 mol dm3t 2.7 2.7 22

*From Wilkinson and Brummer.j ?From Murov et aL9 *From Zahir and H a h 8 #Rate constants measured in pure, air-equilibrated or degassed sol-

vent.

316 PETER DOUGLAS et al.

Table 2. Experimental data using photographic G56 gelatin*

5% gel/HzO 5% gel/D20

k,'/lO6 s ' 2.16 1.53 kLf/106 s-l 1.68 1.08 k3/1OY mol-l dm3 s - ' 1.8 1.7 Initial rate/ 1 O3 s - ~ 2.3 (k0.4) 4.6 (k0.2) First-order rate ~onstant / lO-~ 5.3 (?0.5) 10.6 (k1.1) t *Irradiation conditions were slightly different from those used to

obtain the data given in Tables 3 and 4.

where D20 and H 2 0 refer to values for the appropriate sol- vent. (An analogous equation can be obtained for experi- ments in which different gelatin concentrations are used in the same solvent.)

An alternative, and potentially more accurate, method of analysis can be developed provided k3[02] 5 k2, and [GEL] 2 [O,] and remains constant throughout the irradiation. Un- der these conditions the variation in y as the reaction pro- ceeds is linearly dependent on [O,], and the rate of loss of [O,] becomes first order in [O,], i.e.

d[O,]/dt

= K(k,tGELl/{(ks + kAGEI-1 + h})(k/kd[%]. (14)

In our case for studies in gelatin k3[02]' k2/4 and Eq. 14 can only be used as an approximation (this approach is not valid for the previous studies in methanol, see Table 1). Under these conditions it is more appropriate to use k2 + k 3 [ O 2 P , where [021ave is the average oxygen concentration over the period of analysis, instead of k2 in Eq. 13, i.e.

d[O,]/dt = K{k,[GEL]/(kS + k,)[GEL] + k7)

x (k34k2 + ~3[021ave)~[o,l (15)

because (kL - kLf) 0~ [O,] then (kL - kLf) will decrease with a first-order rate constant, k,,,, given by Eq. 16.

k,,, = K{ks[GEL]/(ks + &)[GEL] + k,)

x {k3/(k2 + k 3 [ 0 2 p ) . (16)

Figure 5 shows first-order analyses for data from irradiations in H 2 0 and D20. Both give reasonable straight-line plots, indicating that Eq. 16 is an acceptable approximation under the conditions used. Tables 2-4 collect data from expen- ments with 5% and 10% solutions of photographic and swine

t

-1.2 -

-1.7 -

-2.2 -

-3.2

0 20 40 60 80 100 120 140 160 180 200 220 240

timelaeconda

-1.2

L -1.4 .c f -1.6 ~

E -1.8 ~

-

A -

X

-2.0 -

-2.2

-2.4

-2.6

-

-

-

0 10 20 30 40 50 tlmelieconds

Figure 5 . Linearized first-order analysis of decrease in decay rate of R ~ ( b i p y ) ~ ~ + * in (top) 5% Wwt gelatin/H,O; (bottom) 5% Wwt gelatin/D,O.

skin gelatins in H 2 0 and D20. As can be seen from these data even though these aqueous gelatin solutions are gelled, oxygen diffusion is still efficient. A similar situation is found in studies of diffusion-controlled electron transfer reactions in gelled solutions.'

As a check on our experimental method we repeated an experiment using 5% gel/D,O using a 9.8% transmittance neutral density filter on the irradiation lamp. The results are shown in Fig. 6, and show the expected = 10-fold decrease in both initial rate and derived first-order rate constant.

An examination of the data in Tables 3 and 4 suggests that (k, + k,)[GEL] is somewhat greater than k in D 2 0 (3.1 x 1 O4 s- I) because increasing gelatin concentration does not

Table 3. Experimental data using photographic E852 gelatin

10% gel/H20 10% gel/DzO 5% gel/H,O 5Yo gel/D20

kL'/106 s-' kLf/106 s-I k3/1O9 mol-I dm3 s-l Initial rate/l03 sv2

2.12 1.52 2.23 1.77 1.07 1.81 1.3 1.7 1.6 7.5 11.8 3.6

1.62 1.12 1.9

13 (1.8)*

First-order rate constant/10-2 s-l 1.7 (k0.5) 3.6 (k0.14) 1.0 (k0.3) 3.93 (50.15) [0.32 (+O.l)]*

Initial ratello' s s 2 (from rate constants) 6.0 16.2 4.2 19.7

*Using a 9.8% neutral density filter.

Use of Ru(bipy),2+ for lo2 reaction rates 317

Table 4. Experimental data using swine skin gelatin

10% geVD20 5% gel/H20 5% gel/D20

kc/106 s-I 1.48 2.12 1.65 kLr/1O6 s-I 1.07 1.72 1.12 k3/109 mol-’ dm3 SKI 1.5 1.5 2.0

Initial rate/103 sx2 (from rate constants) 11.1 1.84 13.3

Initial rate/103 s - ~ 14.4 1.44 14.4 First-order rate constant/10-2 s-I 2.7 (&0.5) 0.46 (k0.2) 2.5 (k0.2)

lead to an increase in rate of decay of kL. In addition, the fact that the rate increases in H 2 0 as gelatin concentration is increased suggests that (k, + k6)[GEL] is less than or com- parable to k in water (4.4 x lo5 s - ~ ) . ~ Gelatin is composed predominantly of six amino acid residues: glycine (25Oh wt fraction), proline (12.5Oh), 4-hydroxyproline (1 1.5Oh), glu- tamic acid (lo%), alanine (9%) and arginine (7.7%), with the remaining 24% made up by smaller amounts of other amino acids.4 The average molar mass of the amino acid residues is 109 g mol-’,’ which gives an average amino acid residue concentration of 0.46 mol dm-3 in a 5% wt/wt gelatin so- lution. Table 5 collects rate constants obtained by application of Eqs. 13 and 16 to the data shown in Tables 2 4 .

The values of (k, + k6) determined here are comparable to those measured previously for pure amino acids: alanine (2.0 x lo6 mol-I dm3 s-l), arginine (5 1.0 x lo6 mol-I dm3 s-]), methionine (2.7 x lo7 mol-I dm3 SKI ) and histidine (3.2 x lo7 mol-I dm3 s - I ) . ~

In principle k, and k6 can be resolved using K derived from Fig. 3. At infinite concentration of 2-methylfuran, every lo2

molecule generated is consumed by reaction with the furan. Under these conditions

d(k,)/dt initial = Kk3(methanol)yi(methanol) (1 7)

and using data in Table 1 and the intercept of Fig. 3, we can calculate K = 2.57 x mol dm-3 s-I. Then k, can be calculated from Eq. 18 if (k, + k6) is known.

initial rate{(k, + k6)[GEL] + k,} [GEL1=3r’

k5 = (18)

Application of Eq. 18 to the data (using (k, + k6) = 3.9 ( i2 .4 ) x los) gives an average value of k, = 4.5 (k l .6 ) x lo5 mol-’ dm3 s-l for swine skin gelatin and (using (k, + k6) = 3.1 (k2.0) x 10,) an average value of k, = 5.9 (k2.6) x 10, mol-I dm3 s - I for photographic E852 gelatin (all initial rates obtained directly and those calculated from the rate constants have been averaged to obtain these data). Within experimental error k, = (k, + k6), and chemical reaction, rather than physical quenching, is the dominant reaction pathway for lo2 and gelatin.

DISCUSSION

A novel method for the determination of lo2 reaction rate constants has been applied to studies of cyclohexadiene in methanol and gelatins in H 2 0 and D20. The technique uses R~(bipy) ,~+ as both lo2 oxygen sensitizer and in situ oxygen concentration monitor during irradiation of sealed samples.

Potential advantages of the method, which arise primarily from the ease of detection of Ru(bipy),*+ emission and the inherent value of lifetime measurements as opposed to in- tensity measurements, are as follows. The method is suitable for small sample volumes. The proportion of R~(b ipy)~*+* quenched by oxygen can be easily obtained directly from the experimental data. Competitive reactions are relatively un-

Table 5. Rate constants for average amino acid in gelatin derived from data in Tables 2 4 *

(k5 + kd k5 Gelatin type and comparison used

G56

(mol-l dm3 s-l) (mol-I cm3 s-l)

5% gel D20/H20 initial rates 5% gel D20/H20 rate constants

1.4 x lo6 2.0 (k0.4) x lo6

E853

5% gel D20/H20 initial rates 5% gel D20/H20 rate constant

10% and 5% gel H20 rate constants Swine skin

5% gel D20/H20 initial rates 5% gel D20/H20 rate constants

9.8 x 105 4.8 (k2.0) x 105 1.4 (k3.0) x los [ave 3.1 (22.0) x lo5]

6.8 x lo4 3.9 (22.4) x los

5.9 (22.6) x lost

4.5 (k1.6) x lost

*Assuming 5% gelatin solution is 0.46 molar in amino acids. ?Average of all values obtained using Eq. 18 and 3.1 (52.0) x los or 3.9 (k2.4) x lo5 for (k, + k6); with all initial rates either measured

directly or calculated from first-order rate constants; within experimental error ks = (ks + k6). Initial rate measurements are less reliable than rate constants.

318 PETER DOUGLAS et a1

1.79

1.89

1.59

& 1.49 3 5 1.39

1.29

1.19

1.09

, , , , , . , , , , , , , , ~ , , , I , , , ,

-

-

-

~

-

l . ( . I . l . l . l . I . I I I I I I I . I

0 100 200 300 400 500 600 700 800 BOO 1000 1100

tlmelseconde

Figure 6. Effect of 9.8% transmission neutral density filter on decay kinetics. (top) Variation in the decay rate of Ru(bipy)32+* in 5% wt/ wt gelatin/D20 following irradiation in a sealed vial. Fitted curve is monoexponential; [(kL - kLf) = e-&]. (bottom) Linearized first-order analysis.

important because it is only those that consume oxygen, or consume or produce quenchers of Ru(bipy)32+*, that are de- tected. The compound Ru(bipy),*+ is photostable in the pres- ence of oxygen. Any loss of sensitizer is easily observed, and any formation or consumption of components that may act as quenchers of R ~ ( b i p y ) , ~ + is apparent from kL when the sample is reopened to air.

The main disadvantages of the method are as follows. The sample must be sealed off from the atmosphere during the experiment. The Ru(bipy),z+ dichloride salt cannot be used in nonpolar solvents because of poor solubility. However, ion pairing with organosoluble anions, or the addition ofalkyl chains onto the bipyridyl ligand gives materials with similar properties with good solubility in nonpolar media.I2

Using this method a rate constant of 2.1 (f0.3) x lo6 mol-' dm3 s-I has been determined for the reaction of '02 with cyclohexadiene in methanol. For two different photo-

graphic gelatins the sum of the reaction and quenching rate constants have been measured as 2.0 (k0.4) x lo6 and 3.1 (f2.0) x los mol- d m 3 SKI; for swine skin gelatin this value is 3.9 (k2.4) x lo5 mol-' dm3 s - ~ . Chemical reaction, rather than physical quenching, is the dominant reaction route for gelatins and lo2.

Acknowledgements- We thank the SERC for studentships for P. C. Evans and K. Henbest and for financial support. We also thank Mike Garley for the computer programs for the emission lifetime mea- surements.

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