Indian Journal of Chemistry Vo1.38A, August 1 999, pp. 760-767

Luminol fluorescence quenching by triethyl amine and non-linear Stern-Volmer

plot : Solvent effect

o Guha, S Mitra, R Das & S Mukherjee* Department of Physical Chemistry,

Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032, India

Received 12 March 1 999; revised 21 May 1 999

The interaction of 3-aminophthalhydrazide (Iuminol) with a quencher, triethyl amine (TEA) has been studied employing steady-state and nanosecond time resolved emission spectroscopy. The bimolecular rate constant for fluorescence quenching in water is found to be about two orders of magnitude higher than that obtained in 1 ,4-dioxane (DIO). The Stern-Volmer (S-V) plot for fluorescence quenching exhibits positive deviation from linearity when water is used as solvent. On the other hand, the S-V plot shows negative deviation from linearity when DIO is used as a solvent. The rate constants obtained in ethanol, dimethyl sulphoxide (DMSO) and DIO are found to be similar to the rate constant for diffusional process. The possible interpretation of the quenching mechanism and nature of deviation from S-V plots have been discussed in relation to the strength of interaction between the colliding species. It is shown that the rate of luminol fluorescence quenching is dependent on the nature of the solvent, being particularly fast in aqueous medium.

It is wel l-known that certain quenching reactions lead to curved Stern-Volmer (S-V) plots. Both positive and negative curvatures from l inearity have been observed and a number of explanations for this have been proposed 1-4,30-35 . A lthough a number of studies on fluorescence quenching have been reported, very few studies have been made on the variation of curvature of the S-V plot and quenching rate constant (kq) with the nature of the solvent. The effect of solvent on the rate of bimolecular quenching and photoprocesses involving excited molecule has received l ittle attention so far.

In some of the works on quenching reaction it is shown that polarity of the solvent medium can be expected to play an important role in the mechanism of fluorescence quench ing reaction5•6. Recently, it has been shown that the rate of fluorescence quenching of aromatic hydrocarbons by olefins is dependent upon the solvent, being particularly fast in aqueous medium. It is suggested that th is type of solvent effect is due to solvophobic interactions or stabi l ization of charge transfer interaction, and it is generally associated with the occurrence of polar structures along the reaction coord inates7-9 . Recently, in the case of dimethoxynaphthalene fluorophore10, it has been shown that the value of quenching rate constant (kq) is dependent upon the nature of the solvent.

Reactions i nvolving luminol participation are widely used for analytical purposes and also for studying the mechanism of transformation of chemical bond energy into electronic excitation energy of molecules. For the last few years, we are engaged in studying the excited state photophysics of

luminol in different solvent medial l , 12 . In this paper we report our observations on the interaction of luminol with triethyl amine (TEA). It is shown that the quenching rate constants are mainly dependent on the solvent and the process is considerably faster in aqueous medium. A study has been initiated here involving quenching of luminol fluorescence by TEA to show the solvent-dependent deviation from Stern­Volmer (S-V) plots.

Materials and Methods

The sample of luminol (98%) was obtained from Fluka AG and used as received. Luminol is sparingly soluble in pure water (- 1 0-5 M), and the concentration of luminol was maintained at that l imit. Triethyl amine (TEA, from E. Merck) was used as received. Solvent ethanol, dimethyl sulphoxide (OM SO) and I , 4-dioxane (DIO) (spectroscopic grade from E. Merck) were dried and disti l led before use. Triply disti l led water was used for solution preparation .

�

Ie)

-

480

GUHA et al. : LUMINOL FLUORESCENCE QUENCHING BY TRlETHYL AMINE

1.0 (a)

'.a

JU

, <

t '.4

•

'.1

•

440 om A

-!

1

J G

!

...

•

510 480 440 DID A

2 3 4 l/(TEA] x lOs mol dm-l

76 1

Fig. I-Absorption (a) and emission (b : water, c : dioxane) spectra for luminol-TEA interaction. [Iuminol] = 4.5 x 1 O-5M; range of

[TEA](a : 0 � 6) = 0 - 3 .8 x l O·sM; (b : 0 � 9) = 0- 4.6 x l O·sM ; (c : 0 � 7) = 0 - 3 .0 x 1 0· IM ; (d) plot of I /(Ac-Ar) vs. I /[TEA] where Ar = absorbance of free donor (luminol) ,Ab = absorbance of the hydrogen bonded complex, Ac = absorbance of a solution of donor where c is the concentration of TEA. '0' stands for the corresponding number of the curve where [TEA]=O.

762 INDIAN J CHEM SEC A, AUGUST 1 999

The electronic absorption spectra were recorded on a JASeO (Model 7850) UV NIS spectrophotometer. Fluorescence emission spectra were recorded on a Perkin-Elmer MPF 44B fluorimeter. Fluorescence l ifetimes were measured with a SP-70 nanosecond spectrometer (Appl ied Photophysics Ltd. ,UK) using a pulsed n itrogen lamp based on the time-correlated single photon counting technique as described earlier' 3, ' 5 . Different solutions were prepared keeping the luminol concentration fixed in each case and varying the quencher [TEA],The temperature was maintained at 300K throughout.

Results and Discussion

The absorption spectra of luminol show two bands, one at 300 nm and another at 360 nm in al l the solvents used, whereas the fluorescence spectra showed bands at 430, 4 1 5, 405 and 390 nm in water, ethanol, DMSO and DIO, respectively as reported earlier" , 1 2 . When the concentration of TEA was increased gradual ly, the emission intensity decreased significantly as a consequence of luminol fluorescence quenching without any appreciable change in posjtion and shape of the emission band (Fig, I ) . It was also observed that by the gradual addition of TEA, the intens ity of the absorption band

increased gradually with a small red shift (- 2.3 nm ) (Fig, I ) and then tended to reach a constant value above a certain concentration of TEA (5 .5 x 1 0-5M), This indicates that intermolecular hydrogen bond formation is almost complete at such concentrations of TEA with the formation of a I : I complex between luminol and TEA molecules, Since water comprises bulk of the solution in which the relative concentration of luminol is low, the effect due to change of polarity, without hydrogen bonding

4 6 8 10 [TEA] • 105 mol/dm3

1 2 14

Fig.2-Stern-Volmer (S-V) plot for luminol fluorescence quenching in aqueous medium. The broken line corresponds to the requirements for the S-V plots.

3.4r------------------------

2.6 � -e r-S ..

-e .. 1.8

0.8 1.6 [ TEA ) x to-l nio) I dm3

Fig.3-Stern-Volmer(S-V) plots tor luminol fluorescence quenching in DIO (e) and DIO:water (2:8) mixture [foil (�), toft (0») . The broken line corresponds to the requirements for the S-V law.

�.

GUHA et al. : LUMINOL FLUORESCENCE QUENCHING BY TRIETHYL AMINE 763 cX:1 , I i � NH

TEA -I cX:1

---TEA

� I I ! - - - TEA

o o

3-arninophthalate 3-aminophthalbydrazide (nonfiuorescent)

(fluorescent in presence of TEA) I (lwninol)

II

Table I -Lifetime (tr ) and bimolecular rate constants (kq, kq') in different solvents for the luminol fluorescence

quenching by TEA

Solvent tr. kq, (kq)* Range of (ns) (MI S· I ) [TEA], (M)

Water 1 0.4 2.4x 1 0 1 2 1 .0-4.9x I O's

010 1 . 8 3 .8x 1 09 0.28- 1 . 7x I 0· 1 (4.0x I 09)

OM SO 2.4 6.5 x 1 09 0.9- 1 .2x 1 0.2

(3 .2x 1 09) Ethanol 5 .5 3 . 5 x 1 0 1O 0.2-5 .2x 1 0.2

(2 .2x I 09) Ethanol: Water 7.2 9.0x 1 01 1 2.2-8.8x 1 0.5 ( 1 : 1 )

*Values in the parentheses . are the kq' values obtained from l ifetime measurements.

interaction wi l l not be of any great significan'ce and the observed spectral changes should presull)ably be attributed to i ntermolecular hydrogen bonding. It can also be said that i ntermolecular hydrogen bonding interaction is almost complete in the l imit of TEA concentration used here (� 1 0.5 M). From the change in absorption spectra, the ground state equil ibrium constant (Kg ) for hydrogen bond formation has been evaluated by Ketelaar's methodl6 using the fol lowing equation

.( I )

Table 2-Lifetime (tr), quenching rate constant (kq ) and effective TEA concentration in luminol fluorescence in DIO/water solvent

Range of [TEA], M 0 .41 -3. l x I 0· 1 0.62-2. 1 x I 0. 1 0.95- 1 .5x 1 0.3 1 .00-2.8>< 1 0.3 3 .90-6.3x I 0-4 O.56-2.8x 1 0.4

mixtures tr, kg,

(ns) (M ·I S· I ) 1 .6 4.5x 1 09 2. 1 7.2x 1 09 3 .8 8 .5x 1 09 4 .6 9.2x l OI I 7 .2 9.8x 1 0 1 1 8 .5 I . I x l 01 2

DIO:Water (v/v) 9 : 1 8 :2 6:4 5 :5 2 :8 1 :9

where Ar and Ab are absorbances of the free donor (luminol) and the hydrogen-bonded complex, respectively and Ac is absorbance of a solution with donor where c is the concentration of TEA. On extrapolation of the straight l ine obtained from plot (Fig. I d) of I /(Ac -Ar ) vs. I /[TEA] to a point where I /(Ac -Ar ) = 0, Kg is obtained as Kg = - I I[TEA] . The value of Kg obtained in water is 2 .2 x 1 03M . 1 . The corresponding quantity in the excited state (Ke ) can be estimated approximately by equation (2) assuming that the entropy change due to hydrogen bond

formation i n the excited state is equal to that in the ground state 10 .

I K I M i l 0.625 · 1 og I' . = log Kel M ' + -- f..yal cm . . (2) T I K Here f..Ya is the l imiting wave number shift in the absorption spectra resulting due to hydrogen bonding

764 INDIAN J CHEM SEC A, AUGUST 1 999

interaction and T (298K) is the temperature in the absolute scale. The value of Ke obtained from Eq . (2) is 7.5 x 1 03 M -I •

The absorption of luminol in any other solvent used both in presence (- J 0-2 M) and absence of TEA does not show any observable change in absorption spectra. The excitation spectra monitored at emission wavelengths are similar to the absorption spectra both in presence and absence of base. We have examined the results of luminol fluorescence quenching by S-V Eqs (3) and (4),

1011= I + kq :to [TEA]

10/T.= I + kq' .T.o [TEA]

. . . (3)

. . . (4)

where 10, I, T.o , and 1 are the intensity and lifetime in absence and presence of TEA respectively; kq and kq' are the bimolecular rate constants obtained from the steady-state and direct l ifetime measurement

h . . I d I ' 1 1 1 5 I . tec IlIque, respective y, as state ear ler - , , t IS

observed from Fig. 2 that the S-V plots are qu ite l inear at relatively lower concentrations of quencher. The kq and kq' values are readi ly calculated from the l inear part of the S-V plots (Figs 2 and 3) and equations (3) and (4). Our results on luminol quenching show that there is interaction between luminol and TEA, particularly in the excited state. Ground state interaction is observed only when water is used as the solvent and in this case we are unable to detect any measurable change in the lifetime values on addition of TEA. The l ifetime value generally

,... E 1 .2

� T_ o

E 1.0 � 0 )(

-ex: w ..... "

0 '""1 " �

I 0.4 ---

0.2

remains unaffected in presence of ground state interaction . Our result shows that TEA quenches the emission of luminol through static mechanism. A quenching process is one which competes with spontaneous emission process and thereby shortens the l ifetime of the emitting molecules. In solution of luminol-TEA the ground state complex is present. There may be juxtaposition of fluorescer and quencher molecules at the moment of excitation, resulting in instantaneous loss of electronic energy, i .e. , static quenching. In the present case there is no competition with the fluorescence process and thus l ifetime value is not affected. Ground state complex formation reduces the intensity by competing with the

uncomplexed molecule by the absorption of incident radiation. The l ifetime and spectral position remain unchanged because the emission is almost exclusively due to those luminol molecules which are sti l l left in the solvent medium. This reflects that added TEA does not change the local environment of the bound luminol appreciably. The decrease in intensity can be explained if TEA causes a decrease in the number of luminol fluorophores I S-2 1 • The fact that luminol fluorescence is quenched by a strong base like TEA ind icates that hydrogen bonding interaction contributes significantly to the change in emission spectra by th� gradual addition of TEA where the luminal fluorophore acts as a proton donor. Because of the presence of electron-deficient nitrogen atom and owing to the high polarity of the imino group (>NH), luminal can act preferential ly as proton donor,

0 0·1 0·2 0·3 0'4 0·5 0·6 0·7 0-6 o·g

1 /10

Fig.4-Plot of 1 -(1//0 )/[TEA] versus 1//0 in water.

....

GUHA et al. : LUMINOL FLUORESCENCE QUENCHING BY TRIETHYL AMINE 765

particu larly in the excited state. It is also noted in this study that TEA is unable to quench the luminol fluorescence in presence of H202 . Recently, it is shown22-25 that luminol can be oxid ized by H202 as shown in Scheme I to form am inophthalate. This confirms our predict ion that im ino proton is involved in this hydrogen bonding interaction between luminol and TEA.

The most striking feature of the bimolecular quenching constants presented in Table I is that the kq value is about two orders of magnitude higher compared to the d iffusion-control led rate constant when water is used as solvent. In this case we have observed distinct positive deviation from l inearity in S-V P lot. Positive deviation also ind icates the presence of ground state complex formation or static quenching in presence of added TEA in water2. 1 5.24.25 . It i s pertinent to mention here that the non-l inearity of the data in Fig.2 could also be due to the formation of a 1 :2 ( luminol :TEA) complex in the ground state, as suggested in Scheme I , that is, two TEA molecules per luminol molecule may be involved . Deviation from S-V plot may also occur even when both static and dynamic quenching components are present. Thus, we have exam ined our quench ing results also by the modified form of S-V equation (5i6

1 1 - -

10 1 1 - W [ TEA] = Kwej;) + [ TEA] . . . ( 5 )

where Ksv (= kt 't o ) is the Stern-Volmer quenching constant and kt is the b imolecular rate constant in the modified equation (5) and other terms have the ir usual mean ings as stated above. According to equation (5 ), only a certain fraction (W) is actual ly quenched in the excited state. If this instantaneous process or static quenching is main ly due to the ground state complex formation, the fraction W should decrease from unity in contrast to that in the case of the simple S-V equation where W = I . It is also l i kely that both static and dynamic quenching processes are responsible for such h igh values of kq and kt26,27 . Moreover, the positive deviation reflects efficient quenching, strong and rapid interaction between the col l id ing species. I n order to get more information i t is reasonable to calcu late W and kt values. The W and kt are calculated from the intercept and slope of the plot of { 1 -(1/10 ) } /[TEA] vs 1/10 plot ( Fig.4). The ranges of W and k values obtained are 0 .52 to 0.65 and 9 .8 x l O

l l

M , I S,I respect ively .

The kt value obtained is sti l l qu ite h igh and this h igh value of kt is not due to the ground state complex formation alone. I t i s l i kely, therefore, that other mechan isms are also operative in this quenching process. Hence, to get complementary evidences in view of the large rate constant, further features remain to be discussed. It is noted in th is study that h igh values of rate constants (kq and kt ) are obtained only when water is used as a so lvent. Th is seems to indicate that there must be some interaction between luminol and water as shown in Scheme 1 22,25 . I nteraction is now expected to be much stronger due to the presence of OH ( I I , Scheme I ) group. Hydroxyl group (OH) is a much stronger proton donor particu larly in the excited state compared to the >N H group. This should explain the stronger interaction, the h igh value of kq and positive deviation from S-V plot in water. It is also observed that the effective concentration for lum inol fluorescence quenching is very low (� I 0'5 M) compared to that requ ired in the case of other solvents used (- I O')M) and we bel ieve that the quencher concentration is somehow related to the value of the rate constant28 .

On the other hand, we observed negative deviation from l inearity in S-V plot when DIO was used as a solvent for luminol fluorescence quench ing: by TEA (Fig. 3) . The kq values are about two orders of magnitude lower and the effective TEA concentrations are two orders of magn itude h igher compared to that required when water is used as a solvent (Table I ) . Al l the kq values obtained in 010, DMSO and ethanol are s imi lar to the diffusion control led rate. It is interesting to note that both positive and negative deviation in S-V plot can be observed simply by changing the so lvent med ium. This, shows that the solvent plays an important role in this luminol fluorescence quenching reaction by TEA.

To get confirmative evidences we have studied luminol fluorescence quench ing in d ifferent DIO/water m ixture and results are shown in Table 2 . It can be seen. from Table 2 that the effective TEA concentration decreases with the increase in kq value when vol% of water is increased in the solvent mixture. It is also noted that l ifetime etf) value decreases gradually as the vol% of 010 is increased in the DIO/water m ixture. The kq values obtained in DIO, DMSO and ethanol are consistent with the observed absence of interact ion between luminol and TEA in the ground state. Negative curvature from S-V plot involves a decrease in kq and is associated with a change in the fluorescence spectra at higher TEA

766 INDIAN J CHEM SEC A, AUGUST 1 999

concentration. The TEA concentrations required here are about three orders of magnitude higher than that required when water is used as solvent. Negative deviation from S-V plot is explained by Rol lefson et af. 3,29 by the assumption that the quencher molecules react with photoactivated luminor to fonn a new activated species which emits l ight of a different energy distribution from that emitted by the original luminol molecules. The fact that photoactivated luminol can undergo quenching reaction with TEA in DIO undergoing negative deviation from S-V plot whereas in water it does not react, is not surprising. It is quite l ikely to observe a high degree of specificity with respect to chemical reactivity. Our experimental studies show that kq values are mainly dependent upon the strength of interaction via a quencher concentration. At this low quencher concentration, steady-state quenching measurements are also guite adequate to describe the luminollTEA interaction2 .26.

[t is pertinent to mention one 11)0re point that polarity of the medium may have some role on the results of luminol fluorescence quenching by TEA. By increasing the vol% of DIO in water/DIO mixture we are actual ly decreasing the polarity of the medium.

The blue shift in the fluorescence spectra of DMSO and DIO from those of ethanol and water indicates weaker interaction of luminol with aprotic solvents. The red shift in water and alcohol wil l enhance the hydrogen-bonding complexation between alcohol and luminol . Stronger hydrogen'-bonding interaction may facilitate the stabi l ization of the emitting state of luminol causing a large red shift in water and alcohol. The relaxation due to solvent interaction in aprotic solvents is expected to be low, reflecting a relatively small Stokes shift in these solvents. The difference in Stokes shift indicates that interaction with the solvent plays an important role in the relaxation of the excited ' states36. Final �, it may be concluded that solvent relaxation in weakly interacting solvents is �xpected

to be low37. In the quenching reaction, a very rapid process may also occur which not only involves the stationary diffusion process but also may have contributions from long range interactions. If long range interactions are present, the charge transfer process can also be assumed to be induced by the hydrogen-bonding interactions. The difference in bimolecular rate constants, obtained by the different methods, may be due to difference in charge transfer interaction processes37• On the addition of TEA, a small blue shift is expected due to the weaker interaction with the basic solvent medium. On the

other hand, we observed stronger interaction in acidic medium as it enhances the hydrogen-bonding interaction between normal alcohols and luminol37•

Conclusions In this work we have shown that in luminol

fluorescence quenching both negative and positive deviations from Stem-Volmer plot can �e observed simply by changing the solvent medium keeping the fluorophore and quencher same. This is mainly due to the variation in reaction abi lity between the col l iding species and solvent medium. We believe that concentration of the quencher is also responsible for the observed order of the rate constants. Since bimolecular quenching rate constant is h igher only when water is used as a solvent, we propose that non­emissive exciplex formation is the cause of luminol fluorescence quenching in which polarity of solvent plays an important role.

Acknowledgement A part of th is work is supported by Dept. of

Science and Technology, Govt. of India, New Delhi. D. Guha thanks the CS[R, New Delhi for providing a senior research fel lowship. R.D is thankful to the CS[R, New Delhi for providing a Research Associateship.

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