Anal. Chem. 1995,67, 174-180
Metal ion Quenching of Fulvic Acid Fluorescence Intensities and Lifetimes: Nonlinearities and a Possible Three=ComponentModel Robert L. Cook and Cooper H. Langford* Department of Chemistry, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4
The quenching of fluorescence of a fulvic acid by metal ions has been revisited in this study. Fluorescence quenching has been used in the past to establish the extent of metal ion binding to fulvic acids. Both emission and synchronous fluorescence show that the relationship between the extent of quenching and fraction of cation sites occupied is not linear. It was also found that synchronous fluorescence is a more revealing measurement than emission. Similar patterns of components can be assignedfrom both time-resolvedfluorescent measurements and synchronous fluorescence. Correlation of these two distinct types of measurements suggests that a three-component treatment of fulvic acid luminescence may be physically meaningful. For this fulvic acid the three components may be assigned lifetimes and wavelength maxima as follows; -50 ps (392 nm), -430 ps (465 nm), and 4.2 ns (512 nm). The three-component model accommodates pH dependence and metal ion quenching. Stem-Volmer plots also support a (minimum) three-component model. The binding of metal ions to humic substances is of great interest in the understanding of metal ion transport and speciation in natural waters and soils. Because of the low concentrations (10-7-10-10 M) of many transition metal ions that are important in natural systems, one cannot use simple solution phase detection methods such as ion-selective electrode USE) measurements. Several exotic instruments including electron spin resonance (ESR)’ and thermal lensing2have been used to study solutions at these low concentrations. Both of these instruments are expensive to purchase and complex to maintain (moreover, neither one could be used in the field with current technology). What is needed is an adequately sensitive, easy to use, compact, relatively cheap, and nondestructive probe giving low detection limits. Fluorescence is such a method, and humic materials are fluorescent. Saar and Webe9 introduced fluorescence quenching as a way of measuring the binding equilibrium between humic substances and metal cation. This was further developed by Ryan and Weber.4The basis of the method is metal ion quenching of humic substance fluorescence. This means that the free ligand concentration a) is being measured and from this the bound (1) Buffle, J. ComplexationReactions in Aquatic Systems, An Analytical Approach; Ellis Harwood Ltd.: Chichester, UK, 1988; Chapter 10. 183-201. (2) Gutzman, D. W.; Langford, C. H. Anal. Chim. Acta 1993,256, (3) Saar, R A; Weber, J. H. Anal. Chem. 1980,52, 2095-100. (4)Ryan, D. K; Weber, J. H. Anal. Chem. 1982,54, 986-90.
174 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
ligand is calculated and the amount of bound metal (ML) is inferred. Weber and co-workers3,4documented this method by use of both model compounds and comparison to ISE measurements. The key assumption that the remaining quenchable fluorescence uniformly measures the remaining free ligand concentration has been questioned? This has led to a debate in the Nevertheless, recent literature shows that the humic fluorescence quenching approach remains popular for measuring the extent of cation binding by humic This paper revisits the assumptions that pertain to the mechanisms of fluorescence quenching. Humic substances are mixtures, and humic fluorophores may not be uniformly distributed so that quenching may not be simply proportional to the free ligand concentration.10In this context, the testing of the method of refs 3 and 4 using model compounds is highly questionable. The model compounds were relatively simple ones, and this means that they have only one type of fluorophore while fulvic acid may have at least three.5J1 Moreover, when the binding site is not the fluorophore itself, some of the binding sites may be partially or fully shielded from the fluorophore. This will not be obvious for a simple model compound, but in a macromolecular system like those of humic substances this may upset the linear relationship first proposed by Weber and co-~orkers.~,~ Throughout this paper, a unique fluorophorewill be considered as a unique fluorescent structure with a certain accessibility for metal ions. ‘Thus, if there are two fluorophores based on the same functional groups but a metal ion has more access to one, because of the structure of the macromolecule, then these two fluorophores are considered to be two separate entities. Two types of steady state fluorescence measurements were used in this study to investigate quenching mechanisms and their relation to metal ion binding. The two fluorescence methods will be called “emission”and “synchronousfluorescence”. The term emission is used to describe experiments in which the excitation wavelength is chosen at a maximum in an excitation spectrum and an emission spectrum is scanned. Synchronous fluorescence is an experiment in which both excitation and emission wave(5) Cabaniss, S. E.; Shuman, M. S. Anal. Chem. 1988,60,2418-21. 1523-6. @)Cabanis, (6) (a) Ryan D. K; Ventry L.S.Anal. Chem. 1990,62, S. E.; Shuman M. S. Anal. Chem. 1990,62,1526-8. (7) Ventry, L. S.; Ryan,D. K; Gilbert, T. R Microchem. J. 1991,44, 201-14.
(8)Gri”, D. M.; Azarraga, L. V.; Carreira, L. A; Susetyo, W. Environ. Sci. Technol. 1991,25,1427-31. (9) Sposito, G.; William, S.Soil Sci. SOC.Am. J. 1990,54, 933-5. (10) Underdown, A W.; Langford, C. H.; Gamble, D. S. Environ. Sci. Technol. 1985,19, 132-36. (11) Power, J. F.; Lesage, R; Sharma, D. K; Langford, C. H. Environ. Technol. Lett. 1986,7, 425-30. 0003-2700/95/0367-0174$9.00/0 Q 1994 American Chemical Society
lengths are scanned synchronously with a small constant wavelength offset (M)between excitation and monitoring,12-14 thus limiting the relaxation pathways available. Emission fluorescence was the method used by Weber and co-worker~~~~ and in subsequent applications of their method.4~~-~ Synchronous fluorescence is exploited because it differentially samples the entire distribution of the fulvic acid macromolecular system more effe~tive1y.l~ Synchronousfluorescencewas also chosen because it gives a higher resolution spectrum, because it is more selective and may allow for the study of differences among fluorophores and binding sites. Finally, dynamic fluorescence (time-resolved) measurements were also made to find out how many components describe the emission decay curve of the fulvic acid used in this work and to study the quenching of these components. The two steady state measurements were compared to the dynamic ones to determine whether synchronous fluorescence gave a picture of the fluorescent properties of the molecule that could be related to the timeresolved measurements. The time scale of these dynamic measurements is picoseconds to nanoseconds. This study exploits the Laurentian fulvic acid (LFA) extracted from a forest podzol from the area controlled by Lava1 University (Quebec). We know something of the elemental composition, acid-base and metal ion titration curves, and IT-IR and NMR spectra of this sample.16-18 Its properties are known in more detail than most humic substances. EXPERIMENTAL SECTION
Reagents. The fulvic acid used in this study was Laurentian Fulvic acid @,FA), it was prepared and purified in accordance with refs 19 and 20. The metal salts used were nickel chloride (BDH Lot 2733500L), and cupric nitrate (Fisher Lot 874505). The water was 18 MQ produced by a Barnstead Nanopure system (unless otherwise stated). Equipment. Both the emission and synchronous fluorescence measurements were made on a Photon Technology Inc. Alphascan fluorometer, with the PMT operating in photoncounting mode. pH-dependence measurements were done for us by Pullin and Cabaniss by methods described by them,2l using a water-cooled 150 W Xe arc lamp source. All slits were set at 5 nm for this set of measuements. We thank them for their help. The metal ion studies used a water-cooled 70 W Xe arc lamp source. For these measurements slits were set at 10 nm, except the emisssion exit slit, which was set at 2 nm. For all the emission measurements the integration time was 0.25 s, and for all synchronous measurements the integration time was 0.5 s. The picosecond experiments were carried out at the Canadian Centre for Picosecond Laser Spectroscopy (at Concordia University). This apparatus uses a mode locked Nd/Yag producing a third harmonic pulse at 355 nm. The half-pulse width is 30 ps, and the pulses measured had energies of 2.5 f 0.5 mJ. The emission decay was (12)Senesi, N.Anal. Chim. Acta 1990,232,77-106. (13)Senesi, N.;Miano, T. M.; Provenzano, M. R; Brunetti, G. Soil Sci. 1991, 152,259-71. (14)Miano, T.M.; Senesi, N. Sci. Total Enuiron. 1992,117/118,41-51. (15)VeDinh, T.Appl. Spectrosc. 1982,36,576-81. (16)Wang, Zd.; Pant,B. C.; h g f o r d , C. H.Anal. Chim.Acta 1990,232,43-9. (17)Wang, Zd.; Gamble, D. S.; Langford, C. H. Enuiron. Sci. Technol. 1992, 26,560-5. (18)Wang, Zd. FkD. Thesis, Concordia University, Montreal, 1989. (19)G a t h , S. M.;Schnitzer, M. Soil Soc. 1975, 120,126-31. (20)Schnitzer, M.; Skinner, S. I. M. Soil SOC.1968, 105,392-6. (21)Pullin, M.; Cabaniss S. E., submitted for publication in Enuiron. Sci. Technol.
measured at 455 nm on a Hammamatsu streak camera with a time resolution of 20 ps. The emitted light was viewed through an interference filter. We thank Dr. A. Vlcek, for conducting quite independent nanosecond laser pulse experiments which can test the assignment of the longer lifetimes. His apparatus used a Lambda Physik excimer laser (308 nm, XeCl). The pulse width is 25 ns. The emission was measured at -455 nm and detected by Applied Photophysics laser kinetic spectrometer LKS20 (the signal was captured by a Philips digital oscilloscope). Procedure. The pH dependence studies were carried out with a stock solution of 10.0 mg/L LFA and 10 mM GHP04 (Fisher) in Milli-Q water. pH adjustment was done with small amounts of HCl and NaOH. Cabaniss and Pullin’s emission fluorescence experiments used 340 nm excitation, while the synchronous fluorescence measurements were made with a constant 20 nm offset (AA)between the excitation monochromator and emission monochromator.21 For the metal ion studies, a stock solution of LFA was made from a 0.175 g/L solution (LF’Ahas been found to have a bidentate complexing capacity of 5.8 mmol/g);18 10 mL of this stock solution was then added to a 100 mL volumetric flask and the necessary amount of metal was added to give stoichiometeric fractions of 0.1-0.9 (1 x 10-5-9 x W5M) metal ion to bidentate binding sites after which the solution was diluted to 100 mL. The solution were then adjusted to a pH of either 4.0,5.0,6.4, or 8.0 with either NaOH or HCl. Fulvic acid selfbuffers. Our emission fluorescence experiments used 355 nm excitation, while the synchronous fluorescence measurements were, like Cabaniss and Pullii’s,21 made with a constant 20 nm offset (M). The solutions were protected from light and allowed to equilibrate for a minimum of 12 h. The same procedure was used for making the stock solution for both the pic0 and nanosecond fluorescence decay experiments except the fulvic acid concentrationwas 0.25 g/L, and all the tested solutions had a pH of 6.4. These solutions were protected from light and allowed to equilibrate for a minimum of 16 h. Data Treatment. All the steady state spectra were smoothed using a 15 point Savitsky-Golay filter. The fitting of the emission decay was done using a three component exponential decay fit. The data were weighted to the relative intensity and the allowable error was 0.1%with the general fit of the KaleidaGraph software package (for the Macintosh), which uses the LevenbergMarquardt algorithm. The nanosecond emission was deconvoluted using an exponential decay fit by in-house software employing Marquardt’s optimization method. The average metal ion binding constant, K4 of ref 22 was calculated in the following way. Consider the equilibrium
SH-
+ M ~ + SM + H+
(1)
where SH- is a singly deprotonated bidentate complexation site and SM is a metal ion complexed to the bidentate site. The average equilibrium constant is defined by
where brackets denote concentrations. This definition corresponds to that used in refs 3 and 4. (22)Gamble, D. S.;Underdown, A. W.; Langford, C. H. Anal. Chem. 1980,52, 1901-8.
Analytical Chemistry, Vol. 67, No. 1, January 7, 1995
175
,
1
i
4.00010'
1
I
3.50010' 3.0001 os
2.500 1os
G
2.000 1os
1.500 1 os
1
400
450
500
550
600
nm
Figure 1. Effect of pH on LFAs fluorescence monitored by emission fluorescence (Cps, counts per second). 7.000 10' 6 000 lo'
,
C
-;
pH 3.50 pH 5.48
,
-
L
I
h,
i
5.000 l o 4
r
4.00010'
T
3.000 l o 4
2.000 10'
o i 300
, 350
I
400
450
500
550
600
nm
Figure 2. Effect of pH on LFAs fluorescence monitored by synchronous fluorescence. 1
io4
I
I
-0.0 M.B.S.
8000
6000
b 4000
2000
0
400
450
500
550
nm
Figure 3. Cu(ll) quenching of LFAs fluorescence monitored by emission fluorescence (M:B.S., ratio of metal to bidentate complexing sites (bidentate complexing sites = 1)).
RESULTS AND DISCUSSION Characterization of LFA by Steady State Fluorescence. p H
Dependence. The fluorescence spectra for LFA are shown in Figures 1and 2. A single broad band is evident in the emission fluorescence spectrum (Figure 1). This broad, but not symmetrical, band has a maximum at -456 nm. The nonsymmetry is evident by the slope in the region from -490 to -550 nm (see Figure 3 for clearer resolution of this feature), which is indicative of another spectra feature, called from here on the -512 nm shoulder. All emission spectra could be fitted by a sum of two Gaussians centred at 456 and 512 nm, confirming the generality 176 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
of the 512 nm shoulder. It was found that this shape altered very little from pH 4.0 to 8.0, and a maximum signal was obtained at a pH of -5.5. These findings are very similar to some reported previously for various fulvic a ~ i d s . ~ ~ The J ~ ,synchronous ~~-~~ spectra of the same fulvic acid sample show much more structure, as can be seen by comparing Figures 1 and 2. First, it can be seen that the synchronous signal is -4.5 times less intense, as is expected since the excitation wavelength is not chosen for optimum emission intensity.25 There are two peaks in the synchronous spectra at -392 and -465 nm and a shoulder at -512 nm. The pH dependence is more complex. A maximum signal was obtained at pH 5.5 for the 465 nm peak while the maximum for the 392 nm feature was found to be at pH 10 (the highest pH studied). The shoulder at 512 nm grew in as the pH increased. There was some change in the shape of the 465 nm peak as pH increased. There was growth of shoulders at either side of the peak (-447 and -478 nm). It should also be noted that the maximum quantum yield is very likely occurring at pH 5.5 and then decreasing with pH. The dependence of synchronous fluorescence on pH can be broken down into four distinct stages. The first of these is a fluorescence increase between pH 3.5 and 5.5. This change can be correlated with the deprotonation of type A carboxylic groups (defined in ref 22, the more acidic carboxylates). There is then a very gradual decrease in fluorescence between pH 5.5 and 6.5 as well as growth of a shoulder on both sides of the 465 nm peak and a simultaneous large increase in the 392 nm peak, this can be related to the deprotonation of type B carboxylic groups (defined in ref 22, the less acidic carboxylates). From pH 7.0 to 8.0 the 465 nm feature decreases drastically and the 392 nm peak also decreases, but to a lesser extent. This change may correlate to deprotonation of relatively strongly acidic phenolic groups or some acid functionality on the carbohydrate moieties of the structure. The final step is an increase in the 512 nm shoulder from pH 8.0 to 10.0. This can be correlated with deprotonation of phenolic groups with normal plcs. One also notices that in this pH range a weak peak to the blue of the 465 nm has developed at -447 nm. From this study it can be seen that synchronous fluorescence resolves distributions of fluorophores that emission fluorescence does not distinguish. Metal Ion Quenching. Cu(II) and Ni(II) quench both types of spectra in a very similar manner; the major difference is that Cu(ID quenches to a greater extent, thus only the CuOD results will be shown in the figures, but the trends are very similar to those for Ni(II). In emission fluorescence the broad main band is quenched as shown in Figure 3. In the synchronous fluorescence, the quenching is greater on the 465 nm peak than on the 392 nm one, and there seems to be much less quenching of the 512 nm shoulder. This is evident by the emergence of the 392 nm peak and of the 512 nm shoulder as the metal quenching increases, as illustrated in Figure 4. By comparing Figures 3 and 4 in can be seen that synchronous fluorescence resolves a broader distribution of fluorophores than does emission fluorescence. In fact, it appears that the broad peak centered at -456 nm may be sampling mainly the same set of fluorophores as sampled by synchronous fluorescence in the two peaks at -392 and -465 nm. (23) Power, J. F. Ph.D.Thesis, Concordia University, Montreal, 1986. (24) Lavinge, J A Ph.D. Thesis, Concordia University, Montreal, 1988 (25) Cabaniss, S. E. Enuiron. Sci. Technol. 1992,26, 1133-9. (26) Cabaniss, S. E.; Shuman M. S. Mar. Chem. 1987,21, 37-50.
,&
4
1000
i
500 I
,
500
550
,
0 1
300
400
350
450
1
600
nm
Figure 4. Cu(l1) quenching of LFAs fluorescence monitored by synchronous fluorescence.
-pH 1.200 10'
5
+pH64
1 .ooo 10'
B
8000 6000
4000 2000
0
0.2
I
I
0.4
0.6
-
0.8
1
Meal:Binding Sites (Binding Sites 1)
Figure 5. Emission fluorescence quenching curves of LFA by Cu(11) at different pHs (binding sites equal bidentate complexing sites). 3000 2500
2000 1500 1000 500
0
0.2
0.4
0.6
0.8
1
McWBinding Sites (Binding rites = 1)
Flgure 6. Synchronous fluorescence quenching curves of LFA by Cu(ll) at different pHs.
Figures 5 and 6 show curves representing the decrease in fluorescence with the increase in ratio of CuaI) to binding sites at different pH's for both types of steady state fluorescence measurements used in this study. The ratio of metal to binding site was based on the report that LFA has a bidentate complexing capacity of 5.8 mmol/g;18 Le., 1 x mol of Cu(II) or N i O is the minimum needed to occupy all the bidentate complexing sites of 17.5 mg of LFA. The synchronous fluorescence curves were obtained by monitoring the quenching of the 465 nm maximum, while 456 nm was monitored for the emission fluorescence curves. The first observation is that at pH 4.0 the quenching curves are linear for both cases. Since, Cu(II) has been shown to compete
only weakly with protons at this pH,Z2 this probably indicates dynamic collisional quenching. Quenching due to Cu(II) complexation, occuring at pH's above 4.0, will be over and above this collisional quenching. We presume (see time resolved for confirmation) that quenching induced by complexation is static and that this is by far the major quenching that occurs at pHs above 4.0. In other words, dynamic quenching takes place when CugD is not complexed, at pH 4.0, and static quenching takes place when CuQ is complexed along with the dynamic quenching of uncomplexed Cu(II), above pH 4.0. However, when one is dealing with macromolecules that can trap but not complex ions, static quenching could be possible even though the metal ion has not been complexed, such static quenching is not complexational. The major part of quenching of the synchronous signal, due to complexation, seems to occur over a narrower range of Cu(II) concentrations than for the emission signal. This can be understood from Figures 3 and 4, recalling that the 465 nm peak is more affected by metal quenching than the 392 nm peak. In emission measurements, the signal is a mixture of these two features. The largest difference in curve shapes between the two methods is observed at pH 6.4. This observation leads to the suggestion that the 392 nm component is quenched over a wider CuQI) range than the 465 nm component. From this study it can be seen that synchronous fluorescence is better at resolving the individual components of the fluorescence signal; the reason is that this technique samples a wider range of fluorophores. The amount of metal ion bound to the fulvic acid may be calculated by use of the proposal from ref 3, i.e., assuming a linear relationship between the extent of metal binding to the fulvic acid and the amount the fluorescence signal quenching. The results of such calculations, for the 0.1 ratio of metal ion to binding sites are given in Table 1. It can also be seen here that there are several factors affecting the quenching of the fulvic acid by the metal ion. The first and most noticeable is the effect of pH. As the pH is increased the amount of quenching increases, up to pH 6.4, and then at pH 8.0 quenching is reduced again. There are two elements in the explanation for this, the first being that at lower pH the metal binding sites are less accessible to the metal ions because of proton competition. The second is an extension of the fust; available sites at lower pH are less important fluorophores than the sites that become available at higher pHs. Also, CuflI) is much more effective at quenching then is NiQI). This probably reflects greater binding constants, &. Under the ref 3 assumption, the results in Table 1 imply that (at pH 6.4 and higher for Ni, and pH 5.0 and higher for Cu) there is more metal available to bind than was added to the system! This despite the smooth quenching curves shown in Figures 5 and 6. A comparison of metal bound and fluorescence quenched is given in Table 2. It is clear from the results in Tables 1and 2 that the linear quenching assumption cannot be maintained! Application of the method of refs 3 and 4 to humic substances in general as reported in refs 7-10 must be viewed with great reservations. This seems to confirm that there is more than one type of fluorophore within the fulvic acid system and that the fluorescence of these different fluorophores is quenched differently. This point of view is also supported by the fact that the synchronous measurements showed a greater deviation from the linear assumption than the emission fluorescence measurements. Since the synchronous measurements sample a wider distribution of fluorophores than the emission measurements, one would Analytical Chernisfry, Vol. 67, No. 1, January 1, 1995
I77
Table 1. Amount of Metal ion Bound in Accordance with Weber and C o - ~ o r k e r s ' ~ Linear * ~ Assumptlon at Different pHs*
metal bound
c u OD
NiOI)
metal added
PH 4.0 5.0 6.4 8.0
M)
Ems
syn (10-5 M)
1.00 1.00 1.oo 1.00
M)
Ems
syn (10-5 M)
b
b
0.50 f 0.15 (n = 3) 2.44 f 0.53 (n = 3) 3.70 f 0.43 (n = 2)
0.60 f 0.25 (n = 3) 1.29 f 0.74 (n = 3) 2.68 f 0.02 (n = 2)
0.85 f 0.12 3.27 f 0.55 5.53 f 0.38 5.04 i 0.16
(n = 3) (n = 3) (n = 3) (n = 3)
M)
0.98 f 0.11 (n = 3) 2.16 f 0.27 (n = 3) 3.17 u 0.23 (n = 3) 3.07 f 0.04 (n = 3)
Syn, synchronous fluorescence; Ems, emission fluorescence. Data not available at this pH because of light scattering. Table 2. Comparlson of the Amount of Cu(li) Added and Bound According to the Weber and C o . ~ o r k e r s ' ~ ~ ~ Assumption
CuQD addedas a frachon of avadable binding sites
Cu (ID bound calcd by the Weber and co-workers assumption
fluorescence
0.0 0. 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0 0.586 0.812 0.908 0.943 0.947 0.960 0.966 0.967 0.968
2174.0 1314.7 860.6 665.1 597.1 590.0 563.6 551.8 541.0 535.5
(%::zx)
expect greater deviation from the linear assumption of ref 3 if there were a distribution of fluorophores. values were calculated when possible but gave no meaningful results. Stem-Volmer Plots. Stern-Volmer plots27and modified Stern-Volmer plots which allow for more than one quenchable fluorophore28were plotted for both emission (456 and 512 nm components) and synchronous (392, 465, and 512 nm components) fluorescent measurements. The components showed unique behavior once again. Both types of fluorescence measure ments showed linear Stern-Volmer behavior at pH 4.0. This is consistent with the idea that the important quenching taking place at this pH is collisional. But at pH 5.0, where complexation takes place, and above, the Stem-Volmer plots deviated from linearity toward the x axis, indicating two or more fluorophores that are not equally accessible to the complexation site of the quencher. Modified Stern-Volmer plots, which accommodate two-component fluorophores, deviated from linearity toward they axis, for pH 5.0 and higher, indicating that there are more than two fluorophores responsible. Thus, it would seem that the SternVolmer plots also indicate that there are at least three components in the fluorescence signal of our fulvic acid. An approach to confirm the above conclusion, that there is a distribution of fluorophores differentially quenched, is to examine the distribution of fluorescence lifetimes. Characterization of LFA by FluorescenceIifelimes. It was found that a minimum of three components were required to fully describe the emission decay of this sample of fulvic acid. The 22 values of the one-, two-, and three-component models are 1.354, 0.241, and 0.232 (as given by Kaleidagraph), respectively,while (27) Lakowicz J. R Principles of Fluovescence Specroscopy; Plenun Press: New York, 1983: Chapter 9. (28) Lehrer S. S. Biochemistry 1971,IO,3254-63.
178 Analytical Chemistry, Vol. 67,No. 1, January 7, 7995
j
j zn
4\
I
o l
550
0
I
4
i
...A
1
1
I
1100
1650
2200
i,.......................................
TLnc UPS)
Figure 7. LFAs time-resolved fluorescence fitted with three components and residuals for this fit. Table 3. Lifetime Components of Two Fulvic Acids
Laurentian fulvic acid preexponential component termn lietime 1 2 3
0.66 0.20 0.14
-50 ps -430 ps 4.2 ns
Armdale fulvic acid preexponential term" lifetime 0.86 0.11 0.03
200 ps 2 ns 7 ns
Normalized to 1.
the fit parameter values (I?) for the one, two-, and threecomponent models are 0.9926, 0.9985, and 0.9986 (as given by Kaleidagraph) , respectively. Figure 7 shows the threecomponent fit to the picosecond data with residuals. The lifetimes and quantities of the three components are tabulated in Table 3. The subnanosecond components were obtained from a series of picosecond time resolution emission experiments while the nanosecond component was found from both the picosecond resolution studies and independent nanosecond resolution emission experiments. It should be noted that in the nanosecond experiments the two picosecond lifetime components will not contribute, because they will have died out, and this third component must be included to fully describe the LFA emission decay. In short, the picosecond results require two components. The nanosecond result demands a third which the picosecond results accommodate. Thus, there must be no fewer than three
200
;
I
,
I
I
t
1600
2000
150 3
; -2
100
so
0 0
400
800
1200 Tune UPS)
Figure 8. Cu(ll) quenching of LFAs fluorescence monitored by timeresolved fluorescence (blank, LFA with no metal ion added).
+Blank
-Ni
10%
150 +Ni 80%
x E
E
-g
100
1 50
0 0
400
800
1200
1600
2000
Time UPS)
Figure 9. Ni(ll) quenching of LFAs fluorescence monitored by timeresolved fluorescence.
components. It is reassuring that a minimum of three components were also found by Power and co-workers" to fully describe the emission decay of an Armdale fulvic acid (AFA) sample as tabulated in Table 3. The most important result to note from Table 3 is that for two different fulvic acid samples a minimum of three lifetimes was needed to describe the emission decay curve. Caution should be exercised when considering these lifetimes, as they are a minimum set of Parameters to fit the decay curves within the experimental error. mere may well be a more complicated distribution of lifetimes. Metal Ion Quenching. A series of experiments were carried out in both the picosecond and nanosecond time domain in which the fulvic acid fluorescence was quenched by metal ions. The results for the picosecond experiments for both Cu(II) and Ni(II) are shown in Figures 8 and 9. From the nanosecond results it was found that quenching of the 4.2 ns component by either metal ion at any metal ion to binding site ratio (up to 0.8) cannot be clearly demonstrated. Because of this, only two components are required to describe the emission quenching, these being the -50 ps component and the -430 ps component. For Cu (II) , the 50 ps component was found to be quenched in the order 0.75 f 0.06,0.77 f 0.06,0.59 f 0.11, and 0.68 f 0.07 preexponential term (the two preexponential terms were normalized to 1) and the 430 ps component was found to be quenched in the order 0.25 f 0.02, 0.14 f 0.01, 0.07 f 0.02, and 0.07 f 0.01 preexponential term, respectively, for the blank, 0.1, 0.4, and 0.8 metal-bidentate complexing sites, respectively. For Ni(II), the 50 ps component was found to be quenched in the order 0.75 f 0.06, 0.80 f 0.18,
0.66 f 0.07, and 0.60 f 0.11 preexponential term (the twocomponent preexponential terms were normalized to 1) and the 430 ps component was found to be quenched in the order 0.25 f 0.02,0.27 i0.01,0.13 i0.01, and 0.12 f0.09 preexponentialterm, respectively for the blank, 0.1,0.4,0.8 metal-bidentate complexing sites, respectively. This would indicate that these two distinguishable fluorophore sets are being quenched by the two metals. It also appears that the -430 ps component is quenched to a greater extent at lower metal to binding site ratios than is the -50 ps component. Comparing these results to the synchronous fluorescence metal quenching study, the following lifetime assignment can be proposed. The 392 nm component is associated with the -50 ps component, the 465 nm component is associated with the -430 ps component, and the 512 nm component, which is not quenched measurably, is associated with is the 4.2 ns component. It is important that fluorescence quenching affects subnanosecond lifetimes, i.e. these processes are faster than collision rates in solution. This is strong evidence that mainly static quenching is occurring and there is little dynamic quenching at pH 5.0 or higher. This is a very important observation because it implies that the nonlinear Stern-Volmer quenching behavior noted above results from only one quenching mechanism, and thus the deviation from linearity must come from the fact that the separate fluorophores quench differentially. Combining the Synchronous Fluorescence and TimeResolved Quenching Results. It can be seen that monitoring the fluorescence of this fulvic acid sample (and the AFA sample) involves monitoring more than one fluorophore. In fact there may be a very complex distribution of fluorophores. But, when one combines the synchronous and time-resolved fluorescence quenching results, there is an emerging case for a physically meaningful three-component first approximation model of fulvic acid. From this point of view, we propose to assign the threecomponent groupings found for LFA in the synchronous spectra the following lifetimes, the 392 nm component -50 ps, the 465 nm component -430 ps, and the 512 nm component 4.2 ns Thus, there cannot be a linear relationship between the amount of metal bound to the fulvic acid and the quenching of that fulvic acid's fluorescence. There are possible explanations for Weber and c o - w o r k e r ~ linear ~~~ relationship. For example, the fulvic acid that they investigated either had only one distinct fluorophore or it had a distribution of fluorophoresthat quenched in a similar manner over the range of metal ion concentrations they used. CONCLUSIONS
The following main conclusions can be drawn from this study: (1) In this study it was found that for LFA there was not a linear relationship between the quenching of the fulvic acid's fluorescence and the amount of metal ions bound. Thus the assumption introduced in ref 3 does not hold for this sample and may well not hold for the majority of fulvic acid samples. (2) By combining synchronous and timeresolved fluorescence quenching results, a physically meaningful three-component first approximation luminescence model of fulvic acid is arising. With the following maxima and lifetimes the three are the 392 nm component -50 ps, the 465 nm component -430 ps, and the 512 nm component 4.2 ns for LFA. Recall that the components described in this paper are a minimal set adequate to fit the decay curves and synchronous spectra and may be a simplification of a more complex distribution. Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
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(3) Different fluorophores are related to different binding sites as shown by the lifetime and synchronous measurements. It may be either that some binding sites are further away from the fluorophore they affect or that these sites are not electronically coupled or electronically coupled to a lesser extent to the fluorophore they affect (in terms of metal ion quenching of the fluorophore). The following general conclusions can be drawn: Synchronous fluorescence is a more sensitive tool than emission fluorescence, since synchronous fluorescence samples the distribution within the molecule in finer detail. Synchronous fluorescence seems to be a more revealing measurement than emission, since it can be correlated with lifetime studies with respect to the number of components and how these components behave.
180 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
ACKNOWLEWMENT We thank The Natural Science and Engineering Research Council of Canada and the University of Calgary for financial support. We also thank Dr. Goujun Liu for the use of his equipment. Dr. D. K. Sharma is thanked for helping with the picosecond time resolved work. We also thank Dr. A. Vlcek, Jr., and Dr. S. E. Cabaniss and M. Pullin for providing nanosecond time resolved spectra and pH measurements, respectively. Received for review July 27, 1994. Accepted October 13, 1994.e
AC940752+ e Abstract
published in Advance ACS Abstracts, November 15, 1994.
