Environ. Sci. Technol. 1982, 16, 613-616
Sims, J. R.; Bingham, F. T. Soil Sei. SOC.Am. Proc. 1968, 32, 364. Sims, J. R.; Bingham, F. T. Soil Sci. SOC.Am. Proc. 1968, 32, 369. Keren, R.; Gast, R. G.; Bar-Yosef, B. Soil Sei. SOC.Am. J . 1981, 45, 45. Keren, R.; Gast, R. G. Soil Sei. SOC.Am. J . 1981,45, 478. Mezuman, U.; Keren, R. Soil Sei. SOC.Am. J . 1981,45,722.
(25) Adams,R. M., Ed. “Boron, Metallo-Boron Compounds and Boranes”; Interscience: New York, 1964. Received for review June 30,1981. Revised manuscript received April 9,1982. Accepted May 3, 1982. Appreciation is extended to the United States Environmental Protection Agency for partial support of this project through Grant R-805935.
Fluorescence Polarization Studies of Perylene-Fulvic Acid Bindingt Paul M. Roemelt* and W. Rudolf Seltz”
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824
w When fulvic acid is added to perylene solutions, perylene fluorescence becomes more polarized. By measuring perylene polarization as a function of added fulvic acid, we have determined an equilibrium constant of 1.5 X lo6 for the binding of perylene to fulvic acid in 75% glycerol. In 40% glycerol perylene forms aggregates. From fluorescence polarization and scattering data, we show that the addition of fulvic acid causes perylene aggregates to disperse into smaller units. However, fulvic acid does not completely solubilize perylene in 40% glycerol, even when present in large excess. The interaction of perylene and fulvic acid in water is thought to be similar to the behavior in 40% glycerol. The interaction of perylene and fulvic acid is the same within experimental error at pH 2,7, and 11in both 40% and 75% glycerol. This study illustrates how fluorescence polarization may be used to follow the interaction of natural organic substances with organic pollutants. Fluorescence polarization is a particularly attractive method for this type of study because it can distinguish free and bound organic substances without a separation.
Introduction Humic substances have been the subject of considerable interest because they can interact with pollutants in soil and water. In particular, the nature and strength of metal complexes with humic substances have been studied because the formation of these complexes may influence the transport and fate of heavy metals. Humics can also interact with organics. The adsorption of s-triazines onto soil humic matter has been widely studied because soil humics modify the herbicidal activity of s-triazines (1-9). Other compounds that have been shown to interact with humic substances include DDT (10-12), bipyridilium herbicides (13-14), PCBs (15),carbaryl and parathion (16, 17))cationic dyes (18))2,4-D and picloran (19),and linuron and malathion (20). Various methods have been used to study the interaction of organic compounds with humics. The most common approach has been to add a known amount of the organic compound to a suspension of undissolved humics. After equilibration, the suspended material is removed by centrifugation, and the amount or organic compound remaining in the supernatant is measured. Free and bound organic compound have also been distinguished by gel chromatography (14) and solvent extraction (2). Most Contribution No. UNH-SG-JR-137. Knolls Atomic Power laboratory, General Electric Co., Schenectady, NY 12301
* Present address:
0013-936X/82/0916-0613$01.25/0
studies have measured adsorption isotherms although it has been shown that dissolved humics substantially increase the solubility of otherwise highly insoluble organic pollutants (10). The interaction of dissolved humics with organic pollutants is a matter of considerable importance. By significantly enhancing the solubility of organic pollutants, humics may modify their transport as well as influence degradation processes. This interaction is, however, quite difficult to study. Methods involving a physical separation of free and bound organic compound are both tedious and highly subject to error due to adsorption of the organic compound to container surfaces. The primary purpose of the work reported here is to demonstrate the use of fluorescence polarization to study the interaction of organic pollutants with humic substances. Fluorescence polarization offers two major advantages for this type of study. It greatly reduces the probability of error because it can distinguish free and bound organic compound without a separation. Also, because of the sensitivity of fluorescence, it is possible to make measurements at the low concentration ranges that occur naturally. Fluorescence polarization has been used to distinguish adsorbed and dissolved polynuclear aromatic hydrocarbons (21) and to study the conformation of soil fulvic acid (22). However, it has not otherwise been applied to environmental chemistry even though it is widely used in the study of proteins and synthetic polymers. The principles of fluorescence polarization measurements are reviewed elsewhere (23-25). A fluorophor is excited with polarized light. Fluorescence intensities are measured in the planes parallel and perpendicular to the excitation radiation, I and I, , respectively. The polarization, p , is calculatedl from these values:
In the absence of depolarizing processes the value of p depends on the orientation of the moments ofdhe transitions involved in excitation and emission. Values range from l/z to -1/3. Under the conditions of this study fluorescence is depolarized by molecular rotation during the lifetime of the excited state, causing a decrease in the measured value of p . The extent of rotational depolarization depends on several factors, including temperature, viscosity, fluorescence lifetime, and molecular size and shape. When a fluorophor is bound to another molecule, its effective size increases and it rotates more slowly. Thus, the polarization value for the bound fluorophor is higher than for the free
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fluorophor. If the polarizations for free and bound fluorophors are known, then the observed polarization can be used to calculate the relative amounts of free and bound fluorophor. We chose to study the binding of perylene to a soil fulvic acid. Perylene is an efficient fluorophor. Because perylene is a polynuclear aromatic hydrocarbon, it is representative of an important class of organic pollutants that enter the aquatic environment via oil spills and emissions accompanying the burning of fossil fuels.
Experimental Section Instrumentation. The polarization fluorometer is a home-built two-detector instrument described in more detail elsewhere (26). For this study the tungsten halogen lamp was replaced by a Canrad-Hanovia 150-W xenon lamp. The excitation wavelength was set at 412 nm by an Aminco grating monochromator with variable entrance and exit slits. The emission wavelength was selected by cutoff filters cutting off at 435 nm. Standard type 1Polaroid f i i polarizers were placed in both the excitation and emission beams. The detectors were RCA 1P21 photomultiplier tubes mounted at right angles to the excitation beam. This arrangement allows simultaneous measurement of Illand II. The photomultiplier currents are converted to voltages by two operational amplifier circuits and are fed directly to a DEC MINC-11 minicomputer for data analysis and storage. Fluorescence spectra were measured on a Perkin-Elmer 204 low-resolution spectrofluorometer. This instrument was also used to measure scatter. Absorption measurements were made on a Cary 219 spectropotometer. Reagents. The soil fulvic acid used in these experiments has been extensively characterized by Weber and co-workers (27-29). It was prepared by extracting the B2 horizon of a podzol soil (Conway, NH) with base. A dissociation-corrected number average molecular weight of 990 has been determined by cryoscopy (30). Perylene was obtained from Aldrich C'remical Co., Inc. The solvents used in these experimer 4 were distilled and deionized water, anhydrous glycerc I, reagent grade methanol, and benzene. A 3.22 X lo4 M aqueous stock solution of fulvic acid was prepared by dissolving 0.0319 g of dried fulvic acid in distilled and deionized water and diluting to 100 mL. A stock solution of perylene was prepared by dissolving 0.1236 g in 100 mL of benzene. A 1-mL portion of this solution was diluted to 100 mL with methanol. This solution was in turn used to prepare the final perylene solutions. The final solutions contain 0.1% methanol and 0.001 % benzene. Phosphate buffers at pH 2, 7, and 11 were prepared accordng to directions given in the CRC Handbook of Chemistry and Physics. Experiments involving glycerol were designed so that an appropriate amount of glycerol was weighed into a scintillation vial. Final solutions were prepared from the above stock solutions to a volume of 20.00 mL in scintillation vials. Fulvic acid and perylene stock solutions were added by use of microburet syringes calibrated to 5.00 pL/division with a syringe microburet. Buffer solutions were added by using a 10-mL calibrated buret. The buffer concentrations were 0.06 M at 40% glycerol and 0.025 M at 75% glycerol. All final solutions were thoroughly mixed. A fluorescence standard solution was prepared by dissolving 0.0041 g of 2,7-dichlorofluorescein and one pellet of sodium hydroxide in 100 mL of water and diluting by a factor of 1000. This solution was used to check instru614
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Flgure 1. Perylene polarization as a function of added fulvic acid in 75% glycerol (4.9X lo-' M perylene): (a) pH 2; (b) pH 7; (c) pH 11.
ment stability during each experiment. Procedures. In making fluorescence polarization measurements the following procedures were followed. Monochromator slit widths were adjusted to get an intensity yielding an output voltage in the range 1-5 V. With a fluorescent sample in the fluorometer, the excitation polarizer was oriented horizontally. In this case, both detectors measure 11,and the relative gains of the two detectors can be established. This must be accounted for when calculating the polarization. The excitation polarizer was then oriented vertically, and Iand llI , were measured for the perylene-containingsample and a blank. The blank is identical in composition with the sample except for perylene. The blank values are subtracted from the total signal. The primary sources of the blank signal are fulvic acid fluorescence and scattering. Because fulvic acid is an inefficient fluorophor, its fluorescence is only significant when it is present in large excess relative to perylene.
Results Effect of Fulvic Acid on Perylene Polarization in 75% Glycerol. Figure 1shows the polarization of perylene fluorescence as a function of added fulvic acid at pH 2,7, and 11in 75% glycerol. In all three curves, the polarization increases with added fulvic acid as expected if perylene
acid causes a 5-fold decrease in scattering. The fluorescence spectra for perylene in 40% glycerol are the same in the presence and absence of fulvic acid when measured on a low-resolution spectrofluorometer with a 10-nm bandbass.
,1301
Discussion Binding of Perylene by Fulvic Acid in 75% Glycerol. The increase in perylene polarization when fulvic
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FULVIC ACID CONCENTRATION ( X 1 0 6 M I
Flgure 2. Perylene polarization as a function of added fulvic acid in 40% glycerol (4.9 X IO-’ M perylene): pH 2.
Table I. Perylene PolarizationQ as a Function of Added Fulvic Acid in 40% Glycerol: Relative Intensities (pH 2 ) [fulvic acid], ZllIllZl11M (total) (blank) (total) (blank) P* 2.705 2.095 0.1271 3.151 0.076 2.456 0.071 0.1264 6.0 X 1.2 X 3.349 0.130 2.649 0,099 0.1160 2.4 X 3.512 0.294 2.735 0.174 0.1137 3.864 0.625 2.993 0.388 0.1085 6.0 X 2.4 X 4.386 1.879 3.216 1.150 0.0964 4.558 2.249 3.318 1.406 0.0941 3.0 X a The polarization is calculated after substracting Zll(b1ank) and Zl(b1ank) from Zll(tota1) and Zl(total), respec tively.
binds to a larger molecule. The intensity of perylene fluorescence is not affected by the added fulvic acid. The higher percentage of glycerol serves two purposes. It increases the solubility of perylene. Perylene was soluble at 4.9 X M in 75% glycerol as evidenced by the electronic absorption spectrum of perylene in this medium. Also, because glycerol is highly viscous, it slows down the rotation of perylene and leads to higher values for the polarizations. In a less viscous medium, both free and bound perylene would rotate so rapidly that fluorescence would be essentially completely depolarized. In this situation the polarization changes are so small that the binding reaction is very difficult to follow. Effect of Fulvic Acid on Perylene Polarization in 40% Glycerol. Figure 2 shows the polarization of perylene fluorescence as a function of added fulvic acid at pH 2 in 40% glycerol. Similar data were obtained at pH 7 and 11. The intensity of perylene fluorescence is an order of magnitude lower in 40% glycerol than in 75% glycerol. The addition of fulvic acid causes a small but distinct increase in perylene fluorescence. This is observed in Table I, which lists the raw data for Figure 2. This table also shows the magnitude of the fulvic acid blanks relative to perylene fluorescence. The decrease in meausred fluorescence intensity from perylene at high fulvic acid concentrations is due to an “inner filter” effect, i.e., the fact that the fulvic acid is absorbing a significant fraction of the excitation radiation. (This effect does not influence the polarization values because Il and l I, are affected equally.) Perylene is not soluble at the 4.9 X M level in 40% glycerol. The electronic absorption spectrum does not show the expected perylene absorption bands. The solution of 4.9 X M perylene in 40% glycerol scatters light much more efficiently than 4.9 X M perylene in 75% glycerol. The addition of excess fulvic
acid is added indicates that perylene binds to fulvic acid. The fact that polarization is constant at fulvic acid levels above 3 X lo4 M indicates that the binding is complete at this point. The relatively small overall change in polarization suggests that the reaction involves one perylene molecule/fulvic acid molecule. If a larger species were formed, we would expect a larger change in polarization. The basic equation for calculating equilibrium constants from polarization data is
where Fb/Ff is the relative amount of bound and free fluorophor, Qb/Qf is the fluorescence efficiency of bound fluorophor relative to free fluorophor, Pf and P b are the polarizations for free and bound fluorophor, respectively, and P is the observed polarization. Since the fluorescence intensity from perylene is not affected by added fulvic acid, the Qb/Qf term must be 1. In this case we can calculate a binding constant for the perylene-fulvic acid reaction directly from the polarizations:
where CSFA and,,C are the total concentrations of fulvic acid and perylence, respectively. The following table lists the binding constants for perylene-fulvic acid in 75 % glycerol from the point where P - Pf = P b - P. pH 2 7 11
‘
binding constant 1.2 x 106 1.8 X lo6 1.5 X l o 6
This is the point where one would expect the greatest precision in the binding constant calculation. The values at the three pHs are the same within the precision of the measurement. The magnitude of the binding constant is quite large. Since the binding constant is a measure of the affinity of the binding molecule for the solute relative to the solvent, the binding constant in water would be considerably larger still. Thus the binding of hydrophobic organic pollutants by natural organic matter is likely to be a significant process in natural systems. The magnitude of the constant for perylene binding is consistent with the observation that the fraction of organic matter in soil correlates strongly with the soil’s tendency to adsorb organic pollutants (31). We also infer structural information from the binding constants. The fact that the binding constant is so large confirms that there are large hydrophobic regions in fulvic acid that provide a stable surface for a highly hydrophobic moelcule such as perylene to associate with. The fact that binding constants are similar at pH 2,7, and 11indicates that the ionization of carboxylate and phenolic OH does not affect the ability of the hydrophobic regions of fulvic Environ. Sci. Technol., Vol. 16,
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acid to associate with hydrophobic organics. The change in perylene polarization upon binding to fulvic acid is greatest at pH 2 and smallest at pH 11. This is consistent with the fact that the conformation of fulvic acid in solution is different at the three pHs (32). However, the conformation of fulvic acid is known to be more spread out at high pH when there are negatively charged carboxylate groups than at low pH. On this basis one would expect fulvic acid to impede perylene rotation at high pH to a greater extent than at low pH. We suspect that this apparent anomaly may arise due to greater internal flexibility in the spread conformation of fulvic acid at high pH. The internal motions of one part of fulvic acid relative to another may be contributing to the depolarization of perylene fluorescence. This would also explain why the polarization changes are relatively small at all three pHs. Interaction of Fulvic Acid with Perylene in 40% Glycerol. Because the solubility of perylene is exceeded, perylene exists as an aggregate in this medium. Perylene molecules associate with each other to form a larger species. The fact that 4.9 X lo-’ M perylene in 40% glycerol scatters light more efficiently than 4.9 X lo-’ M perylene in 75% glycerol is evidence for this. This is also consistent with the high polarization value for perylene in 40% glycerol. This value is higher than the polarization in 75% glycerol even though 75% glycerol has a viscosity of 22.5 CPat 20 “C compared to 3.7 CPfor 40% glycerol at 20 “C. The addition of fulvic acid causes a decrease in both polarization and scattering. The fulvic acid causes the relatively large perylene aggregates to disperse into smaller species. These are probably smaller aggregates. They are almost certainly coated with the hydrophobic part of the fulvic acid while the polar functional groups of the fulvic acid preferentially associate with the solvent, thus forming a micelle. As fulvic acid levels increase, the micelle size decreases. Even at the highest levels of fulvic acid, the perylene polarization remains higher and fluorescence intensities remain considerably lower than in 75% glycerol. Because of this we believe that the fulvic acid is not completely solubilizing the perylene even though it is clearly acting as a surfactant to disperse large perylene colloids into smaller units. We are confident that the effect of fulvic acid on perylene in water is similar to the effect in 40% glycerol. Unfortunately, however, perylene fluorescence was so weak in water that we could not demonstrate this experimentally.
Conclusions We consider our most significant accomplishment is to have demonstrated the use of fluorescence polarization to study the interaction of organic pollutants with natural organic matter without a separation. Because of the limitations of our home-built equipment we chose to study an efficient fluorophor, perylene, which can be excited in the visible range. However, it should be possible to study the interaction of nonfluorescent organics by observing changes in the polarization of fulvic acid fluorescence. This will require an instrument designed for precise, sensitive polarization measurements since fulvic acid is not strongly fluorescent and the polarization changes are likely to be small. We have measured a binding constant for the association of perylene with fulvic acid. The magnitude of the binding constant shows that fulvic acid has a very strong affinity for hydrophobic organics.
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We have also demonstrated how fulvic acid acts as a surfactant to break up large perylene colloids into smaller units.
Acknowledgments We thank Tony Lapen for helping to keep our polarization fluorometer operational.
Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
(16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32)
McGlamery, M. D.; Slife, F. W. Weeds 1966, 14, 237. Sullivan, J. D.; Felbeck, G. T., Jr. Soil Sei. 1968, 106, 42. Hance, R. J. Weed Res. 1969, 9, 108. Weber, J. B.; Weed, S. B.; Ward, T. M. Weed Sei. 1969, 17, 417. Dunigan, E. P.; McIntosh, T. H. Weed Sci. 1971,19, 279. Gilmour, J. T.;Coleman, N. T. Soil Sei. SOC.Am. Proc. 1971, 35, 256. Li, G. C.; Felbeck, G. T., Jr. Soil Sei. 1972, 113, 140. Muller-Wegener, U. Geoderma 1977, 19, 227. Khan, A. U. Pestic. Sci. 1978, 9, 39. Wershaw, R. L.; Burcar, P. J.; Goldberg, M. C. Environ. Sei. Technol. 1969, 3, 271. Ballard, T. M. Soil Sei. SOC.Am. Proc. 1971, 35, 145. Pierce, R. H., Jr.; Olney, C. E.; Felbeck, G. T. Geochim. Cosmochim. Acta 1974, 38, 1061. Damanakis, M.; Drennan, D. S. H.; Fryer, J. D.; Holly, K. Weed Res. 1970, 10, 264. Khan, S. U. Can. J . Soil Sei. 1973, 53, 199. Scharpenseel, H. W.; Theng, B. K. G.; Stephan, S. In “Environmental Biogeochemistry and Geomicrobiology”; Ann Arbor Science: Ann Arbor, MI, 1978; Vol. 2, pp 619-637. Leenheer, J. A,; Ahlrichs, J. L. Soil Sei. SOC.Am. Proc. 1971, 35, 700. Bowman, B. T. Soil Sei. SOC.Am. J. 1978, 42, 441. Banerjee, S. K.; Roy, K. B.; Das, S. C. J . Ind. Chem. SOC. 1973, 50, 549. Kahn, S. U. Can. J. Soil Sei. 1973, 53, 429. McNamara, G.; Toth, S. J. Soil Sei. 1970, 109, 234. Von Wandruszka, R. M. A,; Brantley, S. Anal. Lett. 1979, 12, 1111. Lapen, A. J.; Seitz, W. R. Anal. Chim. Acta 1982,134, 31. Weber, G. In “Fluorescence and Phosphorescence Analysis”; Wiley: New York, 1966; pp 217-240. Dandliker, W. B.; de Saussure, V. A. Immunochemistry 1970, 7, 799. Dandliker, W. B.; Dandliker, J.; Levison, S. A.; Kelly, R. J.; Hicks, A. N.; White, J. U. Methods Enzymol. 1978,48, pp 380-415. Roemelt, P. M.; Lapen, A. J.; Seitz, W. R. Anal. Chem, 1980, 52, 769. Weber, J. H.; Wilson, S. A. Water Res. 1975, 9, 1079. Wilson, S. A.; Weber, J. H. Chem. Geol. 1977, 19, 285. Bresnahan, W. T.; Grant, C. L.; Weber, J. H. Anal. Chem. 1978,50, 1675. Weber, J. H., personal communication, 1981. Karickhoff, S. W.; Brown, B. S.; Scott, T. A. Water Res. 1979, 13, 241. Ghassemi, M.; Christman, R. F. Limnol. Oceanogr. 1968, 13, 4.
Received for review December 24,1981. Accepted April 15,1982. This project was funded in part by the Office of Sea Grant, National Oceanic Administration, U.S. Department of Commerce, through Grant No. RITS-58 to the University of New Hampshire. NSF grant CHE7908399provided partial support f o r the Cary 219 spectrophotometer used in this research.
