Phofochemisfry and Phofobiology. 1972, Vol. 15, pp. 227-237. Pergamon Press. Printed in Great Britain

QUANTUM YIELDS OF THE LUMINOL CHEMILUMINESCENCE REACTION IN AQUEOUS

AND APROTIC SOLVENTS" J.LEE and H. H. SELIGER

Department of Biochemistry, University of Georgia, Athens, Georgia 3060 1, U.S.A.

and

The McCollum-Pratt Institute and Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, U.S.A.

(Received 22 March 197 I : accepted 2 June 197 I )

Abstract - Quantum yields for luminol (3-aminophthalic hydrazide) chemiluminescence reac- tions fall into two classes depending on oxidizing conditions. In aprotic solvents the quantum yield is high and the excitation yield which allows for the fluorescence quantum yield of the product, is 0.09 and is unaffected by changes in solution temperature or polarity, or the presence of quenchers. In aqueous solution under optimum pH conditions (1 1-13), hydrogen peroxide oxidation results in a high chemiluminescence quantum yield with an excitation yield of 0.04 again unaffected by temperature, viscosity or quenchers. Other oxidizing conditions produce lower quantum yields probably by the introduction of competing chemical pathways.

The luminol chemiluminescence light standard has been used to calibrate a spectrofluoro- meter with results in good agreement with the quantum yields of the femoxalate actinometer and the fluorescence of quinine sulfate and diphenylanthracene.

INTRODUCTION THE chemiluminescence oxidation of luminol (3-aminophthalhydrazide, I, Fig. 1) is one of the most studied chemiluminescence reactions in solution[l-3]. White and his coworkers have firmly established that in dimethylsulfoxide (DMSO) the overall stoichiometry requires two moles of base, one of oxygen and one of luminol, with the subsequent elimination of one mole of nitrogen and formation of the electronically excited product, 3-aminophthalate dianion (111, Fig. 1)[3,4]. In water under optimum pH conditions the further addition of sodium hypochlorite or hydrogen peroxide is required for maximum chemiluminescence quantum yield? (Q,)[5,61.

Under many oxidizing conditions in aqueous solution such as ferricyanide, persul- fate and electrochemical oxidation, the Q, is much less than that obtained with peroxide [6-91. In a recent detailed study of the luminol reaction under conditions of dye- sensitized oxidation ('photochemiluminescence') the results can be explained simply by a reaction branching scheme [ 101. Depending on the relative concentrations of un- known oxidizing species the luminol will react along a light or dark path. The dark path in this case does not result in the same final product (111, Fig. 1).

In this present paper we suggest that luminol chemiluminescence reactions can be

*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. Contribution Number 643 of the McCoUum-Pratt Institute.

?The chemiluminescence quantum yield Q, 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.

221

228 J . LEEand H . H . SELIGER

0 0

I 0

1 ii NH2 0

Fig. 1. Mechanism suggested by White and coworkers [4] for the chemiluminescence oxidation of luminol (I) in DMSO.

divided into two types based on the values of Qc. The low Q, reactions are probably the result of competing chemical dark paths. When Q, is high the light path is the sole chemical reaction and Q, can be written:

where QF is the fluorescence quantum yield of the electronically excited species and QE the excitation efficiency, that is the probability of the final light step resulting in an excited vs. a ground state product.

The chemiluminescence emission spectra in water and DMSO correspond to the fluorescence emission of 111 under the same conditions[5,6,11, 121 although a slight difference (200-300 cm-') is observed in DMSO[12]. By comparison with photo- luminescence properties of 111 we can show that Q E is not changed by the addition of certain singlet or triplet state quenchers or by solvent viscosity, polarity or temperature and that the emitting state must be formed directly and not via a proton transfer step as is the case with the fluorescence of 111 in aprotic solvents.

EXPERIMENTAL

Chemicals. Luminol (Eastman Organic Chemicals) was recrystallized several times from dilute acid until colorless. Its properties are compared in Table I with samples from several commercial sources and highly purified samples obtained from several colleagues. These purified samples include the sublimed free base and a six times recrystallized HBr salt. Spectrograde DMSO was from Crown ZellerbachCorp., Camus, Washington; dirnethylformarnide (DMF), acetonitrile (AN) and propylene glycol (PrG) were fluorescence grade from Hartman Ledden Co., Philadelphia, Pennsyl-

Luminol chemiluminescence reaction 229

vania; tetrahydrofuran (THF) was Baker 'analyzed' and was distilled from LiAIH, before use. All solvents were dried over anhydrous Na.$04. Potassium t-butoxide (t-BuOK), potassium superoxide (KO,), and potassium methoxide (MeOK) were from the Mine Safety Appliance Corp., Callary, Pennsylvania and were used withour further purification. All other chemicals used were of the best commercial grade.

Instrumentation. All absorption measurements were made on a Cary 14 spectro- photometer. Emission spectra of fluorescence and chemiluminescence were measured using a 3-port illuminator attachment on the Cary 14 and also with a fluorimeter con- structed of two Bausch and Lomb g4.4 grating single monochromators [ 131. Calibra- tion for spectral sensitivity was made using averaged results for the published spectra of quinine fluorescence as described before [ 121.

Chemiluminescence quantum yields were determined using a photomultiplier tube calibrated for absolute spectral sensitivity. All results are corrected for emission spec- tral overlap with the photomultiplier's spectral sensitivity curve, for self-absorption and for solvent refractive index[6].

Chemiluminescence reactions. Details of the chemical procedures for carrying out these reactions have been given elsewhere[l4]. In the aqueous reaction involving per- oxide optimum light yield was obtained only when the catalyst was added very slowly with rapid mixing. Rapid addition gave consistent results but only about 75 per cent of the optimum light yield. Buffers were prepared with a variety of compositions and salt concentrations without noticeable effect on the light yield. These solutions were pH 7.2 phosphate ( 1 O-, M), pH 7.7 Tris ( lo-, M). pH 8.5 Tris ( 1 M), pH 9 NaHCO, (1 M ) , pH 10 phosphate ( 1 0-2 M), pH 1 1 phosphate ( lo-, M) or NaHCO, (lo-' M), pH 11.6 phosphate (lo-' M) or K,CO, (10-IM). Higher pH values were unbuffered and ob- tained by appropriate concentration of NaOH. Solutions of H,0, and NaOCl were freshly diluted from stock before each measurement. The K,S,O, was used as a lo-, M solution or with Hz02 under the conditions specified by Rauhut and coworkers [7].

The technique for the aprotic reactions was not as critical as to whether solution was added to the oxidant or vice versa or whether rapidly or slowly. The total light in the DMSO reaction was also unaffected as to whether the potassium butoxide was made freshly from reaction of potassium metal with dry t-butanol, or from the commercially available butoxide either solid or in t-butanol solution. The presence of t-butanol merely aids in the solubility. Potassium methoxide was equally effective. A DMSO solution of KO, was also used being made up in a dry box as a saturated solution (lo-, M ) dried on a silica gel column. The reaction with KO, was markedly slow, but resulted in the same final Qc, and could be made more rapid by the addition of K,C03 (anhydrous). The K,CO, by itself was ineffective.

RESULTS

Luminol light standard. We have proposed the use of the luminol chemilumines- cence as a useful secondary light standard[l4]. As an indirect check on the absolute accuracy of Qc we have redetermined this using the fluorimeter calibrated by reference to the fluorescence of 9 : 10-diphenylanthracene in degassed (freeze-thaw) cyclohexane ( Q F = 1) [15]. The excitation intensity (366 nm) was calibrated using the ferrioxalate actinometer[l6]. The hemin catalyzed H,O, oxidation of luminol (pH 11.6) resulted in Q c = 1-1' 0.2 X lo-, in good agreement with our estimate previously made[6].

In order to determine any interference from impurities in the luminol sample the

230 J . LEEand H . H. SELIGER

light output was measured for luminol from different sources and different grades of purity as evidenced by variations in apparent extinction coefficient(€). Table 1 lists these E for solutions of well-dried luminol of different commercial and purified (A-D) grades. The less pure samples are seen to have the lower E but the Qc is reduced in the same proportion to give an unchanged photon yield per optical density unit. It will be shown in a following section that even added impurity quenchers are ineffective in reducing Qc until in quite high concentration.

When .kept in a dark bottle luminol solutions appear to be stable indefinitely. In N NaOH no change in light yield was observed after a period of a year.

High quantum yield reactions. Table 2 lists the luminol Qc in air-saturated aqueous and aprotic solvents. By comparison with the fluorescence yields ( Q F ) of the 3-amino- phthalate dianion, the excitation efficiency Q E can be calculated, assuming unit chemi- cal yield[3,4].

The aqueous reactions were carried out with the pH adjusted to 11.6. The oxidation was carried out with H,O, either alone or with the addition of NaOCl or other catalysts such as hemin or heme enzymes. Myoglobin or cytochrome c however have negligible activity unless denatured by heat treatment. Although the oxidizing conditions affected the rate of light emission over many orders of magnitude, the resulting Qc was the same. The reactions in PrG show also that viscosity has no effect on the Qc, although the rate is very slow and results in a less precise estimate of Q c .

Table I . Effect of source and purity of luminol samples on the total chemi- luminescence emission.

Source

Eastman Aldrich B.D.H.

A B C D

Apparent extinction (W1 cm-I) Water pH 11.6,347 nm DMSO, 359 nm

7625 7650 7410 7500 7300 7530

7575 7600 7900 7510

7900 7957 7897 7860

Average: 9.8 X 10" hv OD-' ~ m - ~

Photon yield (OD-' ~ m - ~ )

9.5 x 10" 10.0 9.8

10.0 9.3

10.0 9.6

9.8

9.7

Table 2. Reactions with high chemiluminescence quantum yield, Qc. Q , is independent of luminol and oxidant concentrations, and

the temperature.

Luminol 3-aminophthalate Excitation Solvent Q C Q F efficiency, Q;

Water 0.0124 0.30 0.04 PrG (50%) 0.012 0.38 0.03 PrG (90%) 0.012 0.38 0.03

DMSO 0.0124 0.14 0.09 DMF 0.004 0.05 0.08 THF 0.003 0.04 0.08

Luminol chemiluminescence reaction 23 1

In aprotic solvents the reaction can be initiated by the addition of KO,, t-BuOK or MeOK without variation in the overall Q , but again with marked variation in rate, being the fastest with MeOK. The Qc decreases with decreasing solvent polarity. This is entirely attributable to an effect on Q F ; and QEis unchanged.

In the hemin catalyzed H202 oxidation the luminol Qc is independent of initial con- centrations of H202 in the range 1 0-5 to lo-' M, provided it remains in 100 times excess over luminol. Outside this range Q , is reduced. Both the aqueous and DMSO reactions give a luminol Qc independent (k 2 per cent) of the initial luminol concentration in the range to 2 x M .

Temperature. Table 3 shows the general lack of a temperature effect on Q c for the high Q , cases. Each result is a mean of three reactions. The aqueous reactions were buffered at pH 11.6 at 20°C and utilized the H,O,/hemin system. The aprotic reactions were made with t-BuOK. The enhancement of Q c at low temperature in DMF is signifi- cant but can be accounted for by an increase in Q F of 3-aminophthalate. At the higher temperatures the PrG system appears to show some reduction but the origin of this effect was not determined.

Effect of p H on the aqueous reaction. Figure 2 shows the pH dependence of the luminol Q , for the high Q c aqueous reaction (c) and compares this with two low Qc

reactions (a) and (b). The pH dependence of Q F of 3-aminophthalate shows a differ- ent behavior. It is constant in the range pH 7-1 1.5 and falls rapidly outside of this. The fall of Q , on the low pH side appears to be the consequence of competing chemical paths since the reaction at pH 8 no longer shows the temperature and concentration independent properties of the high Q , class[l3]. Taken at face value it would appear that QE for curve (c) increases with pH above 11.5. At pH 13 for instance the Qc has dropped to only 75 per cent of its optimum value while Q F is down by a factor of four [ 121. The aminophthalate does not undergo decomposition during the period required for these measurements. The curves (a) and (b) will be discussed in a following section.

Quenching. The quenching studies were of two types - the effect of the addition of proton donors in the aprotic reaction and the effect of radical scavengers and excited state quenchers in the aqueous and aprotic systems.

The addition of proton donors to the chemiluminescence reaction in aprotic solvents produces both a spectral shift and a reduction in Q,. The effect is similar to that noted for the 3-aminophthalate fluorescence but the same proton donor is two or three times less effective in quenching the chemiluminescence as against the fluorescence (cf. Ref. [ 121). Figure 3 shows the reduction of Q, by water added to DMSO. Other proton

Table 3. Effect of temperature on high quantum yield reactions

Solvent T"C Q, Solvent T°C Qc

Water 3 0.0123 PrG(5Wo) -26 0.01 20 0.0124 20 0.012 40 0.0135 35 0.012 54 0.0116 40 0.01

45 0.008 DMF -18 0.06 48 0.005

0 0.063 20 0.05 DMSO 25 0.0124

45 0.0123

232 J. LEE and H. H. SELIGER

I d

I ' O O t

,'-'-+- \

/ ( a ) x I I I I I I 8 9 10 II 12 13 14 15

PH

Fig. 2. pH dependence of the luminol chemiluminescence quantum yield (Qe) for oxidation in air-saturated aqueous solution by (a) K,Fe(CN), without H,O,. (b) NaOCl without H202, (c) NaOCl with H20, or H20, with or without catalysts such as hemin or heme proteins. The

initial luminol concentrations are (a) M ; (b)(c) M.

10

a9

0"

0 5

0

log water concentration. M

Fig. Effect of water concentration on the luminol chemiluminescence quantum yield (QJ for the oxidation in DMSO made basic with t-BuOH (4%) saturated with r-BuOK (-- 10-*M).

Initial luminol concentration is M.

donors such as t-BuOH produce a similar effect on a per mole basis. The quenching rate does not follow a Stern-Volmer relationship and indeed Fig. 3 has the appearance of a pH titration curve. Neither the effectiveness for quenching nor the proportion of the

Luminol chernilurninescence reaction 233

two spectral components produced in the presence of the donor[l 1,121 is affected by changes in temperature in the range -20 to +50"C.

In the first section we showed that the presence of adventitious impurities had no effect on the luminol Q c . I t has been found that the concentration of added quenchers has to exceed lod3 M before a noticeable reduction of Q, can be seen. This again appears to be explicable in terms of the effect of these quenchers on the Q F but the marked slowing of the reactions and large self-absorption corrections required in some cases do not allow a precise statement to be made. Within 230 per cent the effects appear to be the same except for hydroquinone which strongly reduced the 3-amino- phthalate fluorescence at 5 X

Triplet state quenchers tested included oxygen, benzophenone, acetophenone, biacetyl, pyrene, anthracene or iodide and these showed no effect (< M). Addition of the product molecule itself up to M was without effect as were possible energy transfer acceptors such as o-aminobenzoate or 3-amino-2-naphthoate. Free radical scavengers such as butylated hydroxytoluene were also without effect (<

Solvent effects on chemiluminescence spectra. Table 4 compares the maximum of the luminol chemiluminescence spectra (V,) with the fluorescence maximum (i+) of 3-aminophthalate dianion in a number of solvents of decreasing polarity at room temp- erature except as noted. The identity of these spectra in water and the slight red shift of F, from FF in DMSO has previously been noted[l2]. In the other aprotic solvents the level of intensity is insufficient to locate F, or ijF with precision enough to see whether this slight shift also occurs in these solvents.

In the low temperature reaction F~ blue shifts and does not appear to be the same as &, which in the DMF:AN glass at 77°K is located at 20,00O~m-~[12]. Instead it seems to more nearly correspond to the phosphorescence, which at 77°K is located at 20,900 cm-1 and arises from a different ionic species[ 121. However a phosphorescence from 3-aminophthalate in degassed fluid solution of DMF at -60°C has not been detec- ted (1. B. C. Matheson and J . Lee, unpublished observation).

In a like manner to DMSO. addition of proton donor to the chemiluminescence reaction in the other aprotic solvents elicits a bluer emission centered around 25,000 cm-'. corresponding to the readily observable bluer fluorescence of the 3-aminophthal- ate[ 121.

M without noticeable change in Qc[12].

M).

Table 4. Effect of solvent on the maxi- mum of the luminol chemilumines- cence spectrum (&) and the lower energy maximum of the 3-amino- phthalate fluorescence (Ref.[12])

(%F)

Solvent Cc(crn-') FF(crn-')

Water 23250 23250 DMSO 19900 20200 DMF 20050 20100 AN 2oooo 2oooo THF 20 150 2oooo DMF:AN* 21200 20900t

*Ratio 7 : 3 vol, at -50°C ?Maximum of phosphorescence

emission 77°K

234 J . LEE and H. H . SELIGER

Table 5. Low quantum yield reactions. These Qc are maximum values and are all carried out in aqueous solution. The photosensitized result is from Ref. [ 101

Luminol concentration (M) Reaction conditions pH T"C Q C

10-5 K,Fe(CN), 12.2 20 o ~ o 0 O 1 o ~ o ~ m 1 10-5 NaOCl 11.6 20 0~004&0*001 10-3 KzSzOll 11.6 20 0*007~0*001* 10-5 Photosensitized with 20 0.003 ] MeB/O, [:::: 50 0.01

*(Ref.[7]givesQC = 0~008-tO~001)

Low quantum yield reactions. Finally we come to the group of other oxidants which in the aqueous reaction are not as effective as H202 for production of optimum Q,. These are typified by the results shown in Table 5 . The pH dependencies are shown in Fig. 2, curves (a) and (b). The pH dependence of K,S,Oe is similar to that for NaOCI; the methylene blue (MeB) photosensitized reaction parallels that of the K3( Fe(CN),) except that it shows much greater efficiency.

These reactions are also to be distinguished from the high Q , class in that they show pronounced temperature and concentration dependencies [7-9, 17, 181. These features also appear in the catalyzed H,O, reaction at the lower pH's [ 131.

DISCUSSION The optimum quantum yield for the luminol chemiluminescence reaction is much

less than the fluorescence yield of the product, 3-aminophthalate dianion, even though this product is formed in yields approaching 100 per cent[3,4]. The source of the chemiluminescence inefficiency is not clear. It is apparent that in the class of low Qc reactions a competing chemical reaction is probably responsible for the reduced light yield based on luminol. The photochemiluminescence reaction has been analyzed in detail in this regard[lO]. I n this latter case there is a marked temperature and concentra- tion dependence and the chemical yield of 3-aminophthalate is also reduced. I t is pro- bable that the photochemiluminescence Q , based on the product as distinct from the reactant, luminol, is unchanged from the H,O, reaction.

In the high Q c reactions it would seem fortuitous that a dark chemical reaction could be competitive with the light path, having no activation energy difference, concentration dependence, or difference in susceptibility to added quencher.

We propose therefore that there is only one chemical path in the high Q, class and that Q E represents the probability of the final step populating the electronically excited product over its ground state. Whether this final process is physical or a fast chemical re-arrangement of an intermediate is a subject for speculation. The probability is not altered by changes in temperature, solvent viscosity or polarity. concentration or presence of a variety of chemical or physical quenchers.

The fluorescence of I I I is much more readily quenched by proton donors in aprotic solvents or by base or hydroquinone in aqueous solutions, than is the chemilumines- cence. This is to be contrasted with the behavior of singlet and triplet state quenchers such as K1 which show the same behavior in the fluorescence and chemiluminescence. In our study of the spectral properties of 111 we have proposed that the abnormal Stokes shift observed requires that considerable hydrogen bonding re-arrangements

Luminol chemiluminescence reaction 235

and in the aprotic case a complete proton shift, occur in the excited state. Using the nomenclature of our previous work [ 121 we can write for the photoluminescence process in aqueous medium:

(AP= ... H,O)

(AP=) ... H,O - '(AP=) ... H,O

I(AP= ... H,O) - 3(AP=... H,O) I

Quenched t -hv' .1

and in aprotic:

where I(AP') and 3(AP') designate the first excited singlet and triplet states of 3- aminophthalate dianion and the symbols refer to the following hypothetical structures:

H

eo ,"- d

H

'(-AP-)*

0 .H' 'H e: N-H-

H'

'(AP')* ... . H,O

and AP' is structure 111. If the chemiluminescence process populates the emitting states directly and not via

the primary photoluminescence states I(AP=. . . H,O) and TAP=), then the differences in quenching of Q K and Q, can be explained. In aprotic solvents ](APE) reacts more rapidly than I(-AP-) with proton donors to give the non-fluorescent mono-anion. It has to be noted that the proton donor concentration has to exceed 10-I M before an effect is observable in the photoluminescence and this is 20 times higher than the concentra-

236 J . LEE and H. H. SELIGER

tions at which the other quenchers become effective. Assuming a diffusion controlled quenching rate greater than 1O1O M-' sec-' this would put an upper limit on the l(AP=) lifetime of about 1 nsec. If the proton donor reaction is also diffusion controlled a lack of temperature dependence in the relative contribution of the two emissions to the chemiluminescence would also result.

In aqueous medium the primary photoluminescence state reacts faster with base than the emitting state '(AP=). . . HzO, to form the non-fluorescent trianion. A similar argument can be applied here that the higher energy primary photoluminescence state is not accessible to the chemical reaction and that the emitting state is populated directly.

Because of this two-state situation in the photoluminescence it cannot be concluded that the presence of intermolecular hydrogen bonding in the aqueous case reduces Q by two times over the aprotic reaction (Table 2). The QF's are measured for the total photoluminescence process and represent minimum values for the emission probabili- ties from the final excited states. If the probability for3(AP') production is higher in the aprotic medium than the corresponding aqueous triplet then the true excitation effic- iency would be reduced in proportion and approach the measured aqueous value. It appears fortuitous that the chemiluminescence quantum yield in water is identical with that in DMSO. In other aprotic solvents (Table 2) this is not the case.

The effect of singlet and triplet state quenchers on QF for 3-aminophthalate and Qr of luminol is about the same. Assuming diffusion controlled quenching rates this would lead to a lifetime of about 10 nsec for the emitting state, which is the same electronic state in both chemiluminescence and fluorescence. If the primary photoluminescence excited state, I(AP=. . . H,O) is short-lived enough and is quenched at the same rate as the rearranged state, I(AP=). . . HzO its presence would not be detected in these experiments. In the chemiluminescence situation however we are forced to conclude that there can be no quenchable states preceeding the emitting state having a lifetime longer or even comparable to it (1 0 nsec). The chemical reaction then must populate the emitter dir- ectly or, if via an intermediate triplet or radical species its lifetime is too short to be detectable by the present experiments. More precise quenching data would set limits on this lifetime.

The mechanism of Fig. 1 suggests that in aprotic solvents the luminol dianion reacts with molecular oxygen to produce the excited product. It is not readily apparent why the dianion, which is reported to have a pK of 15[19], does not also spontaneously react with oxygen in water, even over a year when stored in N NaOH. Also it is known that the superoxide radical anion, 0,- is formed spontaneously in DMSO containing t-BuOK and oxygen[20,21]. This radical is usually a reactive species and therefore if it leads to a dark reaction with luminol, Qr would be lowered by saturating the solution with KO,. This does not occur suggesting that if it is reacting then it is involved in the chemistry of the light path. Hydrogen peroxide breakdown is known to produce 0,- [22] and the inclusion of this species in the light reaction would provide a common chemical mechanism for the reaction in both aprotic and aqueous solvents. I t could be suggested then that the dianion fails to react with oxygen in aqueous solution since it requires 0,- which is only produced on addition and breakdown of H,O,. The diazo- quinone which White and coworkers have synthesized still requires H,O, for light production[23]. Further chemical work could be usefully made in establishing or eliminating the role of 0,- in these reactions.

237 Luminol chemiluminescence reaction

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