Langmuir 1997, 13, 6089-6094
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Quenching Studies of Hydrophobically-Modified Poly(N-isopropylacrylamides) T. C. Barros,† A. Adronov,† F. M. Winnik,*,‡ and C. Bohne*,†,§ Department of Chemistry, University of Victoria, P.O. Box 3065, Victoria, BC, Canada V8W 3V6, and Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 3M1 Received May 7, 1997. In Final Form: August 27, 1997X Fluorescence quenching by nitromethane of pyrene-labeled poly(N-isopropylacrylamides) (PNIPAMs) which contain alkyl chains was employed to investigate the accessibility of the quencher to the pyrene moieties. The quenching efficiency for monomeric pyrene or excimers was decreased in PNIPAMs when compared to the quenching efficiency of pyrene fluorescence in the aqueous phase, showing that pyrene is always located within the polymeric micelles. The contribution of static quenching was evaluated by comparing the quenching efficiency determined from steady-state measurements with that obtained from time-resolved data. Static quenching was present for all PNIPAMs studied. The decrease of the quenching efficiency was related to the polymeric micelle structure. The highest degree of protection was observed for the excimer emission of the polymer containing randomly-substituted alkyl chains and with the pyrene bound to the same polymeric unit as the alkyl chain.
Introduction Compounds capable of self-aggregation can alter dramatically the properties of aqueous solutions. Amphiphilic polymers, which are constituted of well-defined hydrophilic and hydrophobic parts, often undergo aggregation in water. There are two broad classes of amphiphilic polymers. They can have either a high content of hydrophobic groups, as in the case of alternating copolymers of ionic and hydrophobic monomers (polysoaps), or a low content of hydrophobic groups, as for example in hydrophobically-modified cellulose ethers or polyacrylamides. The latter polymers exhibit rheological features markedly different from those of the unmodified polymers. Interchain aggregation of the hydrophobic groups leads to an enhancement in viscosity and reversible shear sensitivity. These materials have found numerous practical applications as thickening additives in water-borne industrial fluids, such as paints, foods, and cosmetics.1,2 More recently they have attracted much interest with respect to biotechnological and pharmaceutical applications.3 Fundamental questions related to their aggregation mechanisms need to be answered to help in designing new polymers. The properties of aqueous solutions of amphiphilic polymers are dictated by the chemical structure of the hydrophilic and hydrophobic portions, the content of hydrophobic groups, and their distribution along the hydrophilic component.4-6 In the present work, we * To whom correspondence should be addressed. † University of Victoria. ‡ McMaster University. § Phone: (250) 721-7151. Fax: (250) 721-7147. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, October 15, 1997. (1) Polymers in Aqueous Media: Performance through Association; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1989; Vol. 223. (2) Hydrophilic Polymers, Performance with Environmental Acceptability; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1996; Vol. 248. (3) Macromolecular Complexes in Chemistry and Biology; Dubin, P., Bock, J., Davis, R., Schultz, D. N., Thies, C., Eds.; Springer-Verlag: Berlin, 1994. (4) Hwang, F. S.; Hogen-Esch, T. E. Macromolecules 1995, 28, 3328. (5) Morishima, Y.; Nomura, S.; Ikeda, T.; Seki, M.; Kamachi, M. Macromolecules 1995, 28, 2874. (6) Akiyoshi, K.; Deguchi, S.; Tajima, H.; Nishikawa, T.; Sunamoto, J. Macromolecules 1997, 30, 857.
S0743-7463(97)00477-0 CCC: $14.00
address the issue of the impact of the molecular architecture of the hydrophobically-modified polymer on its aggregation properties, by studying the fluorescence quenching of previously characterized polymers. We have selected the poly(N-isopropylacrylamide) (PNIPAM) backbone as the hydrophilic portion and the n-octadecyl group as the hydrophobic component, together with pyrene, used as a fluorescent label. The hydrophobic group was either linked randomly and in small amounts along the main chain or specifically at one chain end. The structures of the polymers are shown in Figure 1. PNIPAM-C18Py consists of a PNIPAM chain to which are attached a few (on average 0.5 mol %) N-[4-(1-pyrenyl)butyl]-N-n-octadecyl groups. In this sample, the fluorescent label is linked to the same amide nitrogen as the octadecyl group. The second polymer, PNIPAM-C18-Py, also consists of a PNIPAM main chain carrying a small number of noctadecyl and 4-(1-pyrenyl)butyl groups, but the fluorescent label and the octadecyl chain are attached to different amide nitrogen atoms and are thus kept apart from each other. In the third polymer, b-PNIPAM-C18Py, a N-[4(1-pyrenyl)butyl]-N-n-octadecyl group is linked to one end of the PNIPAM chain. In contrast to the unmodified PNIPAMs, the polymers containing hydrophobic substitution aggregate below the lower critical solution temperature. The hydrophobic alkyl chains of different polymer molecules form the viscous core of these polymeric micelles surrounded by a looser envelope of PNIPAM chains.7,8 The fluorescence spectra of aqueous solutions of the pyrene-labeled polymers studied here all present contributions from excimer and monomer emission. The formation of pyrene excimers can be a measure of the spatial proximity of pyrene groups in a restricted environment. In homogeneous solution, excimers are formed by the diffusional interaction of a singlet excited pyrene with a ground-state molecule.9 However, in confined environments two ground-state pyrene molecules can be close enough so that they can be viewed as dimers; i.e., (7) Ringsdorf, H.; Simon, J.; Winnik, F. M. Macromolecules 1992, 25, 7306. (8) Winnik, F. M.; Davidson, A. R.; Hamer, G. K.; Kitano, H. Macromolecules 1992, 25, 1876. (9) Birks, J. B. Rep. Prog. Phys. 1975, 38, 903.
© 1997 American Chemical Society
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Figure 1. Structure of the PNIPAM polymers.
excimer emission will be virtually instantaneous ( 0.927), but the quenching efficiency for each species varied (Figure 4). The overall dynamic quenching expressed as a ratio of intensities, (I0/I)dyn, was calculated employing eq 4 (Table 3). In the case of PNIPAM-C18Py the lifetime for the growth of the excimer emission did not change in the presence of nitromethane. For this reason, this lifetime component was not included in the calculations of the overall dynamic quenching. In the absence of static quenching the values of I0/I (steady-state measurement) and (I0/I)dyn (time(20) Winnik, F. M. Macromolecules 1990, 23, 1647.
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Table 3. Efficiencies of Quenching of Pyrene-Labeled PNIPAM Polymers by Nitromethanea polymer PNIPAM-C18-Py PNIPAM-C18-Py PNIPAM-C18Py PNIPAM-C18Py b-PNIPAM-C18Py b-PNIPAM-C18Py
monomer excimer monomer excimer monomer excimer
(KSV + Keq)b (M-1)
KSVb (M-1)
Keq (M-1)
percent static quenching/%
87 ( 5 (2) 40 ( 5 (2) 40 ( 4 (2) 27 ( 1 (2) 37 ( 4 (3) 12 ( 1 (3)
33 ( 13 (4) 22 ( 5 (4) 23 ( 12 (3) 8 ( 6 (2) 23 ( 3 (2) 6 ( 1 (2)
54 ( 14 18 ( 7 17 ( 13 19 ( 6 14 ( 5 6(1
62 ( 16 45 ( 18 43 ( 33 70 ( 22 38 ( 14 50 ( 9
a The static quenching expressed as K was obtained from the difference between the overall quenching efficiency (K eq SV + Keq) measured in steady-state experiments and the dynamic quenching (KSV) obtained from time-resolved experiments. b The number in parentheses corresponds to the number of experiments performed. For experiments performed twice, the errors were calculated as average deviations, while, for experiments performed more than twice, the errors correspond to standard deviations.
Figure 5. Schematic representation of the polymeric micelles.
resolved measurement) should be the same. For all polymers a smaller slope was observed for the dependence of (I0/I)dyn on nitromethane concentration, indicating that static quenching played a role in the excimer and monomer quenching processes (Figure 3 and Table 3). Discussion The observation of excimer emission in the pyrenelabeled PNIPAM fluorescence spectra indicates that two, or more, pyrene molecules are located in close proximity. From previous studies, there is strong evidence that the incorporation of pyrene groups along the PNIPAM chain occurred randomly and that the pyrenes are part of multipolymeric micelles.11,13,21 In the case of the endlabeled sample, b-PNIPAM-C18Py, if only one polymer chain was responsible for micelle formation, no excimer fluorescence should have been observed.14 The pyrene molecules which lead to excimer emission are probably located in an environment where the volume they explore is relatively small, since no growth or a short-lived growth (PNIPAM-C18Pys20 ns) was observed for the excimer emission. The structure of the polymeric micelles will be primarily determined by the position of the alkyl chains on the PNIPAM framework. A schematic representation of these micelles is given in Figure 5. For PNIPAMC18-Py the alkyl chains are located on different monomers of the PNIPAM framework than the pyrene, and the alkyl chains can form microdomains that do not contain pyrene. Pyrene groups will be distributed randomly, and microdomains containing two or more pyrene molecules will lead to excimer emission. The fact that monomer and excimer emission decays could be fitted by a smaller (21) Winnik, F. M. Macromolecules 1990, 23, 233.
number of exponentials for this copolymer, compared to the cases for the other PNIPAMs, also suggests that the distribution of chromophores in PNIPAM-C18-Py is relatively homogeneous. It is important to note that the molar content of alkyl chains is slightly higher for PNIPAM-C18-Py and that this copolymer should have a larger capacity for micelle formation. PNIPAM-C18Py has a slightly smaller content of alkyl chains than PNIPAM-C18-Py but they are also randomly distributed on the polymer framework. However, for PNIPAM-C18Py the pyrene moiety is always located close to the alkyl chains, and most of these moieties will be located in the polymeric micelles. As a result the IE/IM values are higher for PNIPAM-C18Py than for PNIPAM-C18-Py. The end substitution of b-PNIPAM-C18Py leads to the highest IE/ IM values observed, indicating that the structures of these micelles are different from those formed for the randomlysubstituted PNIPAMs. Nitromethane has previously been employed for quenching studies in microheterogeneous systems other than polymers,19,22 and a decrease in the quenching efficiency was observed for probes incorporated in the organized assemblies. However, no data on the partitioning of nitromethane are available. Static quenching was observed for the monomer and excimer emission of all copolymers. This mechanism occurs when pyrene and nitromethane are solubilized within the same microdomain in the copolymers. Thus, the values for Keq are proportional to the partitioning of nitromethane between the aqueous and polymeric phases. We cannot further evaluate the magnitude of the Kaq values derived from the static quenching, since we do not know if nitromethane just partitions between different phases, i.e. aqueous and polymeric phases, or if the polymeric micelles provide domains for binding. In any event, since static quenching is significant, we have to take it into account before analyzing the accessibility, which is related to dynamic quenching, of the quencher to the pyrene moieties in the PNIPAMs. A measure of the degree of accessibility of the quencher to the probe in the polymeric micelles can be obtained when the quenching rate constants (kq) are compared to the value for this rate constant in the homogeneous phase (kq°). The values of kq for all PNIPAMs in methanol are only a factor of 1.2 lower than the quenching rate constant for pyrene in this solvent. This result indicates that the quenching rate constant is not affected significantly when no polymeric micelles are formed, as is the case in methanol. On the basis of the similar quenching rate constants in methanol, we will adopt the quenching rate constant measured for pyrene in water (8 × 109 M-1 s-1) as the value for kq° for the pyrene monomer and excimer emissions in PNIPAMs. In order to obtain a rate constant from the Stern-Volmer constant corresponding to the (22) Chen, M.; Gra¨tzel, M.; Thomas, J. K. J. Am. Chem. Soc. 1975, 97, 2052.
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Table 4. Average Quenching Rate Constants for the Pyrene Monomer and Excimer Emission in PNIPAMs and Ratio for the Quenching in the Homogenous Phasea polymer PNIPAM-C18-Py PNIPAM-C18Py b-PNIPAM-C18Py a
〈kq〉 (×109 M-1 s-1)
kq°/〈kq〉
0.5 0.3 0.6 0.1 1.6 0.6
16 27 13 80 5 13
monomer excimer monomer excimer monomer excimer
kq° ) 8 × 109 M-1 s-1.
dynamic quenching, we have to know the lifetime of the fluorophore in the absence of quencher. The decays of the PNIPAMs are in most cases not monoexponential, and the quenching for each individual lifetime was different. We are employing an average lifetime in order to calculate average values of 〈kq〉. This is preferable over calculating a quenching rate constant for each lifetime, since the number of exponentials for each fit may not have a direct correlation to different environments in the polymeric micelles. The 〈kq〉 values (Table 4) for monomer and excimer quenching were calculated from KSV (Table 3), and the average lifetime values were calculated by
〈τ〉 )
∑Aiτi ∑Ai
(5)
where Ai is the pre-exponential factor for the species with lifetime τi. While the excimers can be expected to be protected from quenching by the surrounding polymer chains, the pyrene monomers may either be located in protected microdomains within micelles or be exposed to the aqueous phase. In the latter case one would expect a ratio for the quenching rate constants (kq°/〈kq〉) smaller than 1.5, as was observed for the quenching in methanol. For all PNIPAMs studied the monomer is protected from nitromethane quenching, indicating that the monomers are located within the polymeric micelles. The protection efficiencies are similar for PNIPAM-C18-Py and PNIPAM-C18Py, suggesting that the randomly distributed alkyl chains of the polymer framework form micelles
with similar structures. However, for b-PNIPAM-C18Py the protection efficiency is much smaller than that for the other two polymers. This effect could be explained by a micellar structure that is much less compact, since each polymer has only one alkyl chain. For all polymers the quenching efficiency of the excimers by nitromethane is smaller than that observed for the respective monomers, suggesting that the excimers are located in environments that are better protected by the polymer from the nitromethane in the aqueous solution. In addition, the kq°/〈kq〉 value for PNIPAM-C18Py is significantly higher than that for PNIPAM-C18-Py. In the former PNIPAM the pyrene moieties are bound close to the alkyl chains, and the better protection from quenching indicates that this molecular arrangement leads to more compact micelles for the excimers, in contrast to the observation for the pyrene monomers. The behavior for b-PNIPAM-C18Py excimers parallels the behavior observed for the quenching of the monomer; i.e., the value for kq°/〈kq〉 is lower than that for the other PNIPAMs. In conclusion, the quenching experiments with nitromethane have shown that both the pyrenes leading to monomer and excimer emission are located within polymeric micelles and that the access by the quencher is restricted. The structures of the micelles formed for PNIPAMs randomly substituted with alkyl groups are more compact, leading to a higher protection from the quencher in the aqueous phase. In the case of the end group substitution for b-PNIPAM-C18Py, the protection of the excited state pyrene is smaller, although a higher relative emission intensity for the excimer was observed. These facts indicate that for b-PNIPAM-C18Py the micelles provide an easier access for nitromethane although most micelles contain enough pyrene molecules for excimers to be observed. Acknowledgment. F.M.W. and C.B. would like to thank NSERC (Natural Sciences and Engineering Research Council of Canada) for support of their research programs, through their respective research grants. A.A. thanks NSERC for a summer student award, and T.C.B. thanks CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, Brazil) for a postdoctoral fellowship. The authors at the University of Victoria thank L. Netter for supports in software development. LA970477X
