Indian Journal of ChemistryVol. 35A, August 1996, pp. 633-638

Spectroscopic properties and fluorescence quenching of luminolin aqueous medium

U Bhattacharjee. S Mitra. R Das & S Mukherjee-

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

Received 1 December 1995; revised 16 February 1996

The interaction of 3-aminophthalhydrazide (luminol) with aqueous solution of NaOH and H2S04has been studied employing steady-state fluorescence quenching measurements and time-correlatedsingle photon counting technique. From nanosecond lifetime measurements and quantum yields of fluor-escence, the radiative and nonradiative decay rate constants are calculated at different pH. It is shownthat both the fluorescence quantum yield and rate constants are pH dependent. The quenching of lumi-nol fluorescence is analyzed by using simple Stern-Volmer equation. It is proposed that quenching is dueto some exciplex type of hydrogen bonding interaction.

Luminol is unique in that its reaction with bloodresults in the production of light as a blue whitelurriinescence. Luminol has been employed as lu-minescent probe and its applications in analyticalchemistry are now becoming important 1-6. Klimoret al? pointed out that the photoexcitation of theground state complex of luminol does not lead toformation of the' free radical. This is probably ow-ing to static quenching. Stanley and KrickaR ex-plored the mechanism of luminol oxidation byH202• Their observation reflects a complex natureof acid-base equilibria in solution and they con-cluded that luminol oxidation by H202 does notneed the presence of a second substrate enhancer.

The most significant advances in the study ofsuch donor-acceptor interaction or quenchingreaction have been achieved with the aid of time-resolved spectroscopy. However, at very lowquencher concentration steady state quenchingmeasurements are also quite considerable in de-scribing the fluorescer / quencher interaction. Wereport in this paper the results of both steady stateand kinetic measurements by time resolved singlephoton counting on luminol fluorescence quench-ing in water at different pH. It is also shown thatboth fluorescence quantum yield and decay rate ofluminol fluorescence are pH dependent. .

The existence and magnitude of the quenchingconstant can be understood in terms of the com-peting process of luminol fluorescence andquenching as given below:

(luminol) ~ (luminol)"

(luminol)" (k1( = Ih(~(luminoI)+ h v

(luminol)" + Q !swluminol) + Q*

where To is fluorescence lifetime in the absence ofquencher (NaOH, Q), kq is the rate constant forthe quenching reaction. The Stern-Volmer plot willbe linear as long as kq is independent of quench-er". We have examined the fluorescence quenchingby Stern-Volmer relationship.

It is now well known that certain quenchingreactions lead to curved Stern-Volmer (S-V) plotsand a number of explanations for this have beenproposed. Keizer? explained the deviations fromS-V equation by postulating association of thequencher with fluorescer in the ground state.However, deviation from S-V plots are also ex-pected even when the static quenching or groundstate complexation is absent!":". Recently, a num-ber of works of both theoretical and experimentalnature have been carried out on the fluorescencequenching and the observations are explained bydifferent models starting from conventional Smol-uchowski formulation to mean field approach 12-15.

If non-emissive exciplex formation is the cause offluorescence quenching, the polarity of the sol-vent medium is expected to play a role in the me-chanism and also on rate constant for quenchingreaction. Moreover, if the fluorescence is pro-duced by more than one excited state the quench-

634 INDIAN J CHEM, SEe. A, AUGUST 1996

IIIC

'"C

1.0

0.8

(b) ~(.( '"0.6 uc0J .DL

0III.D

0.4 -r

'">-o'"<r 0.2·

A, nm.!

Fig. I-Absorption (a) and emission (c) spectra of luminol inalkaline aqueous medium. [Iuminol] = 5.4 x 10- 5 mol dm - -' andrange of [NaOH] = 2.5-9.8 x 10- 5 mol dm -.1 from .(I to 9) and0(0); (b) in the excitation spectra corresponding to 430 nm

emission.

ing constants for the various states should differ.In this case the intensity of the fluorescenceshould be a function of the quencher concentra-tion".

Materials and MethodsLuminol is sparingly soluble in pure water ( -

10-5 mol dm r ') and the concentration was main-tained at that limit. Luminol was obtained fromFluka AG and used as received. AR grade NaOH,H2S04 and triply distilled water have been usedthroughout. Fluorescence emission and excitationspectra were recorded on a Perkin-Elmer MPF44B fluorimeter. The electronic absorption spectrawere scanned with JASCO UV/Vis spectropho-tometer, model 7850. The transient fluorescencelifetimes (.0) were measured with an SP-70 nanos-cond .spectrometer (Applied Photophysics Ltd.,England) using a pulsed nitrogen lamp based ontime correlated single photon counting technique.Relative quantum yields were determined from430 nm emission as described earlier!"!".

Results and Discussion

A. Spectral properties and luminol fluorescencequenching

The absorption and emission spectra of 3-amin-ophthalhydrazide (Iuminol) are shown in Fig. 1.

9

Fig. 2-Stern-Volmer plot for the quenching of luminol byNaOH.

The absorption spectra show two peaks, one at300 ,nm and another at 360 nm in pure water(Fig. l a) and-even in the presence of dilute acidand alkali. The two bands are probably due to twovibronic states. The large E (Emax - 5000) impliesthat luminol absorption is due to a not" tran-sitiorr'". The fluorescence spectra, on the otherhand, exhibit a single band at 430 nm region(Fig. lc) in aqueous medium. The excitationspectra of luminol (Fig. 1b) when monitored at430 nm agree reasonably with the absorption

.spectra. This indicates that emission is originatedfrom the main absorbing species. It is seen in ourstudy that the intensity and quantum yield of flu-orescence gradually increase with the increase inexcitation wavelength (Aexc,) as shown in Fig. 2.However, whatever be the excitation wavelengththe emission maxima remain always at the sameposition. It is also observed that the Franck-Con-don emission envelopes and excitation spectra ofluminol fluorescence did not show any measurablechange even at 77K. This implies that the structu-ral properties of luminol in the ground state arethe same as that in the excited state even at 77K.The fact that the excitation spectra of Iuminol aresimilar to its absorption spectra, indicates that theabsorbing species is fluorescent i.e. emission isoriginated from the ground state conformer. In ex-citation spectra the long wavelength band (360nm) is always stronger thanthe shorter wavelength(300 nm) band. This reflects that the emission isoriginated mainly from 360 nm region. The singlebroad fluorescnece band does not have mirror im-age symmetry relationship with its absorptioncounterpart. The consequence of this is that thetwo vibronic states must have coupled in the excit-ed state' or are very close togetherv". The largeStokes shift ( - 5000 cm - 1) shows a larger stabili-zation of the excited state relative to the groundstate. Since the ~r's are Aexc dependent and twobands are observed both in absorption and excita-tion spectra, it is quite possible that two close-ly-

BHATIACHARJEE et af.: FLUORESCENCE QUENCI;IING STIJDIES OF LUMINOL 635

¢NH2 °

l (luminal)!H202

~~:NH2 °

)-aminophthalal<

ONa+'

~~I" -...:.~yyNNH ONat2 -

1\1

OH

H20.'~

NH2 OH

\I

ing excited states are present in the broad emis-sion band of luminol (SI S2)24.25. The emitting stateof luminol relaxes from S2 state by interactionwith water molecules and ultimately emits frommore stabilized SI state. The absorption spectra ofluminol in water and in weakly polar aprotic sol-vent like 1,4-dioxan (010) are similar?". However,the emission spectra of luminol in 010 appearedat 390 nm. This is because the relaxation due tosolvent interaction in weakly interacting solventlike DIO is expected to be low reflecting a relat-ively small Stokes shift in 010 (2500 em -I). Thisdifference in Stokes shift in water and DIO isquite high and indicates that solvent relaxationplays a predominant role23.27.The relative quan-tum yield of fluorescence (tPr) at 77K seems todecrease markedly from that of the room tempera-ture yield. The tPr at room temperature and 77Kare determined to be 0.86 and 0.35, respectively.

The fluorescence emission intensity is found todecrease continuously by the gradual addition ofvery dilute solution of NaOH without any appreci-able change in position of the band (Fig. 1). Weare unable to detect any measurable change in theabsorption spectra at the concentration of NaOH( - 10- 5 mol dm - 3) used here. Since the quenchingis observed at very low quencher concentrationthe quenching process is supposed to be very effi-cient. Luminol fluorescence quenching is, there-fore, due to some exciplex type of interaction be-tween luminol and NaOH. Because of the pres-ence of electron deficient nitrogen atom and ow-ing to the high polarity of the imino group (> NH)luminol can act preferentially as proton donor,particularly in the excited state (Scheme 1). Thus,it is reasonable that luminol is acting as a protondonor in this excited state interaction between lu-minol and a strong base like NaOH. It is also ob-served in this study that NaOH is unable toquench luminol fluorescence in the presence ofH202• Recently, Yeshiorr" showed that H20t can

oxidize luminol to form aminophthalate as shownin Scheme 1 and concluded that luminol oxidationdose not need the presence of a second substrateenhancer. This carries further support to the factthat lumino, can act as proton donor and this willshow the possible sites of interaction i.e. > NHgroup (present in luminol) is involved in this hy-drogen bonding interaction.

The results of fluorescence quenching measure-ments by steady state and single photon countingtechnique can therefore be interpreted in terms ofa simple Stern-Volmer mechanism by Eqs (1) and(2).

]011 = 1 + kq To[NaOH]

T(l/ Tr= 1 + r; To[NaOH]

... (1)

... (2)

where ]011 and T°/rr are the ratios of the emissionintensity and lifetime in the absence and presenceof quencher (NaOH), respectively; kq and k~ arethe respective biomolecular rate constants. Fromthe linear part of the S-V plots (Fig. 2) and Eqs(1) and (2) the rate constants are readily calculat-ed. The values obtained for kq and k~ are1.2x1012 and 9.8xlOlI drrr'mol "!s " ', respect-ively. The striking feature is that these values arequite higher, higher than' the diffusion controlledrate constant (kd), which is of the order of - 109

dm-mol" 's - '. Moreover, results of steady statemeasurements show positive curvature from line-arity in S-V plot at relatively higher quencher con-centration. These observations reflect staticquenching or ground state complex formation'i".However, a variety of quenching reactions havebeen reported which exhibit curvature in S-Vplots, yet which show no evidence of molecularassociatiorr'". On the other hand, if non-emissiveexciplex formation is the cause of fluorescencequenching, the polarity of the solvent medium isexpected to play a role in the high value of kq anddeviation from S-V mechanisrrr'", Positive devia-tion from the S-V plot may be due to staticquenching which is discussed using the groundstate complexv-" or the sphere of action33,34staticquenching model. In the sphere of actionmodeP3,34 for static quenching, the coexistence offluorophore and the quencher within a certainreaction distance "R" of one another in a "sphereof action" of volume V in solution is necessary. Aquencher molecule within this interaction spherewill quench the fluoroescence due to photoexcitedfluorophore without requiring a collisional interac-tion". Since no specific interactions between thefluorophore and quencher have been detected in

636 INDIAN J CHEM, SEe. A, AUGUST 1996

Table l+-The quantum yield (;f), lifetime (1'f) and decay rate constants (kf, kr' and kf) at different pHpH <PI 1'f,ns lO--skf,s-1 lO-skr',s-1 1O-7k;,s-1

3.0 0.02 5.2 1.9 1.8 0.44.6 0.30 6.0 1.7 1.2 5.05.6 0.88 10.6 0.94 0.11 8.35.8 0.88 10.5 0.95 0.11 8.46.1 0.81 9.5 1.1 0.2 8.56.7 0.58 8.7 1.2 0.53 6.77.8 0.56 6.8 15 0.68 8.28.6 0.44 6.4 1.71.0 6.99.5 0.03 5.6 1.8 1.7 0.5

1.0 -,

~<18

.e:0.6

<14

•°3~1~O~--~~~~~~L-~~-UAnc ,nm

Fig. 3-rIuorescence quantum yield (<Pf) at different pH (e)and excitation wavelength (0).

both absorption and fluorescence spectra even atvery high quencher concentrations we concludethat the "sphere of action" model best suits oursystem similar to that observed by Zeng et al.",Here, kq> kd such that the reaction would be dif-fusion-controlled. For, further knowledge on thoseexcited state reactions, the influence of diffusion isconsidered. Several theoretical models of diffusioncontrolled reactions have been put forward". For,a diffusion controlled bimolecular reaction, therate coefficient is time dependent and is given bythe Smoluchowski-Collins-Kimball (SCK) model!'.

Since kq> kd , a dynamic model for quenchingis also required. The long time SCK model wasused by Zeng et aL36 but it was not fruitful in dis-criminating rates. of several quenching reactionswhere the reactions are diffusion influenced. So,they used the ''finite'' sink approximation"modeP8,39 which successfully explained the discri-mination of reaction rates between differentclasses of quenching reactions. Furthermore, if arapid quenching occurs this may not involve sta-

----------- --

tionary diffusion process but instead involve longrange interaction. It should be noted here that thequencher concentration for effective luminol fluor-escence quenching is low and if the quencher isefficient, the quencher concentration is usuallysmall enough to make the steady state measure-ments valid". Thus, one can see that the presentquenching process is remarkably stronger. It isshown in our earlier work on carbazoles that inthe case of a strong quencher, quenching effect islarge,kq values are high and distinct positive devi-ation from S-V plots are observed'":". A numberof features seems to be responsible for such highvalue of kq and deviation from S-V plots and webelieve that quencher concentration is somehowrelated with high value of kq (refs 18, 19). Thequenching experiment by nanosecond lifetimemeasurements (Fig. 2) does not show any devia-tion from linearity in S-V plot. This indicates thepresence of effective collisional quenching andthese collisions would obviously be very sensitiveto the emission Iifetimes":",

B. Luminol fluorescence quantum yield and decayrates at different pH

Luminol is highly fluorescent (~= 0.86) betweenpH 5 to 6.5. The pH of pure water containing lu-minol is 5.8. We measured the fluorescence quan-tum yields as a function of NaOH and H2S04

concentration. The relative quantum yields at dif-ferent pH are shown in Fig. 3. We have also mea-sured luminol fluorescence decay at different pH.The emission showed a single exponential decayboth in the presence and absence of base and ac-id. This indicates that luminol emission originatesfrom same environment both in the presence andabsence of base. The fluorescence decay rate con-stants (kr) are given as the sum of the radiative (kf)and nonradiative (k?) decay rate constants. Fromnanosecond measurements and quanttum yields of

BHATIACHARJEE etal.: FLUORESCENCE QUENCHING STUDIES OF LUMINOL 637

2.0

1.5

I",'"5? 1.0

-""0.5

0.00.0 5.0 10.0

[NoOH] • 10~mol dm-315.0

Fig. -l-~l.uminol fluorescence quenching by NaOH and rela-tion of k, with NaOH concentration.

fluorescence the rate constants are calculated fromEq.(l).

... (3)

Some of the results at different pH are shown inTahle 1. From the data in Table 1 it can be seenthat the radiative decay rates are always lowerthan the nonradiative decay rates and clearly de-pendent upon the pH of the medium. Thus nonra-diative decay process is dominant in the decayprocess of the excited state of luminol, at pH 9.5rate is faster. Thus one may conclude that thehase enhances the intermolecular interaction ef-fectively in aqueous solution. The rate constantsshown in Table 1 are of the order of diffusioncontrolled rate constant.

Recently" it is shown in the case of diphen-ylmethyl radical fluorescence quenching by a goodnumber of quenchers (Q) such as carbon tetrach-loride, methyl benzoate, chloroform etc., that theobserved decay rate constant (kr) is related to thequenching rate constant (kq) and to the decay rateconstant in the absence of quencher (k?) accordingto Eq. (4).

... (4)

We have examined our results of luminol fluor-escence quenching by NaOH with Eq. (4). Theplots of k, versus NaOH concentration are quitelinear with a slope of kq, the quenching rate con-stant. The plots obtained are shown in Fig. 4. Thisshows that as the lifefimes in the absence ofquencher. 1/ k?), decreases the rates of quenchingwill decrease. The kq value obtained in this way is1.2 x 1012 drrr'mol ~ 1S - 1. This kq value is similar to

-_._--- --------

the value obtained from S-V plots which confirmsour results. Such high k" value and positive dcvia-tion reflect efficient quenching, strong and rapidinteraction between the colliding species.

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638 INDII\N J CHEM. SEe. 1\. I\UGUST 1996

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