Fluorescence Quenching Kinetics of Anthracene and Naphthalene

9366

J. Phys. Chem. B 1999, 103, 9366-9377

Fluorescence Quenching Kinetics of Anthracene and Naphthalene Covalently Bound to the Terminus of Poly(methacrylic Acid) John H. Clements and S. E. Webber* Department of Chemistry and Biochemistry and Center for Polymer Research, The UniVersity of Texas at Austin, Austin, Texas 78712-1167 ReceiVed: May 26, 1999; In Final Form: August 3, 1999

Poly(methacrylic acid) (PMA) end-tagged with anthracene, bromoanthracene, and naphthalene fluorescence probes have been prepared by anionic polymerization methods, and the fluorescence quenching by a simple monovalent ion (Tl+) has been studied at pH 11 as a function of ionic strength. Under equivalent conditions the efficiency of fluorescence quenching has been found to be approximately an order of magnitude lower than for an anthracene probe located in the center of the PMA chain (Clements, J. H.; Webber S. E. J. Phys. Chem. A 1999, 103, 2513). This is believed to reflect the relatively low density of condensed ions near the chain ends. The steady-state fluorescence quenching data has been analyzed using several models (SternVolmer, hindered access, a modification of a model proposed by Morishima et al. (Morishima, Y.; Ohgi, H.; Kamachi, M. Macromolecules 1993, 26, 4293)). The latter model suggests a preferential binding of the Tl+ ion in the vicinity of the hydrophobic probe. The relation of the fitting parameters of these models to the fluorescent lifetime of the probes is discussed as is the relative importance of static quenching. The pH dependence of the fluorescence spectrum, intensity, and quenching by Tl+ was also measured for the anthracenetagged PMA, and it was found that at low pH the fluorescence intensity was diminished and the chromophores were almost completely protected from the Tl+ ion.

Introduction The behavior of polyelectrolytes in solution remains an active area of research.1 One of the most enlightening methods to study polyelectrolyte systems has been through the use of fluorescent probes.2 The coiling of poly(methacrylic acid) (PMA) in solution as a function of pH has been investigated by fluorescence measurements of anthracene3 and pyrene4 probes covalently bound to or solubilized by PMA. More recently, Liu et al.5 have studied the dimensions of PMA chains as a function of pH using Fo¨rster energy transfer between chromophores covalently attached at different locations along the polymer chain and at the chain ends. It is well-known that the electrostatic potential associated with these materials greatly influences the rate of chemical reactions that take place in their vicinity.2 An understanding of this phenomenon for simple systems may prove useful in elucidating the roles that more complex biological polyelectrolytes such as DNA play with regard to biochemical processes.6 The fluorescence of a probe covalently attached to a polyelectrolyte may be quenched by free ions in solution which in turn provides information about the equilibrium distribution and dynamics of quencher ions in the vicinity of the polyion. Morishima and co-workers have published numerous studies dealing with copolymers of sodium acrylate and acrylamide with pendant attachment of probes such as phenanthrene and 4-(4-hydroxyphenyl)ethenylpyridinium bromide.7 In earlier work from our laboratories, Morrison et al.8 have focused their attention on phenanthrene and anthracene pendant-tagged to PMA. The typical synthesis of tagged polyelectrolytes has utilized free-radical polymerization of the polyelectrolyte monomer with a low mole fraction of a monomer with the probe attached as * Corresponding author.

a pendant group. This method incorporates an inexact number of chromophores per chain and the spatial distribution of these chromophores along the polymer chain is expected to be random. Better-defined polymer-fluorophore systems have been achieved using anionic polymerization techniques.9 We have used this technique to synthesize PMA with a single 9,10dimethylanthracene probe in the center of the polymer, linked along the polymer backbone (referred to as A-m-PMA, see Scheme 1).10 We report here the synthesis of PMA labeled at the end of the chain with three different probes. Although other researchers have studied PMA using probes attached at the chain ends,3-5 the prior work has emphasized PMA coiling. As is well-known, the excited-state lifetime (τ0) strongly affects the quenching efficiency. To characterize this effect, PMA end-labeled with 9-methylanthracene probe (τ0 ) 9.6 ns), 9-methyl-10-bromoanthracene (τ0 ) 6.2 ns) and naphthalene (τ0 ) 28.4 ns) were prepared. The fluorescence quenching by Tl+ at pH 11 as a function of ionic strength was studied. This pH was chosen to completely ionize the polymer so that we may concentrate on polyelectrolyte effects rather than the complex behavior of PMA as a function of pH that has preoccupied the authors cited above. Very little work has been done on the theoretical modeling of the ion distribution around the end of a polyelectrolyte11 and so far as we know no work exists on how the end-effects might influence the kinetics of quenching. Experimental Section I. Synthesis. (a) Terminating Agents. (1) 9-Bromomethylanthracene (1). An amount of 2.80 g (1 equiv) of triphenylphosphine was dissolved in 20 mL of dry acetonitrile. The solution was placed in a round-bottom flask capped with a septum and bubbled with nitrogen for 20 min while being stirred to purge

10.1021/jp9917345 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/15/1999

End-Labeled PMA: Fluorescence Quenching

J. Phys. Chem. B, Vol. 103, No. 43, 1999 9367

SCHEME 1

SCHEME 2

the system. To this was slowly added 0.55 mL of bromine (2 equiv) via an airtight syringe. The resulting mixture appeared light yellow and contained a milky precipitate. An amount of 2.07 g of solid 9-methanolanthracene (purchased from Aldrich and used as received) was added slowly over a period of a few minutes via the opened round-bottom neck according to the procedure developed by Bullpitt and co-workers (method 2).12 A steady flow of nitrogen into the system was maintained during the addition to minimize the introduction of oxygen. After the addition was complete the mixture cleared. The neck was recapped and the solution allowed to react with stirring for 1 h under a steady nitrogen flow and at room temperature. The solution was cooled to 0 °C and cold-filtered. The dark yellow residue obtained was twice recrystallized from chloroform (15 mL of chloroform per 750 mg material) to obtain pure 1 (yellow needles). The material was freeze-dried from dioxane to obtain the product in a dry powder form. MS: (M+) m/z 192, 271, 273. HNMR: d 5.54 (s, -CH2-), 7.45-7.68 (m, 2,3,6,7-H), 8.00-8.06 (d, 4,5-H), 8.26-8.32 (d, 1,8-H), 8.49 (s, 10-H) in agreement with Bullpitt.12 MP: 130 °C (char onset), 134-136 °C (melt) in reasonable agreement with Stoffel13 (MP: 141142 °C).

(2) 9-Bromomethyl-10-Bromoanthracene (2). This procedure was very similar to that outlined for 1 with the exception that 1.70 mL bromine (6.2 equiv) was used in place of 0.55 mL (2 equiv). After the addition of bromine to the solution of triphenylphosphine in acetonitrile, a dark yellow color was observed. No distinctive change in the color or consistency of the mixture was observed after the slow addition of solid 9-methanolanthracene. After 1 h of reaction at room temperature the mixture was cold-filtered and allowed to air-dry in a fume hood to remove excess bromine vapors. The residue was dissolved in dioxane and rotoevaporated to further remove excess bromine. This dark yellow residue was twice recrystallized from chloroform (40 mL of chloroform per 750 mg of material) to obtain pure 2. The material was freeze-dried from dioxane to obtain the product in a dry powder form. MS: (M+) m/z 191, 269, 270, 271, 272 (parent molecule signals 349, 350, 351 not present in significant quantities). HNMR: d 5.46 (s, -CH2-), 7.56-7.70 (m, 2,3,6,7-H), 8.22-8.32 (d, 4,5-H), 8.56-8.64 (d, 1,8-H) in agreement with Wang. MP: 178 °C (char onset), 193-195 °C (melt) (Wang:14 200-202 °C). (3) 2-Methylbromonaphthalene (3). 3 was purchased from Aldrich and freeze-dried from dioxane in order to produce a dry powder that was easy to handle. No further sample treatment was carried out. (b) Preparation, Termination, and Purification of Polymers. All polymers were prepared by anionic polymerization of tertbutylmethacrylate in THF at -78 °C using cumylpotassium as the initiating species. After all monomer had been consumed, a solution containing the desired fluorophore in THF was added in order to obtain the labeled polymers (Scheme 2). Poly(tertbutylmethacrylate) end-labeled with bromoanthracene (BA-ePtBuMA) was obtained by the addition of 0.9 equiv of terminating agent 2 while 1.1 equivalents of the terminating agents 1 and 3 were used to prepare A-e-PtBuMA and N-ePtBuMA, respectively. Details of the synthetic and purification procedures, including the preparation of cumylpotassium initiator and cumylmethyl ether precursor, have been described at length in a previous paper using the bifunctional terminating species 9,10-dimethylbromoanthracene.10

9368 J. Phys. Chem. B, Vol. 103, No. 43, 1999 All labeled PtBuMA materials were injected into GPC immediately after termination without prior purification. Monitoring the chromophore absorption and fluorescence revealed the presence of unbound chromophore in both A-e-PtBuMA and N-e-PtBuMA polymer samples. This is expected since an excess of the appropriate terminating agent was added to the reaction mixture in both cases. Conversely, the BA-e-PtBuMA material showed no unbound chromophore, which is consistent with the fact that 2 was added as a limiting reagent. We suggest that allowing the terminator to be the limiting reagent is preferable because the tagging efficiency is excellent (90%) and purification of the resulting polymer product is greatly simplified.15 BA-e-PtBuMA was dialyzed in acetone before deprotection to remove the small quantities of cumene formed as a consequence of the reaction of cumyl potassium with trace impurities.10 The BA-e-PtBuMA number average molecular weight was 17 100 (target: 20 000), and the polydispersity (PD) was 1.09. There was a much larger amount of free chromophore in A-ePtBuMA and dialysis in acetone proved to be a rather ineffective purification method. It proved to be much more efficient to deprotect A-e-PtBuMA and purify this material by dialysis in aqueous solution. This yielded a polymer sample in which the unbound chromophore accounted for 30 mM) could not be obtained by the addition of small aliquots of the Tl+ stock solution because the dilution effect would be significant. Therefore for Tl+ concentrations higher than 30 mM individual solutions with identical polymer concentrations were prepared for each quencher concentration of interest. Solutions used for phase modulation studies were prepared as above except the polymer concentration had to be increased

9370 J. Phys. Chem. B, Vol. 103, No. 43, 1999

Clements and Webber

TABLE 1: Fluorescence Lifetimes and Yields for Labeled Polymers and Model Chromophores speciesa A-m-PMA 9,10-DMA BA-e-PMA BA-e-(MA)1-2 9-M-10-BA A-e-PMA 9-MA N-e-PMA 2-MN

solvent

φ

MeOH H2Ob MeOH MeOH H2Ob MeOH H 2O b MeOH H2Ob MeOH H2Ob 95/5 EtOH/H2O

0.90 0.85 0.63 0.17 0.39 0.20 0.33 0.10c 0.29c 0.13 0.16c

τ (ns) 10.7 11.0 6.2 7.4 2.1 9.6 5.2 28.4 47.0

a 9,10-DMA: 9,10-dimethylanthracene, 9-M-10-BA; 9-methyl-10bromoanthracene, 2-MN; 2-methylnaphthalene. b pH 11 KOH. c Birks, J. B. Photophysics of Aromatic Molecules; John Wiley & Sons Ltd., New York, 1970.

to provide adequate signal-to-noise ratio. For the same reason only a modest range of quencher concentration could be used. Results and Discussion I. Photophysical Characteristics. Quantum yields, φ, and fluorescence lifetimes, τ, for all labeled polymers including the anthracene midchain labeled polymer (A-m-PMA) previously studied10 have been determined and are given in Table 1. Values for model chromophores in similar solutions are shown for comparison. Although there are significant differences in the values for the polymer-bound chromophores and the analogous model species, certain similarities are obvious. Both A-m-PMA and 9,10-dimethylanthracene (9,10-DMA) have higher fluorescence yields and longer lifetimes than 9-methylanthracene (9MA), as is typical of many 9,10 di-substituted anthracenes.20 In addition, both BA-e-PMA and 9-methyl-10-bromoanthracene (9-M-10-BA) have reduced yields compared to 9-MA owing to the enhanced rate of intersystem-crossing, as is common with brominated substituents.21 As discussed above, we were not able to obtain quantum yields for A-e-PMA. II. Steady-State Fluorescence Quenching. We observed spectral changes as a function of quencher concentration for all labeled-polymers studied. Both A-e-PMA and BA-e-PMA exhibit slight red-shifting of the fluorescence maxima and enhancement of the long wavelength tail with increasing quencher concentration. Similar effects were observed for A-mPMA.10 This suggests that a fraction of chromophores may be in a specific environment that is inaccessible to quencher ions, as described by the “hindered-access model” (see later). We will argue later that this model does not seem to be adequate to linearize all quenching data. N-e-PMA has a relatively low fluorescence quantum yield and was quenched more efficiently than the anthracene polymers, as expected from its longer lifetime. At higher quencher concentrations the fluorescence intensity was reduced to such a degree that Raman scattering from water became a prominent feature of the spectrum. When the concentration of quencher exceeded 2.5 mM, two distinct peaks at 481 and 515 nm appeared (Figure 1), which correspond to the phosphorescence peaks of naphthalene. This emission feature was quenched by bubbling the solution by air. We note that 2-bromonaphthalene is a well-known example of fluid-phase phosphorescence.22 Evidently the quenching of our 2-substituted naphthalene by Tl+ is analogous to the internal heavy-atom effect.23 Quenching data are usually expressed as the ratio of unquenched to quenched fluorescence intensity, i.e., the well-

Figure 1. Fluorescence spectrum of N-e-PMA in pH 11 KOH at different quencher concentrations: (a) 0.386 mM, (b) 2.37 mM, (c) 5.41 mM, (d) 16.5 mM.

known Stern-Volmer (SV) plot of Io/I vs the concentration of quencher. Values for I and Io were obtained by integrating the fluorescence spectra after subtraction of the dark counts. The spectra of the anthracene-labeled polymers were integrated from 395 to 520 nm while those of N-e-PMA were integrated from 323 to 400 nm. Although a small amount of naphthalene fluorescence is present below 323 nm, this integration limit was chosen to avoid inclusion of the Raman scattering peak discussed above. The upper integration limit of 400 nm for the naphthalene spectra was chosen to avoid inclusion of the phosphorescence at long wavelength. An empirical nonlinear SV expression for fluorescence quenching is

Io/I ) 1 + KSV[Q] + A1[Q]2

(2)

where [Q] is the quencher concentration. In our use of eq 2 A1 is an empirical coefficient representing the deviation from SV linearity, and KSV is the SV constant.24 At low quencher concentrations, the higher order SV term is negligible and KSV can be calculated from the initial slope of the data. The apparent second-order quenching rate constant, kq, can be obtained from the relationship KSV ) kqτo, where τo is the fluorescence lifetime in the absence of quencher.25 The quenching curves for all labeled polymers in the absence of excess salt are presented in Stern-Volmer format in Figure 2. The corresponding KSV and kq constants at various ionic strengths are collected in Table 2. In our previous work a kq value of 1.41 × 1012 and 0.94 × 1012 M-1 s-1 was found for A-m-PMA and 9EA-PMA, respectively.8a,10 These values are 5-10 times larger than that for any of the anthracene end-labeled polymers, illustrating the dramatic effect of chromophore position within the polyelectrolyte. In the absence of excess salt, the KSV and kq values for the BA-e-(MA)1-2 model compound were found to be approximately two times larger than for BA-e-PMA, as is expected from the Smoluchowski equation for diffusion controlled reactions.26 kq for BA-e-PMA is smaller than A-e-PMA, which we believe reflects steric hindrance by the bromine substituent.



Figure 2. Steady-state fluorescence quenching of labeled polymers: (a) N-e-PMA, (b) A-m-PMA, (c) A-e-PMA, (d) BA-e-(MA)1-2, (e) BA-e-PMA.

TABLE 2: Stern-Volmer and Quenching Rate Constants for All Labeled Polymers at Various Ionic Strengths polymer A-m-PMA A-e-PMA BA-e-PMA BA-e-(MA)1-2 N-e-PMA

c

IS (mM)

KSVa × 10-3 (M-1)

kqb × 10-11 (M-1 s-1)

kq∞c × 10-9 (M-1 s-1)

1.83 1.83 9.31 18.3 1.72 13.0 20.2 1.12 12.4 20.4 1.76 8.45 15.7

12.2 1.42 1.00 0.495 0.161 0.0666 0.0605 0.414 0.304 0.250 6.45 3.55 2.34

14.2 1.48 1.04 0.515 0.261 0.108 0.0979 0.560 0.412 0.338 2.27 1.25 0.822

5.21 0.483

Figure 3. Tl+ quenching of A-e-PMA at various ionic strengths: (a) IS ) 1.87 mM, (b) IS ) 9.62 mM, (c) IS ) 19.0 mM. The smooth lines through the data represent the fit to the modified Morishima model (see text).

0.345 0.336 0.591 0.411

a Based on the initial slope of the SV plot. b Based on k ) K /τ . q SV 0 Based on the limiting slope at high quencher concentration.

Using the same estimate of the molecular sizes used previously8a,27 and the Smoluchowski expression, the diffusioncontrolled rate constant for quenching in water at 25 °C is calculated to be 1.29 × 1010 M-1 s-1 (assuming that the polymer to which the chromophore is bound is immobile). Correcting for the Coulombic attraction between the Tl+ cation and the polyanion (assuming a charge of -1 in the vicinity of the anthracene) yields a quenching rate constant of 1.91 × 1010 M-1 s-1. This is approximately the quenching rate constant we observe for BA-e-PMA but approximately an order of magnitude lower than the value for A-e-PMA (see Table 2). Increasing the effective negative charge near the anthracene or naphthalene group to 14 or 21 yields approximately the observed rate for A-e-PMA and N-e-PMA, respectively (cf. 113 for A-m-PMA10). Quenching curves for A-e-PMA at various ionic strengths are presented in the SV format in Figure 3. Addition of salt to the system systematically decreases the degree of quenching. The quenching data for all other labeled polymers depend similarly on ionic strength and are not shown. The strong dependence of the quenching efficiency on the ionic strength of the system can be interpreted in two ways. First, the presence of excess K+ counterions reduces the effective electrostatic

Figure 4. The quenching data from Figure 3 plotted as a function of quencher mole fraction: (a) IS ) 1.87 mM, (b) IS ) 9.62 mM, (c) IS ) 19.0 mM.

attraction of the polyanion for the quencher ion through Debye shielding. Second, the excess counterions displace quencher ions within the vicinity of the polyelectrolyte, thereby lowering the local quencher concentration. This has been referred to as “statistical dilution” in our previous paper.8a In the case of ideal statistical dilution Io/I plotted as a function of the quencher mole fraction (based on all cations) should collapse into a single curve. As can be seen from Figure 4, the quenching curves plotted in this manner remain quite distinct, much more so than our earlier results for A-m-PMA or 9EA-PMA.8a,10 We conclude that there is a much less obvious “polyelectrolyte effect” for the endtagged PMA. If the effect of ionic strength on Tl+ quenching is strictly electrostatic in origin, then the quenching by a neutral species will be independent of ionic strength. However, increased Debye


Clements and Webber

TABLE 3: Acrylamide Quenching of Labeled Polymers polymer

IS (mM)

KSV (M-1)

kq × 10-9 (M-1 s-1)

A-m-PMA

1.65 9.28 16.6 1.87 19.0 1.67 16.5 1.72 14.7

16.0 15.4 14.7 10.5 10.2 4.50 4.55 97.7 80.7

1.50 1.44 1.37 1.09 1.06 0.729 0.737 3.44 2.84

A-e-PMA BA-e-PMA N-e-PMA

shielding may result in large-scale polymer conformational changes such as chain coiling, especially in the vicinity of the hydrophobic chromophore. In this case, quenching by a neutral species will be dependent on the ionic strength of the solution. We tested this effect using acrylamide as the neutral quencher. For the two anthracene-labeled polymers studied the rate of quenching by acrylamide was found to be independent of ionic strength (Table 3). On the other hand, acrylamide quenching of the naphthalene-labeled material, N-e-PMA, does show a significant dependence on ionic strength, with kq decreasing from 3.44 × 109 M-1 s-1 at IS ) 1.72 mM to 2.84 × 109 M-1 s-1 at IS ) 14.7 mM. We also find that the fluorescence intensity of N-e-PMA in the absence of quencher depends on the ionic strength of the solution. The fluorescence intensity with a K+ concentration of 17.4 mM is only 73% of the value at 2.63 mM, whereas the fluorescence of the anthracene-labeled polymers decreased no more than 2% over the same K+ concentration range. It is our experience that the substituted naphthalene chromophore is quite sensitive to its environment.28 Evidently there are environmental modifications induced by the addition of salt that can be detected by the 2-methyl naphthalene chromophore. At very high quencher concentrations, the quenching data for all anthracene-labeled polymers roll over and approach a constant slope that is nearly independent of ionic strength (data not shown). The limiting slope at a high concentration of Tl+ was determined in a separate experiment ([Tl+]max ≈ 90 mM) and yields a second-order rate constant on the order of one-third the diffusion-controlled rate constant (denoted kq∞ in Table 2). Similar experiments for A-m-PMA yield a kq value approximately an order of magnitude larger.10 Data were not obtained for N-e-PMA in the high quencher concentration regime because of the poor signal-to-noise, as noted previously. III. Inaccessible Fluorophores: The Hindered-Access Model. The negative deviation from linearity in the SV plots for all polymers studied (Figure 2) suggests that a fraction of chromophores may be inaccessible to quencher ions. In such cases a two-state model is often employed in which the population of probes is divided into an accessible fraction, fa, and an inaccessible fraction, fb ) 1 - fa. In the usual application of this model it is assumed that as quencher is added to the system the fluorescence intensity of the accessible probes decreases according to the linear SV relation (we denote the SV constant for the accessible probes by KSVa) while the fluorescence intensity of the b probes remains unchanged. This yields the following expression:24

Io/(Io - I) ) 1/fa + 1/(KSVafa[Q])

(3)

where Io ) Ioa + Iob. Therefore, a plot of Io/(Io - I) vs 1/[Q] should yield a linear function in which fa can be obtained from the intercept and KSVa from the ratio of the intercept and slope.

Figure 5. The quenching data from Figure 3 plotted according to the hindered-access model: (a) IS ) 1.87 mM, (b) IS ) 9.62 mM, (c) IS ) 19.0 mM.

TABLE 4: Apparent Stern-Volmer Constants and Accessible Fractions Obtained by Analyzing the Quenching Data for All Labeled Polymers in Terms of the Hindered-Access Model polymer A-m-PMA A-e-PMA BA-e-PMA BA-e-(MA)1-2 N-e-PMA

IS (mM)

KSVa × 10-3 (M-1)

fa

1.83 1.87 9.62 19.0 1.72 13.0 20.2 1.12 12.4 20.4 1.76 8.45 15.7

13.0 1.83 0.899 0.664 0.587 0.347 0.312 1.05 0.794 0.732 6.85 3.86 2.60

0.945 0.634 0.646 0.623 0.216 0.194 0.175 0.385 0.377 0.350 0.962 0.943 0.922

Figure 5 shows the A-e-PMA quenching data plotted in this fashion. This plot is similar for all the polymers studied so only this one is shown explicitly. The values of KSVa for A-e-PMA and N-e-PMA match those obtained from the initial slope of the SV plot very well (KSV in Table 2), and the fraction of accessible probes was found to be within the range 0.62-0.65 or 0.92-0.96 for A-e-PMA and N-e-PMA, respectively (Table 4). The KSVa values for BA-e-PMA, and BA-e-(MA)1-2 are significantly higher than their corresponding KSV values, and the fraction of accessible probes was found to be much less than unity (0.18-0.22 and 0.35-0.39 for BA-e-PMA and BAe-(MA)1-2, respectively). We will argue later that this is a consequence of their shorter fluorescence lifetimes. Yekta et al.29 have proposed a model that is analogous to the hindered-access model but focuses on the dynamics of quencherfluorophore encounters. They consider the quenching reaction between fluorophores residing within a spherical microdomain of radius R and quenchers that have congregated at the microdomain boundary. Note that for this model chromophores are not isolated from quencher molecules by rigid encapsulation, as would be the case for extensive chain coiling around the chromophore, but are isolated “dynamically”; i.e., the probability that the probe will diffuse into contact with the quencher during



the lifetime of the excited state is much less than unity. After a fairly elaborate mathematical development Yekta et al. obtain an equation of the form of eq 3 in which fa increases with dimensionless parameter Dτo/R2, where D is the diffusion constant of the chromophore inside the spherical microdomain. From this perspective, the larger value of fa for N-e-PMA as compared to A-e-PMA is a natural consequence of the longer lifetime of the naphthalene species, which allows more time for quenchers to diffuse toward the probe. In contrast, the lower values of fa obtained for BA-e-PMA and BA-e-(MA)1-2 are a natural result of the shorter lifetime. As discussed earlier, steric protection provided by the bromine substituent may also play a role in the quenching efficiency and hence the value of fa. While the qualitative trends of our fa values follow the model of Yekta et al., the values of fa we obtain change much more drastically with τ0 than predicted (see Figure 2 of ref 29). This is not surprising given the very different geometry of end-tagged polyelectrolytes and their spherical model. It would be very interesting to develop a model along these lines that reflects the cylindrical geometry of mid- and end-tagged polyelectrolytes. Upon closer inspection of the fit (Figure 5), we find that the model linearizes data fairly well at low quencher concentration but not near the intercept (at higher quencher concentrations). A similar result was found for A-m-PMA10 and for all other polymers studied herein. Nevertheless, the model does provide insight into the empirical “accessibility” of the chromophore. IV. Combined Stern-Volmer and Perrin (Morishima) Analysis. The sphere-of-action quenching (Perrin) model was invoked by Morishima et al. to deal with quenching by counterions that are “condensed” within the vicinity of a polyelectrolyte.7b The Perrin model assumes that quenchers residing within a “sphere of action” surrounding the fluorophore have a quenching efficiency of unity.18,24 Furthermore it is assumed that the probability of finding n quenchers within this region can be described by a Poisson distribution, P(n) ) exp(〈nQ〉)〈nQ〉 n/n! where 〈nQ〉 is the average number of quenchers that reside within the volume of the sphere of action. Therefore, the probability that the probe will not be quenched is equal to P(0) ) exp(-〈nQ〉).30 Morishima et al. assume that ions which reside outside the condensation zone (so-called “atmospheric” ions) quench by a dynamic mechanism described by SV kinetics. In polyelectrolyte systems both mechanisms are expected to be operative such that quenching can be described by

Io/I ) (1 + KSV,app[Tl+]atm) exp[〈nQ〉]

(4)

where KSV,app is the “apparent” SV constant, [Tl+]atm is the “atmospheric concentration” of Tl+, and 〈nQ〉 is the average number of quencher ions in the active quenching sphere of the chromophores. In our modification of the Morishima model we assume that the number of ions that reside near the chromophore is fixed (〈N〉) and when quencher ions are added the average number of quenchers is given by 〈N〉xQ where xQ is the mole fraction of quencher ions. This assumption is in the spirit of Manning condensation theory31 and also was the basis of a simplified Poisson-Boltzmann model applied in earlier work.8a One would expect xQ to be computed on the basis of the total concentration of all cations, but if the quencher ion is preferentially bound to the polyelectrolyte in the region near the chromophore the apparent value of the quencher mole fraction may be higher. Preferential binding of Tl+ near the chromophore can be accounted for by considering the displacement of K+ bound to the PMA (K+(b)) by bulk Tl+ (Tl+(aq))

Tl+(aq) + K+(b) ) Tl+(b) + K+(aq)

(5)

with an equilibrium constant Kb. Assuming that [Tl+]atm ∼ [Tl+]o, eq 4 becomes

[{

+

Io/I ) (1 + KSV,app[Tl ]o) exp 〈N〉

[Tl+]o

[Tl+]o + [K+]/Kb

}]

(6)

with the fitting parameters 〈N〉, Kb, and KSV,app.10 For every polymer studied, eq 6 failed to fit the data unless Kb was allowed to vary, in which case excellent fits were achieved (Figure 3). The values of the fitting parameters are listed in Table 5. The Kb values for all polymers were found to be greater than unity for all ionic strengths studied which means there is preferential binding of Tl+ compared to K+.10 The KSV,app values are much lower than those obtained from the initial slope of the SV plot (KSV in Table 2) because the exponential function in eq 5 makes a large contribution to the initial slope of the Io/I plot. We have no explanation for the increase of Kb with ionic strength. In general, the 〈N〉 values from our fits do not change appreciably with ionic strength. There is a very strong nonlinear correlation between the 〈N〉 values and the accessibility fractions (fa) that were found from the hindered-access model (Figure 6). 〈N〉 values within the range 2.4-2.5 were obtained for A-m-PMA10 which is much higher than the range 0.760.88 found for A-e-PMA. We presume this result reflects the differences in the electrostatic potential of the two regions. 〈N〉 also depends on the excited-state lifetime, as can be seen in the 〈N〉 values for N-e-PMA (2.3-2.6) and BA-e-PMA (0.110.19). The diffusion length of a quencher during the excitedstate lifetime is proportional to (DQτ0)1/2 where DQ is the diffusion constant of the quencher species. If the quenching volume surrounding a chromophore is related to the cube of the diffusion length, then a proportionality of 〈N〉 to (τ0)3/2 is expected. Qualitatively this is observed, albeit for only three data points.32 As discussed in the preceding, fa is expected to be related to τ0, so it is reasonable that 〈N〉 and τ0 are strongly correlated. V. Time-Resolved Quenching. The lifetime and steady-state quenching of A-e-PMA, BA-e-PMA, and BA-e-(MA)1-2 are shown in Figure 7. Due to signal-to-noise limitations, the lifetime measurements were limited to relatively low quencher concentrations. The fluorescence decays were fit to a sum of exponentials:

I(t) )

Ri exp(-t/τi) ∑ i)1

(7)

where I(t) is the fluorescence intensity as a function of time (normalized to unity at t ) 0) and Ri τi are the preexponential factor and lifetime of the ith component, respectively. We define the average lifetime as:

〈τ〉 )

Ri τi ∑ i)1

(8)

where 〈τ〉 represents the integral of I(t) from 0 to ∞, which is directly comparable to the steady-state intensity.33 The lifetime quenching is characterized by the value of 〈τo〉/ 〈τ〉 where 〈τo〉 and 〈τ〉 are the average lifetimes in the absence and presence of quencher, respectively. In the case of “static quenching” (i.e., a quenching process faster than we can measure) then we expect 〈τo〉/〈τ〉 to lie below the corresponding


Clements and Webber

Figure 6. The correlation of fa and 〈N〉.

TABLE 5: KSV,app, 〈N〉, and Kb Values Obtained from Fitting the Quenching Data in Figure 3 to the Modified Morishima Model polymer A-m-PMA A-e-PMA BA-e-PMA BA-e-(MA)1-2 N-e-PMA

IS (mM)

KSV,app (M-1)

〈N〉

Kb

1.83 6.63 14.8 1.87 9.62 19.0 1.72 13.0 20.2 1.12 12.4 20.4 1.76 8.45 15.7

26.2 9.38 17.4 9.04 9.09 11.1 4.44 5.35 6.34 8.60 6.85 7.38 54.0 51.8 64.4

2.50 2.52 2.42 0.882 0.838 0.759 0.188 0.156 0.110 0.382 0.416 0.393 2.58 2.47 2.26

12.6 15.0 20.8 3.49 7.04 9.95 1.76 5.23 9.95 1.79 8.53 11.4 5.78 11.1 14.6

steady-state value Io/I. We should note that the models used to fit the data in the previous sections do not directly calculate the dynamics of quenching but rather the integral of I(t). In our previous study of A-m-PMA we found that a large fraction of quenching occurs via the dynamic mechanism.10 In contrast, from Figure 7 we see that the quenching of A-e-PMA and BA-e-PMA is largely static. The fact that there is almost a complete absence of a dynamic quenching process for these end-labeled materials is probably a result of the nonuniformity of the electrostatic potential near the chain ends. Static quenchers are presumed to be condensed along the polyelectrolyte in the vicinity of the chromophore, and dynamic quenching arises either from atmospheric ions or condensed ions relatively distant from the chromophore that have migrated into the “sphere of action” following excitation.34 It is unfortunate that more precise time-dependent data could not be obtained for these systems because in principle this provides a test of any dynamical model of ion motion near a polyelectrolyte. The quenching of the oligomeric model compound BA-e(MA)1-2 is largely dynamic. The fact that very little static quenching occurs for this species reinforces the belief that static quenching is a result of counterion condensation. VI. Effect of pH on Fluorescence and Quenching Efficiency for Anthracene-Tagged PMA. Many researchers have studied the fluorescence spectroscopy of PMA-solubilized or covalently attached probes as a function of solution pH. Chen and Thomas have shown that pyrene is solubilized fairly well

Figure 7. Steady-state (a) and time-resolved (b) quenching data for A-e-PMA, (middle) BA-e-PMA, and (bottom) BA-e-(MA)1-2 (no added salt).

by protonated PMA but as the pH is increased the coil expands, releasing free pyrene into the aqueous phase and reducing the fluorescence intensity.35 Similar observations were made for pyrene covalently attached to the polymer, which demonstrated that exposure of the probe to the aqueous environment reduces the pyrene fluorescence.4 Tl+ quenching experiments were carried out in the latter case and revealed that at low pH the



probe is almost completely inaccessible to quencher ions while expansion of the coil at higher pH greatly increases the Tl+ quenching efficiency. We measured the fluorescence spectrum and intensity for all anthracene-tagged PMA samples as a function of added HNO3, from pH 11 down to pH e 3, at which point >95% of the methacrylic acid residues are protonated.36 Then a reverse titration was carried out by adding a sufficient amount of KOH to re-ionize the polymer and obtain a final solution with pH ∼ 11. The results of these titrations are presented in Figure 8. The data have been presented as the ratio of the fluorescence intensities to the initial value (Io). The vertical dashed line denotes the equivalence point. We find that the fluorescence intensity of all the anthracene-labeled polymers discussed in this paper decreased with added acid, whereas Chu and Thomas observed the opposite effect for pyrene bound to PMA.4 This result is not of itself surprising because anthracene behaves very differently from pyrene in solution.18 Several groups have synthesized end-tagged PMA which is similar to the polymer studied herein. For example, 9-methylanthracene has been attached at the chain ends through chain transfer in free-radical polymerization by Tan and Treloar and they report the fluorescence lifetime as 7.30 ns in dilute HCl (neutral polymer),37 which is shorter than we find for A-e-PMA at pH 11 (11.0 ns, Table 1). We find the fluorescence intensity of A-e-PMA at low pH is diminished by approximately the ratio of the lifetimes. There is a modest but abrupt spectral change that occurs near the equivalence point (see Figure 9 for BA-e-PMA, which is typical of the others). We believe the spectral change is due to a significant degree of polymer coiling near the equivalence point as the repulsion between methacrylic acid residues decreases sharply. Using an energy transfer method Liu et al. report that the radius of gyration, Rg, for PMA changes very little at high pH (compare 7.7 nm at pH 11 to 7.8 nm at pH 8.7) and much more abruptly near neutrality (compare 5.2 nm at pH 7.7 to 3.5 nm at pH 7.5).5 Because of the small number of methacrylic acid residues in BA-e-(MA)1-2 aggregation occurs for pH < 7 and what appears to be an anthracene excimer appears in the fluorescence spectrum (Figure 9).38 HNO3 titrations were also performed in the presence of Tl+, and the ratio of the unquenched to quenched fluorescence intensity as a function of added HNO3 is plotted in Figure 10. Clearly, the fluorescence intensity of the probe is independent of the presence of quencher at low pH. Since the hydrophobic fluorescent probe seeks to maximize stabilizing interactions with the polymer chain, it is reasonable to expect that the probe will be protected by the interior of the coil at low pH, even for our end-tagged polymers. It is interesting that the quenching efficiency drops sharply before the equivalence point is reached. Presumably this is because the linear charge density is below the critical charge density required for counterion condensation.39 Summary The primary objective of this research was to compare the local ionic environments at the end and middle of a polyelectrolyte chain using fluorescence quenching by Tl+. The fluorescence quenching of A-m-PMA is considerably more efficient than that for A-e-PMA, which we believe is the result of the “end-effects” associated with the electrostatic potential of the polyelectrolyte. Our modification of a model due to Morishima7b suggests that the concentration of condensed quencher ions falls off sharply at the chain terminus. Time-resolved measurements

Figure 8. Fluorescence intensity as a function of HNO3 added (solid line) and KOH added (dashed line) for (a) A-m-PMA, (b) A-e-PMA, and (c) BA-e-PMA.

indicate that quenching of A-e-PMA and BA-e-PMA is dominated by immobilized condensed quencher ions whereas a significant fraction of dynamic (“diffusional”) quenching by atmospheric ions was observed for A-m-PMA.10 While the position of the probe on the polyelectrolyte is extremely important, dynamic factors are of considerable importance as well. Therefore we also examined a bromoanthracene and naphthalene probe which have a shorter and longer lifetime than


Clements and Webber

Figure 10. The ratio of the fluorescence intensity in the absence of quencher (Io) to that in the presence of quencher (I) as a function of acid addition. (a) A-e-PMA, (b) BA-e-PMA, (c) BA-e-(MA)1-2, (d) A-m-PMA, (e) N-e-PMA.

SCHEME 3

Figure 9. Fluorescence spectra for (top) BA-e-PMA and (bottom) BAe-(MA)1-2: (a) before titration, pH 11, (b) after titration, pH e 3, and (c) after back-titration, pH ∼ 11.

A-e-PMA, respectively. In qualitative agreement with hindered geometry models, we find that the “apparent accessibility” (fa) of the chromophore increases with the fluorophore lifetime. Similarly the number of “neighboring ions” in the context of the modified Morishima model (〈N〉) increases approximately as τ03/2, which suggests that 〈N〉 reflects the diffusive motion of ions in the vicinity of the chromophore. The fact that 〈N〉 is much larger for A-m-PMA than A-e-PMA, despite their similar τ0 values suggests that A-m-PMA has more than twice as many nearest neighbors (see Scheme 3). Additionally the concentration of condensed ions may decrease as one approaches the PMA terminus. On the basis of our modification of Morishima’s model we find an apparent preferential binding of the Tl+ ion compared to K+ in the vicinity of the hydrophobic chromophore for all end-labeled polymers.40 The magnitude of the preferential binding constant (Kb) is on the order of half that of the midtagged case. We have no explanation for this preferential binding, the difference between end- and mid-tagged polymers

and the systematic increase of Kb with ionic strength (Table 5). Of course, it is possible that Kb is an artifact of the kinetic model. The fluorescence intensity of all anthracene-labeled polymers decreases as the pH of the solution is reduced. This dependence takes the form of a relatively abrupt decrease near the equivalence point (Figure 8). Similarly the efficiency of Tl+ quenching decreases at low pH (Figure 10). Both effects could be the result of the polyelectrolyte coiling below a critical degree of ionization (that other researchers have estimated to be 2025%3,35). Alternatively this could represent the linear charge density at which no counterion condensation occurs, which also sharply diminishes the efficiency of Tl+ quenching. At low pH, the coiled polymer provides a nearly impenetrable barrier to quencher ions, indicating that both end and midchain labeled probes are buried deep within these coils. For all polymers, the coiling process is reversible upon back-titration. Acknowledgment. This research has been supported by the Office of Naval Research (Grant N00014-91-J-1667) and the Robert A. Welch Foundation (Grant F-356). This support is

End-Labeled PMA: Fluorescence Quenching gratefully acknowledged. The authors also thank Prof. G. C. Willson for many helpful discussions regarding the deprotection procedure. References and Notes (1) For a review of the current state of the theory of polyelectrolytes, see Barrat, J.-L.; Joanny, J.-F. AdV. Chem. Phys. 1996, 94, 1. (2) (a) Morawetz, H. Acc. Chem. Res. 1970, 3, 354; (b) Morawetz, H. Acc. Chem. Res. 1994, 27, 174; (c) Morawetz, H. J. Polym. Sci.: Part A: Polym. Chem. 1999, 37, 1725. (3) Ghiggino, K. P.; Tan, K. L. Polymer Photophysics; Phillips, D., Ed.; Chapman and Hall: New York, 1985; Chapter 7. (4) Chu, D.-Y.; Thomas, J. K. Macromolecules 1984, 17, 2142. (5) Liu, G.; Guillet, J. E.; Al-Takrity, E. T. B.; Jenkins, A. D.; Walton, D. R. M. Macromolecules 1991, 24, 68. (6) Wensel, T. G.; Meares, C. F.; Vlachy, V.; Matthew, J. B. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 3267. In this work, the excited-state lifetime of the various Tb+3 chelates used was on the order of milliseconds such that the results are dependent only on the equilibrium distribution of ions near the DNA, not the dynamics of ion transport. (7) (a) Morishima, Y.; Higuchi, Y.; Kamachi, M. J. Polym. Sci. 1991, 29, 677; (b) Morishima, Y.; Ohgi, H.; Kamachi, M. Macromolecules 1993, 26, 4293; (c) Morishima, Y.; Higuchi, Y.; Kamachi, M. J. of Polym. Sci. 1993, 31, 373; (d) Morishima, Y.; Sato, T.; Kamachi, M. Macromolecules 1996, 29, 1633; (e) Morishima, Y.; Sato, T.; Kamachi, M. Macromolecules 1996, 29, 3960. (8) (a) Morrison, M. E.; Dorfman, R. C.; Clendening, W. D.; Kiserow, D. J.; Rossky, P. J.; Webber, S. E. J. Phys. Chem. 1994, 98, 5534; (b) Morrison, M. E.; Dorfman, R. C.; Webber, S. E. J. Phys. Chem. 1996, 100, 15187. (9) (a) Procha`zka, K.; Kiserow, D. J.; Webber, S. E. Acta Polym. 1995, 46, 277. (b) Quirk, R. P.; Kim, J.; Rodrigues, K.; Mattice, W. L. Makromol. Chem., Macromol. Symp. 1991, 463, 42. (c) Tcherkasskaya, O.; Ni, S.; Winnik, M. A. Macromolecules 1996, 29, 610. (10) Clements, J. H.; Webber, S. E. J. Phys. Chem. A 1999, 103, 2513. (11) Manning, G. S.; Mohanty, U. Physica A 1997, 247, 196. (12) Bullpitt, M.; Kitching, W.; Doddrell, D.; Adcock, W. J. Org. Chem. 1976, 41, 760. (13) Stoffel, W.; Michaelis, G. Hoppe-Seyler’s Z. Physiol. Chem. 1976, 357, 7. (14) Wang, H.; Wen, Z. B.; Cao, Y. J. Photochem. Photobiol. A 1995, 92, 29. (15) Untagged PMA does not cause any difficulties in these experiments so long as it is taken into account in computing ionic strengths and so on. Untagged PMA is equivalent to having a higher degree of polymerization of the tagged PMA so long as it is the concentration of ions in the vicinity of the chromophore that effects the fluorescence quenching. (16) Programs written by T. J. Martin. (17) Procedure referenced in Aldrich catalog under the trade name Diazald.

J. Phys. Chem. B, Vol. 103, No. 43, 1999 9377 (18) Birks, J. B. Photophysics of Aromatic Molecules; John WileyInterscience: New York, 1970; Chapter 4. (19) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991. (20) Cherkasov, A. S.; Molchanov, V. A.; Vember, T. M.; Voldaikina, K. G. SoV. Phys. Dokl. 1956, 1, 427. (21) McGlynn, S. P.; Azumi, T.; Kinoshita, M. Molecular Spectroscopy of the Triplet State; Prentice Hall: Englewood Cliffs, NJ, 1969; Chapter 7. (22) Winnik, M. A.; Pekcan, O. Can. J. Chem. 1985, 63, 129. (23) That is to say, the T1 f S0 radiative rate is enhanced more than the radiationless rate. See ref 21, Table 7.2, p 272. (24) Lakowicz, J. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; p 260. (25) We use the average lifetime, 〈τo〉 (see eq 7 later) for nonexponential decay, although in fact the fluorescence decay for these systems was dominated by a single lifetime component that is close to the 〈τo〉 values quoted in Table 1. (26) Levine, I. Physical Chemistry, 3rd ed., McGraw-Hill Book Co.: New York, 1988; p 559. (27) CRC Handbook of Chemistry and Physics, 66th ed.; Weast, R., Ed.; CRC Press: Boca Raton, FL, 1985; pp D 167. (28) Martin, T. J.; Webber, S. E. Macromolecules 1995, 28, 8845. (29) Yekta, A.; Duhamel, J.; Winnik, M. A. J. Chem. Phys. 1992, 97, 1554. (30) This is clearly an oversimplification because ions in the sphere of action are not necessarily 100% effective at quenching. However the simple Perrin model captures the main physical features of “contact quenching”. (31) Manning, G. J. Chem. Phys. 1969, 51, 924. (32) A plot of 〈N〉 vs (τ0)3/2 is fit to a straight line with R2 of 0.966 and a fit of log(〈N〉) vs log(τ0) yields a slope of 1.57 with an R2 of 0.891. (33) Webber, S. E. Photochem. Photobiol. 1997, 65, 33. (34) If we assume that we could detect a component with 0.2 ns lifetime (feasible with our phase-modulation system) and take the diffusion constant for Tl+ (2.0 × 10-5 cm2/s-1), then the sphere of quenching would have a radius on the order of 0.63 nm, which corresponds to 2-3 PMA repeating units. Therefore it is plausible that Tl+ ions located near the chain terminus will act as static quenchers. (35) Chen, T. S.; Thomas, J. K. J. Polym. Sci. 1979, 17, 1103. (36) Leyte, J. C.; Mandel, M. J. Polym. Sci.: Part A 1964, 2, 1879. (37) Tan, K. L.; Treloar, F. E. Chem. Phys. Lett. 1980, 73, 234. (38) The intensity of quasi-elastic light scattering increases by a factor of 4 at pH < 3 relative to pH ∼ 11. (39) The critical charge density for condensation is ξc ) 1 for univalent ions, where ξ ) e2/ekTb and b is the linear charge density and the other symbols are defined as usual. For fully ionized PMA b ) 2.55 Å and if we propose that at the transition point that ξ ) ξc ) 1, then b ) approximately 7.14 Å, which implies that ca. 0.36 of the PMA groups are ionized. (40) We note that in our prior work no preferential binding of Tl+ was found for PMA as a whole (ref 8b). Therefore Kb must be interpreted as the preferential binding in the vicinity of the chromophore.

Fluorescence Quenching Kinetics of Anthracene and Naphthalene

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