780 Volume 53, Number 7, 1999 APPLIED SPECTROSCOPY0003-7028 / 99 / 5307-0780$2.00 / 0

q 1999 Society for Applied Spectroscopy

Interactions between Methylene Blue and Sodium DodecylSulfate in Aqueous Solution Studied byMolecular Spectroscopy

MARY K. CARROLL,* MARC A. UNGER, ANDREW M. LEACH,MELISSA J. MORRIS, CHRISTINE M. INGERSOLL, and FRANK V. BRIGHTDepartment of Chemistry, Union College, Schenectady, New York 12308 (M.K.C., M.A.U., A.M.L., M.J.M); and Department of

Chemistry, Natural Sciences Complex, State University of New York at Buffalo, Buffalo, New York 14260-3000 (C.M.I., F.V.B.)

The interactions of methylene blue (MB, a cationic redox indicatorand biological stain) and sodium dodecyl sulfate (SDS, a micelle-

forming, anionic surfactant) in aqueous solution have been exam-

ined by using Rayleigh scattering, UV-visible absorption, and ¯ uo-rescence spectroscopy. At SDS concentrations signi® cantly below

the critical micelle concentration (cmc), MB forms noncovalent di-

mers and aggregates with SDS that scatter light but do not ¯ uo-resce. For solutions containing 1 m M MB and , 3± 5 mM SDS, shifts

in the absorption spectrum characteristic of the formation of MB

H-aggregates are noted. There appears to be little effect on the¯ uorescence emission spectrum, indicating that these MB aggre-

gates do not ¯ uoresce appreciably. At and above the known SDS

cmc, MB is observed to interact with the micelles. The MB excited-state ¯ uorescence lifetime (380 ps) remains constant until SDS mi-

celles form, then increases to 615 ps. The MB rotational reorien-

tation time similarly increases from 105 to 500 ps between 6 and 8mM SDS. This ® nding suggests that the MB is encountering, on

average, a microenvironment in the SDS micelles that is 5-fold more

viscous than liquid water or the molar volume of the MB/SDS spe-cies that is reorienting is 5-fold larger than MB in water.

Index Headings: Methylene blue; Sodium dodecyl sulfate; Pre-mi-

cellar aggregates; Fluorescence spectroscopy; Fluorescence lifetime;

Rotational correlation time.

INTRODUCTION

The self-association of methylene blue (MB), a com-mon redox indicator and biological stain, has been in-vestigated by several researchers over the past half cen-tury.1±5 The oxidized (blue MB) and reduced (colorlessleuco-MB) forms of this dye are cationic. MB is stablein aqueous solution between pH 2 and 12.1 The maximumin the MB absorbance is at 662 nm, and MB exhibits a¯ uorescence emission maximum at 680 nm. This char-acteristic renders MB particularly well suited for spectro-scopic studies with 670 nm diode lasers. NoncovalentMB dimers and multimers are known to form in aqueoussolution.1±5 MB dimers, which have a 1 2 charge, exhibita shift in the electronic absorption spectrum; the dimerabsorbs most strongly at 610 nm.1,4

Sodium dodecyl sulfate (SDS) is a common anionicsurfactant, and its interactions with chromophores suchas ruthenium complexes6 and acridine orange7 have beenstudied by many researchers. The SDS critical micelleconcentration (cmc) in water is reportedly 8 mM.8

Several types of interactions are known to occur be-tween cationic dyes and anionic surfactants in solution, 9

Received 19 December 1998; accepted 19 February 1999.* Author to whom correspondence should be sent.

including dye±dye dimer formation,2,3 dye±surfactant in-teraction (including ion pair formation and dye±surfac-tant aggregates),6,7,10,11 and dye±solvent interaction.1,12,13

Molecular absorption and ¯ uorescence spectroscopy haveproven themselves useful tools to elucidate interactionsbetween species. Several review articles have appearedsummarizing much of the work on dye±surfactant inter-actions9 and the use of molecular spectroscopy to studymicellar solutions.8,14

It is well known that the local microenvironment sur-rounding a dye molecule (its so-called cybotactic region)in¯ uences its electronic structure and thus its photophys-ics. Changes in this local microenvironment, as the dyeinteracts with other species in solution, including mi-celles, can produce measurable spectral shifts 9,14 whichcan, in turn, be monitored spectroscopically. Moreover,the excited-state anisotropy and intensity decay kineticsof certain ¯ uorescent dyes often depend on their localenvironment. For example, the interactions between MBand DNA are known to affect the MB excited-state ¯ uo-rescence lifetime ( t F ).15

This paper focuses on the Rayleigh scattering, static¯ uorescence emission, and time-resolved ¯ uorescence ofdilute aqueous MB solutions as a function of added SDS.These results demonstrate that MB interacts in ion pairsand clusters with SDS anions at surfactant concentrationsbelow the SDS cmc and with SDS micelles at and abovethe SDS cmc.

EXPERIMENTAL

Except where noted, distilled deionized water was usedas the solvent. SDS (Sigma) was recrystallized twicefrom ethanol; all other reagents were used without furtherpuri® cation. Methylene blue (Aldrich) stock solutionswere prepared in 1 mM HCl, to minimize adsorption ontothe container walls and to lower the likelihood of pho-todecomposition during storage.3

Electronic absorption spectra were recorded by usinga Hewlett-Packard HP8452A diode array spectrophotom-eter or a Milton±Roy Spectronic 1201.

Rayleigh scattering measurements were performed ina 90 8 geometry with a compact, home-built spectrometerthat uses a small diode laser as the source. 16

Steady-state ¯ uorescence spectra were collected by us-ing a Perkin-Elmer LS-5B or an SLM 8100 luminescencespectrometer. Steady-state total ¯ uorescence intensitydata were collected with the use of one of the aforemen-tioned instruments or a compact spectrometer, built in

APPLIED SPECTROSCOPY 781

FIG. 1. Absorption spectra for 1 m M MB dissolved in aqueous solu-tions containing 0 to 8.0 mM SDS. Inset: Ratio of absorbance at 662to absorbance at 610 nm (A662 /A610) as a function of SDS concentration.

house.16 The latter spectrometer uses a small 7 mW, 670nm diode laser as the excitation source and photodiodesas detector elements. The total ¯ uorescence signal waspassed through a high-pass, thin-® lm ® lter (#83 MediumBlue, Roscolux, Edmund Scienti® c), which blocks theexcitation light.

Steady-state ¯ uorescence anisotropy measurementswere performed by using the SLM 8100, with excitationat 662 nm and emission at 700 nm, and excitation andemission bandpasses of 2 and 16 nm, respectively. Allsteady-state ¯ uorescence anisotropy measurements werebackground-subtracted; each blank contained SDS, of thesame concentration as the sample, in deionized water.

The excited-state intensity decay kinetics were mea-sured by using frequency-domain ¯ uorescence.17 Forthese particular experiments we used an SLM 48000MHF spectro¯ uorometer in concert with a 670 nm diodelaser (Photon Technology, Inc., 40 mW maximum con-tinuous-wave output) as the excitation source. The ¯ uo-rescence was monitored in the typical 90 8 geometrythrough a 690 nm high-pass ® lter, and the total ¯ uores-cence intensity was detected with a Hamamatsu R928photomultiplier tube. Phase-modulation data were ac-quired at 5 MHz intervals between 5 and 200 MHz. Mag-ic angle excitation conditions were maintained for all ex-cited-state ¯ uorescence lifetime measurements. 18 Allphase-modulation data sets were analyzed by using acommercially available software package from GlobalsUnlimited. In all cases, the true uncertainty in each datumwas used as the measurement imprecision.

The rotational reorientation times were calculated fromthe steady-state anisotropy and the excited-state ¯ uores-cence lifetime by using the Perrin equation.19

r 5 r 0 /(1 1 t / f ) (1)

which rearranges to

f 5 (r t )/(r 0 2 r ) (2)

where f is the ¯ uorophore rotational reorientation time, tis the average MB excited-state ¯ uorescence lifetime, r isthe steady-state ¯ uorescence anisotropy, and r 0 is the MBlimiting ¯ uorescence anisotropy. The r 0 value for diluteMB dissolved in glycerol at 2 55 8 C under the aforemen-tioned excitation/emission conditions is 0.37 6 0.03.

RESULTS AND DISCUSSION

At 662 nm, the MB absorbance vs. concentration plotfollows Beer’ s law from 0 to . 10 m M. Similarly, the total¯ uorescence intensity is related linearly to the MB con-centration up to 3 m M. Aqueous solutions of 1 m M MBwere chosen for all our work, because this level is withinthe linear region of Beer’ s law and ¯ uorescence intensityvs. MB concentration plots, and because MB exists pri-marily in its monomeric form at this concentration.4

Absorption spectra for MB (1 m M) in aqueous solu-tions containing 0 to 8.0 mM SDS are shown in Fig. 1.Electronic absorption spectra were acquired at 0.5 mMSDS intervals over this range, but for clarity we showabsorption spectra at four representative SDS concentra-tions only. The trends in all spectra are systematic, andthe four concentrations presented in Fig. 1 illustrate thesalient features over the entire concentration pro ® le. For

MB in the absence of any SDS, the absorbance maximumis observed at approximately 664 nm, with a shouldernear 610 nm. This observation is consistent with litera-ture reports.1,3,4 The absorbance feature at 610 nm arisesfrom MB H-aggregates (noncovalently associated di-mers) 2±4,20 as well as MB monomers. Upon addition ofSDS, the MB monomer absorbance (664 nm) decreasessigni® cantly until one reaches 3±4 mM SDS, where theabsorbance plateaus. At approximately 5 mM SDS themonomer absorbance begins to increase, and for solutionsat SDS concentration at or above the known SDS cmc(8 mM),8 there is a slightly higher absorbance value thanfor the MB alone in water without SDS. The absorbancevalue at 610 nm (H-aggregate) does not change signi® -cantly as the concentration of SDS is varied; however,the band shape changes from a shoulder (primarily mono-mer contributions to A610) in pure water to a separate peak(primarily H-aggregate contributions to A610) at 3±5 mMSDS. For comparison, if one increases only the MB con-centration in the absence of SDS and forms more dimers(K f 5 5900 M 2 1),2 one observes a trend similar to theone we see as we add SDS to a dilute MB solution. Infact, in aqueous solutions that contain 10 m M MB, thesespectral changes are readily observed visually: solutionscontaining no SDS or SDS at concentrations near orabove the cmc appear turquoise in color, whereas solu-tions containing 3±5 mM SDS appear lavender.

A similar response vs. added SDS is noted for ¯ uores-cence signal (see Fig. 2). Speci® cally, as the SDS concen-tration is increased from 0 to 10 mM, the MB ¯ uorescence® rst decreases until one reaches ; 4 mM SDS, then increas-es steadily between 4 and 8 mM SDS. Once one is abovethe reported SDS cmc, the ¯ uorescence levels off. Abovethe SDS cmc there is an approximately 2-fold enhance-ment in the MB ¯ uorescence signal relative to the ¯ uo-rescence from MB dissolved in water alone. No shiftsin ¯ uorescence emission maximum or emission pro® leare observed as we add SDS. We also do not observeany changes in the MB excitation spectra on adding SDS.At MB concentrations above 1 m M, we note signi® cantself-absorption, which results in an observed ``red-shift’ ’

782 Volume 53, Number 7, 1999

FIG. 2. MB ¯ uorescence intensity as a function of SDS concentration.Data from three replicate sets of measurements are shown.

FIG. 3. Rayleigh scatter signal, from solutions containing MB andSDS, as a function of SDS concentration. Data from three replicate setsof measurements are shown.

FIG. 4. MB ¯ uorescence lifetime as a function of SDS concentration.The error bars represent 6 1 standard deviation for three replicate mea-surements.

of the emission spectra. At or below 1 m M MB, self-absorption and spectral distortions are not a problem.

When the Rayleigh scatter signals ( l 5 670 nm) fromthe solutions containing 1 m M MB and 0 to 8.0 mM SDSare measured (Fig. 3), two features are readily apparentin the scattering pro® les. First, there is a strong, relativelynarrow scattering signal near 3 mM SDS. This peak inthe scattering pro® le is coincident with the aforemen-tioned decreases in the A662 /A610 (Fig. 1) and ¯ uorescence(Fig. 2). Second, there is an additional, albeit less prom-inent and more broad, scattering feature that is coincidentwith the known SDS cmc.

The excited-state ¯ uorescence lifetime that we deter-mined for dilute MB dissolved in aqueous solution in theabsence of SDS (380 6 15 ps) agrees with the valuereported previously by Fujimoto et al.15 Figure 4 presentsthe mean MB excited-state ¯ uorescence lifetime as afunction of SDS concentration. The error bars denote 6 1standard deviation for the measurement of three discretesamples at each SDS concentration. In all cases, the avail-able phase-modulation data between 5 and 200 MHz arebest described by a single exponential decay law (averagex 2 5 1.09). However, because these decay times are allwell below 1 ns, we cannot, at this time, rule out mul-tiexponential intensity decay kinetics or continuous life-time distributions with two or more subnanosecond com-ponents. However, when we performed simultaneousglobal analysis 21,22 on all available phase-modulation datasets at all SDS concentrations, we were unsuccessful atidentifying any linking scheme that involved double ortriple exponential decay models where we could link the``lifetimes’ ’ across all SDS concentrations.

Inspection of Fig. 4 illustrates several important fea-tures. First, below 6 mM SDS, the apparent MB excited-state ¯ uorescence lifetime is constant at ; 380 ps. Sec-ond, there is no detectable change in the MB excited-state ¯ uorescence lifetime between 3 and 5 mM SDS thatcorresponds to the observed changes in absorbance (Fig.1), total ¯ uorescence intensity (Fig. 2), or Rayleigh scat-tering (Fig. 3). Third, between 6 and 8 mM SDS, thereis a sharp increase in the MB excited-state ¯ uorescencelifetime from 380 to 615 ps. This increase is coincidentwith the known SDS cmc. Finally, for solutions at orabove 8 mM SDS, the MB excited-state ¯ uorescence life-time remains constant at ; 615 ps.

We used the excited-state ¯ uorescence lifetime data(Fig. 4) and steady-state ¯ uorescence anisotropy (notshown) to estimate the MB rotational reorientation time asa function of added SDS (Eq. 2). The results are sum-marized in Fig. 5, where we present the effects of addedSDS on the MB rotational reorientation time at 20 8 C.Interestingly, the observed rotational reorientation time (f )follows the same trend as the ¯ uorescence lifetime data.Speci® cally, at SDS concentrations between 0 to 6 mMSDS, f remains constant at ; 105 ps. Above the knownSDS cmc, f is substantially longer, approximately 500 ps,and also independent of added SDS. Between 6 and 8 mMSDS, the rotational reorientation time increases systemat-ically from 105 to 500 ps as we add SDS. This increaseis again coincident with the known SDS cmc.

The results obtained in this study cannot be explainedby a single type of interaction between MB and SDS.The decrease in the MB monomer absorbance and ¯ uo-rescence intensities at SDS concentrations below the cmcsuggest that MB monomers may be interacting with one

APPLIED SPECTROSCOPY 783

FIG. 5. Rotational correlation time for MB as a function of SDS con-centration. The error bars represent 6 1 standard deviation for threereplicate measurements. The standard deviation from the lifetime datawas propagated through the Perrin equation.

or more SDS anions or are somehow exhibiting self-ag-gregation2±4,20 that is modulated by the addition of SDS.Changes in absorbance, ¯ uorescence intensity, excited-state ¯ uorescence lifetime, and rotational reorientationtimes at concentrations at and above the cmc of SDSsuggest that MB interacts with SDS micelles in a straight-forward manner.

Interactions at SDS Concentrations below the Crit-ical Micelle Concentration. The spectral changes ob-served when the SDS concentration is below the cmc arenot fully consistent with MB dimer or multimer forma-tion. The change in absorbance pro ® le shown in Fig. 1indicates an initial decrease in monomer concentrationand increase in dimer concentration. (At the concentra-tions of MB used in these studies, there is reportedlynegligible trimer and tetramer formation).4 However, ifwe assume that all MB is present in either monomer ordimer form and then calculate the expected absorbancevalues for each solution by using the MB monomer anddimer extinction coef® cients reported by Spencer andSutter,4 we calculate that the A610 value should, in fact,increase for those samples that have a signi® cantly lowerA660 than the solution containing 1 m M MB in the absenceof SDS. Speci® cally, if the measured absorbance valuesare due to monomer and dimer only, and if the extinctioncoef® cients of the MB species are unaffected by the pres-ence of SDS, then not all of the loss in monomeric MBabsorbance can be explained completely by an increasein the number of MB dimers. For example, for the so-lution containing 2.5 mM SDS, the result of these cal-culations is that we cannot account for more than 15%of the initial amount of MB. Moreover, formation of therelatively small dimer alone cannot be used to explainthe prominent Rayleigh scatter peak seen at 3 mM SDS.Lack of spectral changes in the ¯ uorescence excitationand emission spectra indicates that the MB dimers or ag-gregates do not contribute signi® cantly to the observedtotal ¯ uorescence signal.

Interestingly, a standard method of analysis for surfac-tants in water is based on extraction of ion pairs of MBand anionic surfactants, including SDS, into an organicsolvent.23 This method suggests that ion pairs betweenMB and surfactants form in aqueous solution, as well. Ifinsoluble ion pairs were forming, we might expect to seea precipitate and/or an increase in scattering signal, aswell as a decrease in absorbance and ¯ uorescence inten-sity. We were unable to visually detect any precipitate,and our solutions are not turbid; however, the concentra-tion of MB is so low that visible precipitate would notbe anticipated. A peak in the Rayleigh scattering vs. SDSpro® le is observed near 3 mM SDS. Ion pair or clusterformation between MB and SDS could account for suchan increase in scattering signal observed below the SDScmc.9 Other researchers have observed that tris(2,2 9 -bi-pyridyl)ruthenium(II),6 ethidium,7 and acridine orange10

form clusters (premicellar aggregates) with SDS belowthe cmc. Our absorption and ¯ uorescence spectral dataindicate that MB in the ion pair and/or cluster is lesslikely to absorb light and this species does not ¯ uorescesigni® cantly if at all. This ® nding is consistent with otherwell-known dimer systems.

Turner and co-workers12,13 have shown, through kineticstudies, that the presence of an alcohol as the solventreduces dimer formation. Speci® cally, Turner demonstrat-ed that ethanol±solute interactions, rather than changes insolvent properties, were responsible for the lack of thio-nine12 and pro¯ avin13 dimer formation in aqueous solu-tions containing ethanol. In our experiments, we havebeen careful to keep the MB concentration low to avoidsigni® cant MB dimer formation. However, to address thisissue more directly, we investigated the effects of addedethanol on the MB/SDS solutions. At ethanol concentra-tions of 25% or higher, MB absorbance is no longer afunction of SDS concentration and the MB absorptionspectrum does not change (results not shown). Further-more, a plot of MB ¯ uorescence as a function of SDSconcentration in 25% ethanol shows that the MB ¯ uo-rescence is also constant (results not shown). A constant¯ uorescence signal would be expected if MB was notinteracting with SDS or forming MB multimers as theconcentration of SDS is increased. Consequently, we con-clude that the likelihood of formation of both MB-SDSand SDS-induced MB-MB aggregates decreases as eth-anol concentration in the solvent increases, due to inter-action between MB and ethanol in solution.

MB dimers/aggregates do not apparently ¯ uoresce.This ® nding is most clearly evident from the excited-state¯ uorescence lifetime data (Fig. 4), in which there is ab-solutely no detectable change in the excited-state ¯ uores-cence lifetime where we see the greatest changes in ab-sorbance, emission intensity, and Rayleigh scattering.The data suggest that the species that do ¯ uoresce areunaffected by the added SDS until one approaches theSDS cmc; however, the number of those species that ¯ uo-resce is somehow lowered as SDS is added between 0and 3±5 mM SDS. Moreover, there is no decrease in theMB excited-state ¯ uorescence lifetime as one adds SDSup to 5 mM, which would be expected if MB specieswere in close enough proximity to cause dynamicquenching of the MB emission. Clearly, only a staticquenching scenario 24 (i.e., a MB-SDS complex of unde-

784 Volume 53, Number 7, 1999

® ned stoichiometry that does not ¯ uoresce) can explainall the available data below 5 mM SDS.

If MB and SDS form ion pairs/clusters with a singlestoichiometry (i.e., 1:1, 1:2) and no self-aggregation ofthe SDS anions or MB cations occurs, one would expectto observe a linear relationship between SDS concentra-tion and MB monomer absorbance and ¯ uorescence in-tensity, at concentrations below the SDS cmc. No suchtrend is observed. As the SDS concentration is increased,spectral changes indicate that a different sort of interac-tion is occurring. As the SDS concentration approachesthe cmc, and the SDS reorganizes into micelles, we spec-ulate that the MB is ``released’ ’ from the ion pairs orclusters back into solution.

Interactions at SDS Concentrations at or above theCritical Micelle Concentration. Explanation of the in-teraction between MB and SDS at and above the SDScmc is more straightforward. A subtle, but clearly de-tectable, increase in the Rayleigh scattering signal (Fig.3) is observed near the literature value for the SDS cmc,8 mM.8 This ® nding is fully consistent with SDS micelleformation. In contrast to its interaction with one or a fewSDS anions, when MB interacts with SDS micelles, thereis a slight enhancement in the absorbance relative to MBdissolved in water alone (Fig. 1), a 2-fold increase in¯ uorescence (Fig. 2), and a 1.6-fold increase in the ex-cited-state ¯ uorescence lifetime (Fig. 4). The 5-fold in-crease in MB rotational reorientation time (Fig. 5) impliesthat the MB dynamics slow as it becomes part of the SDSmicelles and thus part of a larger rotating unit (i.e., themicelle). The bulk solvent viscosity does not increase sig-ni® cantly as we add SDS, so viscosity alone cannot ex-plain the increase in rotational reorientation time notedbetween 6 and 8 mM SDS. In terms of a simple Debye±Stokes±Einstein hydrodynamic model ( f 5 h V /RT; h isthe viscosity encountered by the MB, V is the MB molarvolume, R is the gas constant, and T is the Kelvin tem-perature), the observed rotational dynamics suggest thatthe MB is encountering, on average, (1) a microenviron-ment in the SDS micelles that is 5-fold more viscous thanliquid water, or (2) a molar volume of the MB/SDS spe-cies that is reorienting which is 5-fold larger than MB inwater. Each of these observations is fully consistent witha strong interaction between MB and the SDS micellesat and above the SDS cmc.

Because MB is positively charged, it should undergostrong electrostatic intermolecular interactions with thepolar head groups of the SDS micelle. Moreover, absorp-tion and ¯ uorescence enhancement observed when MBis in the less dipolar micelle microenvironment is con-sistent with the known behavior of MB in dipolar organicsolvents; however, it must be noted that MB is unlikelyto be found entirely within the central, nonpolar regionof the SDS micelle.

CONCLUSION

MB and SDS undergo completely different intermolec-ular interactions in aqueous solution, depending on theSDS concentration. At SDS concentrations below theknown SDS cmc, MB-MB dimers and/or MB-SDS ionpairs or clusters form, and a corresponding shift in ab-sorption spectrum and decrease in ¯ uorescence intensity,

consistent with a static quenching process, are observed.As the SDS concentration is increased, micelle formationoccurs so that (1) the MB-MB dimers are disrupted; (2)MB-SDS ion clusters (mixed micelles)9 are incorporateddirectly into micelles; and/or (3) the dissociation of ionclusters is driven by the added SDS, micelles form, andlone MB monomers become associated with these mi-celles. In solutions of SDS concentration at or above theknown cmc, the interactions between MB monomers andSDS micelles result in enhanced MB absorption and ¯ uo-rescence, as well as a longer MB excited-state ¯ uorescencelifetime and rotational reorientation time, indicating thatthe MB in SDS micelles is better protected from quench-ing than when it is free in aqueous solution. Finally, theMB rotational reorientation dynamics suggest that the MBis encountering, on average, a microenvironment in theSDS micelles that is 5-fold more viscous than liquid wateror a molar volume of the MB/SDS species that is reori-enting which is 5-fold larger than MB in water.

A full, quantitative explanation of the Rayleigh scat-tering data is lacking at this time. We are currently in-vestigating this issue in more detail in our laboratories.

ACKNOWLEDGMENTS

This work was supported through Union College Faculty ResearchFund Grants to M.K.C. and by the National Science Foundation (pri-mary award to F.V.B.; ROA supplement to F.V.B. and M.K.C.), Of® ceof Naval Research (to F.V.B.), and Department of Energy (to F.V.B.).A.M.L. and M.J.M. were supported by Union College UndergraduateSummer Research Fellowships. A.M.L. acknowledges an Internal Ed-ucation Fund grant from Union College. C.M.I. acknowledges Silbertand Allied Fellowships from SUNY-Buffalo.

1. E. Rabinowitch and L. F. Epstein, J. Am. Chem. Soc. 63, 69 (1941).2. K. Bergmann and C. T. O’ Konski, J. Phys. Chem. 67, 2169 (1963).3. P. Mukerjee and A. K. Ghosh, J. Am. Chem. Soc. 92, 6403 (1970).4. W. Spencer and J. R. Sutter, J. Phys. Chem. 83, 1573 (1979).5. M. Liska, L. Bartos, and J. Valasek, J. Chem Papers 43, 303 (1989).6. J. H. Baxendale and M. A. Rodgers, J. Phys. Chem. 86, 4906

(1982).7. S. J. Atherton and C. M. G. Dymond, J. Phys. Chem. 93, 6809

(1989).8. J. Georges, Spectrochim. Acta Rev. 13, 27 (1990).9. M. E. Diaz Garcia and A. Sanz±Medel, Talanta 33, 255 (1986).

10. S. H. Chou and M. J. Wirth, J. Phys. Chem. 93, 7694 (1989).11. S. Hamai, Bull. Chem. Soc. Jpn. 58, 2099 (1985).12. T. G. Dewey, P. S. Wilson, and D. H. Turner, J. Am. Chem. Soc.

100, 4550 (1978).13. T. G. Dewey, D. A. Raymond, and D. H. Turner, J. Am. Chem.

Soc. 101, 5822 (1979).14. R. von Wandruszka, CRC Crit. Rev. Anal. Chem. 23, 187 (1992).15. B. S. Fujimoto, J. B. Clendenning, J. J. Delrow, P. J. Heath, and

M. Schurr, J. Phys. Chem. 98, 6633 (1994).16. A. M. Leach, M. A. Unger, M. J. Morris, and M. K. Carroll, un-

published results.17. F. V. Bright, Appl. Spectrosc. 49, 14A (1995).18. R. D. Spencer and G. Weber, J. Chem. Phys. 52, 1654 (1970).19. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum

Press, New York, 1991), p. 132.20. S. Otsuki and K. Adachi, Polym. J. 27, 655 (1995).21. J. M. Beechem and E. Gratton, Proc. SPIE 70, 909 (1988).22. J. M. Beechem, E. Gratton, M. Ameloot, J. R. Knutson, and L.

Brand, in Topics in Fluorescence Spectroscopy, J. R. Lakowicz,Ed. (Plenum Press, New York, 1991), Vol. 2, Ch. 5, p. 241.

23. Standard Methods for the Examination of Water and Wastewater(American Public Health Association, American Water Works As-sociation, Water Environment Federation, Maryland, 1992) 18thed., 5540C.

24. M. R. Eftink, in Topics in Fluorescence Spectroscopy , J. R. Lak-owicz, Ed. (Plenum Press, New York, 1991), Vol. 2, Chap. 2, p.53.