The Effect of Micelles on the Steady-State and Time-Resolved Fluorescence of Indole, 1-Methylindole, and 3-Methylindole in Aqueous Media

3-Methylindole (skatole) is a component of animal waste and is, consequently, a primary component in odor problems arising in livestock management, notably swine production. The ability to probe and to exploit the interactions of 3-methylindole with micelles has important implications for monitoring and controlling odor problems. The effect of a surfactant (Brij-35) on the fluorescence properties of indole, 1-methylindole, and 3-methylindole in aqueous solutions is reported. Steady-state fluorescence spectra reveal a blue shift in the emission as the surfactant concentration is increased, while the absorption spectra are practically unaffected. Time-resolved fluorescence measurements reveal shorter average lifetimes for 3-methylindole (3-MI) as the Brij-35 concentration is increased. The fluorescence decay of 3-MI in water is described well by a single exponential, whereas, at the highest Brij-35 concentration, a triple exponential is necessary to describe the fluorescence decay. The contributions of each component in the fluorescence decay are used to determine the extent of 3-MI partitioning into the micelle phase. It is found that 93% of the 3-MI molecules partition into the micelle at the highest Brij-35 concentration used. The equilibrium constant for the association between the micelles and the 3-MI molecules is determined to be 2.6 × 104 M-1. In addition, the reduction of 3-MI in the vapor phase by addition of a dry surfactant, lecithin, is also demonstrated. Disciplines Analytical Chemistry | Chemistry Comments Reprinted (adapted) with permission from Analytical Chemistry 69 (1997): 1925, doi: 10.1021/ac9611632. Copyright 1997 American Chemical Society. This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/chem_pubs/689 The Effect of Micelles on the Steady-State and Time-Resolved Fluorescence of Indole, 1-Methylindole, and 3-Methylindole in Aqueous Media K. D. Ashby, K. Das, and J. W. Petrich* Department of Chemistry, Iowa State University, Ames, Iowa 50011-3111 3-Methylindole (skatole) is a component of animal waste and is, consequently, a primary component in odor problems arising in livestock management, notably swine production. The ability to probe and to exploit the interactions of 3-methylindole with micelles has important implications for monitoring and controlling odor problems. The effect of a surfactant (Brij-35) on the fluorescence properties of indole, 1-methylindole, and 3-methylindole in aqueous solutions is reported. Steady-state fluorescence spectra reveal a blue shift in the emission as the surfactant concentration is increased, while the absorption spectra are practically unaffected. Timeresolved fluorescence measurements reveal shorter average lifetimes for 3-methylindole (3-MI) as the Brij-35 concentration is increased. The fluorescence decay of 3-MI in water is described well by a single exponential, whereas, at the highest Brij-35 concentration, a triple exponential is necessary to describe the fluorescence decay. The contributions of each component in the fluorescence decay are used to determine the extent of 3-MI partitioning into the micelle phase. It is found that 93% of the 3-MI molecules partition into the micelle at the highest Brij-35 concentration used. The equilibrium constant for the association between the micelles and the 3-MI molecules is determined to be 2.6 × 104 M-1. In addition, the reduction of 3-MI in the vapor phase by addition of a dry surfactant, lecithin, is also demonstrated. 3-Methylindole (skatole) is an important component of animal waste. Because of its noxious odor, 3-methylindole is a primary problem in livestock management, especially in swine production.1-7 In this article, we consider the effect of surfactants on the steadystate and time-resolved fluorescence of indoles in the aqueous and vapor phases. Micelles can be used as model, and perhaps practical, systems capable of trapping small molecules such as indoles. Understanding under what conditions 3-methylindole can be sequestered and developing means of detecting it are of great practical interest. The fluorescence properties of indoles (Figure 1a-c) are extremely sensitive to the environment. This sensitivity arises from the presence of two closely spaced excited singlet states (Figure 2), which are traditionally denoted La and Lb. The energy of the Lb state is insensitive to the solvent, and it is the lower lying of the two excited singlet states in nonpolar solvents. In nonpolar solvents, Lb is the emissive state.16 On the other hand, the La state interacts strongly with the solvent. In nonpolar media it lies above Lb, but in polar media the La state strongly interacts with the solvent and subsequently lowers its energy relative to that of Lb, thus becoming the emissive or fluorescent state. Because of the interaction of these two excited states with their environment (and with each other), shifts of emission spectra to higher energies (blue shifts) are excellent signatures of the probe molecule moving to a nonpolar environment.17,18 Also, as the indole moves to a nonpolar environment, the fluorescence lifetime shortens, due largely to the different radiative properties of the Lb state with respect to those of the La state15 (Figure 2). Organic molecules are generally hydrophobic in nature. The interior of micelles consists of a nonpolar region, which offers an ideal environment into which organic molecules may partition from the aqueous phase. Consequently, the micelle phase can induce substantial changes in the fluorescence properties of (1) Muehling, A. J. Swine Housing and Waste Management; Department of Agricultural Engineering, College of Agriculture, University of Illinois: UrbanasChampaign, IL, 1969; pp 65-85. (2) Smith, A. T.; Lawrence, T. L. Pig Housing and the Environment; BSAP Occasional Publication No. 11; The British Society of Animal Production: Edinburgh, UK, 1987; pp 41-51. (3) Nielson, V. C.; Voorburg, J. H.; La’Hermite, P. Volatile Emissions From Livestock Farming and Sewage Operations; Elsevier Science Publishing Co., Inc.: New York, 1988; pp 54-58. (4) Leffel, R. E.; Parthum, C. A. Odor Control for Wastewater Facilities; Manual of Practice No. 22; Lancaster Press Inc.: PA, 1979; pp 6-12. (5) Jensen, M. T.; Cox, R. P.; Jensen, B. B. Appl. Environ. Microbiol. 1995, 61, 3180-3184. (6) Jensen, M. T.; Cox, R. P.; Jensen, B. B. Anim. Sci. 1995, 61, 293-304. (7) Hansen, L. L.; Larsen, A. E.; Jensen, B. B.; Hansen-Møller, J.; Barton-Gade, P. Anim. Prod. 1994, 59, 99-110. (8) Platt, J. R. J. Chem. Phys. 1951, 19, 101-111. (9) Strickland, E. H.; Horwitz, J.; Billups, C. Biochemistry 1970, 25, 49144921. (10) Callis, P. R. J. Chem. Phys. 1991, 95, 4230-4240. (11) Valeur, B.; Weber, G. Photochem. Photobiol. 1977, 25, 441-444. (12) Ruggiero, A. J.; Todd, D. C.; Fleming, G. R. J. Am. Chem. Soc. 1990, 112, 1003-1014. (13) Eftink, M. R.; Selvidge, L. A.; Callis, P. R.; Rehms, A. A. J. Phys. Chem. 1990, 94, 3469-3479. (14) Rich, R. L.; Chen, Y.; Neven, D.; Negrerie, M.; Gai, F.; Petrich, J. W. J. Phys. Chem. 1993, 97, 1781-1788. (15) Meech, S. R.; Phillips, D.; Lee, A. G. Chem. Phys. 1983, 80, 317-328. (16) This statement must be qualified. The La and Lb levels are typically closely spaced, so the lower state can thermally populate the higher one. Furthermore, on a fast (subpicosecond) time scale, the emission from the higher lying state is detectable. These two phenomena contribute to the low limiting anisotropy values of most indoles.12 (17) Eftink, M. R.; Ghiron, C. A. J. Phys. Chem. 1976, 80, 486-493. (18) Hill, B. C.; Horowitz, P. M.; Robinson, N. C. Biochemistry 1986, 25, 22872292. (19) Lakowicz, J. R.; Keating, S. J. Biol. Chem. 1983, 258, 5519-5524. Wimley, W. C.; White, S. H. Biochemistry 1993, 32, 6307-6312. Anal. Chem. 1997, 69, 1925-1930 S0003-2700(96)01163-8 CCC: $14.00 © 1997 American Chemical Society Analytical Chemistry, Vol. 69, No. 10, May 15, 1997 1925 indoles. In the nonpolar micelle phase, the indole emits from the Lb state, whose fluorescence will be blue shifted and shorter lived with respect to those of molecules in the polar aqueous phase having no interaction with the micelle and which emit from the La state. The chromophore may also be located between the inner core of the micelle and the aqueous phase (water/micelle boundary),20,21 which would be expected to afford different fluorescence behavior which is intermediate to those of the two just mentioned (Figure 3). The distinct fluorescence properties of the indoles in these different environments permit their association with the micelle to be quantified. Given the biological importance of indoles, that indole is the chromophoric moiety of the amino acid tryptophan, and the considerable effort that has been devoted to understanding their fluorescence properties and exploiting these properties as probes of environment (e.g., see refs 22-25), it is remarkable that, with only a few exceptions of which we are aware (e.g., see refs 1719 and 22), quantitative investigations of the interactions of indoles with micelles have been ignored. (20) Schick, M. J. Nonionic Surfactants; Surfanctant Science Series 1; Marcel Dekker, Inc.: New York, 1966; Chapter 17. (21) Shinoda, K.; Nakagawa, T.; Tamamuchi, B.; Isemura, T. Colloidal Surfactants; Academic: New York, 1963; p 140. (22) Beecham, J. M.; Brand, L. Annu. Rev. Biochem. 1985, 54, 43-71. (23) Creed, D. Photochem. Photobiol. 1984, 39, 537-562. (24) Szabo, A. G.; Rayner, D. M. J. Am. Chem. Soc. 1980, 102, 554-563. (25) Petrich, J. W.; Chang, M. C.; McDonald, D. B.; Fleming, G. R. J. Am. Chem. Soc. 1983, 105, 3824-3832. Figure 1. Structures of (a) indole, (b) 1-methylindole, (c) 3-methylindole, and (d) Brij-35 (poly(oxyethylene)23 lauryl ether). (e) A general chemical structure of lecithin (phosphatidylcholine). The long-chain fatty acid groups, by convention, occupy positions 1 and 2 of the glycerol bridge, while the phosphorylcholine headgroup occupies position 3. The hydrocarbon chains vary in length and saturation, with the more unsaturated chain in position 2, as represented by the double bond in the chain at this position. Figure 2. Representation of the two low-lying excited states of indole and its derivatives and the effect of solvent on the fluorescence. The figure is adapted from that in the article by Meech et al.15 In nonpolar solvents, the Lb state is the lower lying of the two excited states. Consequently, it is the fluorescent state. The La state is very sensitive to solvent polarity, whereas the Lb state is not. As the polarity of the solvent increases, the energy of the La state lowers, and it can become the fluorescent state. The specific behavior of the fluorescence lifetimes of the Lb and the La states is determined largely by their radiative rates. Meech et al. note that the increase of fluorescence lifetime of 3-MI with increasing polarity cannot be attributed to the lowering of the energy of the La state but must take into account nonspecific interactions with the solvent that can arise because the dipole moment of the La state is greater than that of the Lb state. The ability of the La state to lower its energy in polar solvents is referred to as “solute-solvent interaction” (SSR). The ability of the La state to interact with the solvent in such a way as to modify its radiative properties is referred to as “internal charge transfer” (ICT). These processes are denoted in the figure. Figure 3. Schematic drawing of a Brij-35 micelle and possible depictions of the indole-micelle complex. The indole chromophore could be located within the nonpolar core of the micelle, solubilized between the inner core and the water phase in the hydrated oxyethylene phase or in the aqueous phase with no interaction with