Environ. Sci. Techno/. 1995, 29, 2162-2165
Solubility Enhmernellt and Fluorescence Quenching of pvnSae by Humic Substances: The Effect of Dissolved Oxygen on Quenching Processes -
KARLIN M. DANIELSEN,' YU-PING CHIN,*,' JEFFREY S. BUTERBAUGH,' TERRY L. GUSTAFSON,+ AND SAMUEL J. TRAINAS Department of Geological Sciences, Department of Chemistry, and School of Natural Resources, The Ohio State University, Columbus, Ohio 43210
Introduction Humic substances play an important role in the fate and transport of nonpolar organic compounds (NOCs). Measuring accurate values of pollutant-humic substance partition coefficients(&c,hs) is central toward understanding the role of these natural materials in the facilitated transport of contaminants in subsurface and surface waters and sedimentary porefluids (1-8). One reliable approach for elucidating accurate values of &c,hs is to measure the apparent aqueous solubility of the organic contaminant in the absence and presence of humic materials (7,s).The solubility enhancement (SE) method, however, requires copious amounts of humic substances (as much as 5 mmol L-l as organic carbon) and may be insensitive to those substances that possess liquid (or subcooled liquid) mole or less (7). fraction solubilities of Recently, fluorescence quenching (FQ) has become a popular technique for measuring polycyclic aromatic hydrocarbons (PAH) &c,hs values (1-6'). This approach is fast and requires only small quantities of sample, but can only be used to measure Koc,hsvaluesfor fluorescent organic compounds. Moreover, fluorescence quenching-derived PAH &hs values reported in the literature are generally larger than those measured using other methods (Le., reverse-phase chromatography, dialysis) (9). Thus, it is plausible that another radiative process (e.g., dynamic quenching) could be responsible for quenching nonpolar fluorescent probes. For example oxygen has been demonstrated to be an efficient quencher for a number of fluorescent substances (particularlyPAHs) (10). The effects of these dynamic quenchers on static processes, however, are presumably accounted for in separate control experiments. In this study, we measured the binding of a PAH probe (pyrene) to a humic acid and a fulvic acid from the Suwannee River in Georgia using both the solubility * To whom correspondence should be addressed. FAX: 614-2927688; e-mail address:
[email protected]. +
Department of Geological Sciences.
* Department of Chemistry.
School of Natural Resources.
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enhancement approach and fluorescence quenching. We have also measured the fluorescence lifetime of our probe in the presence of humic substances under oxic (Le., in equilibrium with the atmosphere) and anoxic conditions. Our objectives were to (a) to compare pyrene-humic substance partition coefficients measured by the SE and FQ techniques and (b) to measure (if any) nonstatic quenching processes using fluorescence lifetime data in an effort to explain any observed differences between FQand SE-derived &c,hs values.
Methods Aquatic humic and fulvic acids were isolated from the Suwannee River (Georgia). Both materials have been thoroughly characterized and have been used by others in pyrene binding studies (1, 2, 5). Studies evaluating the solubility enhancement of pyrene in the presence of humic substances used a modified method originally developed by Chiou and co-workers (7). Briefly, pyrene dissolved in a volatile carrier (acetonitrile)was plated onto the surfaces of a 25-mL Corex centrifuge tube (VWR) using argon gas. Buffered aqueous solutions (1 mmol L-l KH2P04and 1 mmol L-' K2HP04) containing various concentrations of humic material (0-40 mg/L organic carbon) were added to each tube. Subsets of humic solutions were assayed by total organic carbon analysis (Shimadzu TOC 5000) to determine the precise concentrations of humic material in each reactor. Each tube was covered, capped in aluminum foil, and allowed to equilibrate on a reciprocating shaker for 1week. Upon equilibration, the samples were centrifuged at 1000 rcf or greater for 2 h. Our samples were assayed by direct aqueous injection (100 pL sample loop) onto a isocratic reverse-phase HPLC system (Waters Associates) that utilized a UV detector set at a wavelength of 240 nm (Waters). The mobile phase was comprised of 90: 10 methano1:water (vlv). The length and efficiency of the column (Waters Nova-pak) and the composition of the mobile phase coupled with relatively slow flow rates (-1 mL/min) allowed us to completely separate total pyrene from the humic materials. Fluorescence quenching experiments were conducted using the techniques developed by other investigators ( I , 4) on a SLM Aminco SPF 500C scanning fluorescence spectrophotometer (lex= 230 nm, A,, = 373 nm). Saturated pyrene solutions were titrated with a solution of concentrated (71 g/L) humic acid or Mvic acid. A blank was titrated in the same manner to q u a n t a background humic substance fluorescence and a control containing only the probe was titrated with Milli-Q water to ascertain changes in fluorescence caused by dilution effects. Optically matched quartz cuvettes were used and inner-filter effect measurements were conducted on a Cary 1 spectrophotometer at the respective excitation and emission wavelengths of 230 and 373 nm. Finally, the total organic carbon of the titrated blank solution was measured to determine the final concentration of the humic substances in the cuvettes. We used this value to extrapolate the concentration of humic substance in the cuvette after each titration point. Lifetime measurements were conducted using the timecorrelated single-photon counting (TCSPC) method. Ex-
0013-936W95/0929-2162$09.00/0
C 1995 American Chemical Society
TABLE 1
t2
2i
!
0.8 0,00000
t 0.8 0.00001
0.00002
0.00003
Organic Carbon Normalized Humic Substance-Pyrene Partition Coefficients (&,hs) Determined by Fluorescence Quenching and Solubility Enhancement fluorescence quenching
solubility enhancement
sample
(I(oc,hr)
(Koc.ht)
Suwannee River fulvic acid
27540 100000~ 15400-6000’ 33880 31 500-21 500’
10230
O.ooOo4
Suwannee River Humic Acid (KgC L )
FIGURE 1. Binding of pyrene by Suwannee River humic acid as determined by the solubility enhancement and fluorescence quenching methods.
citation near 283 nm was achieved by frequency doubling the output of a synchronously pumped Rhodamine-6Gdye laser (Coherent, 500 kHz). Fluorescence at 373 nm was isolated using an American Holographic DB- 10s subtractive double monochromator (5 nm bandpass) and was detected with a Hammamatsu R2809U-07 microchannel-plate photomultiplier tube. While pyrene exhibits a straightforward single-exponential decay in aqueous solution (i.e., the time-dependent fluorescence intensity, It,is equivalent to Ioe-t’T where Io is the initial fluorescence intensity and z is the lifetime of the fluorescent probe), data analysis was complicated by the presence of intense, multi-exponential fluorescence interference originating from the fulvic and humic acids. Fortunately, the fluorescence lifetimes of humic materials are much shorter than those of pyrene. Consequently, we were able to fit each pyrene decay to a single exponential (plusa constant background)by simply restricting the fitting window to the portion of the decay where the fluorescence contribution of the humic substances was insignificant. This was achieved by collecting a decay for the humic substance alone and noting a channel (time position) at which fluorescence had decayed to near-zero intensity. We then collected decays for pyrene in the presence of various concentrations of the humic substances. The experiments were repeated under anoxic conditions by sparging argon through the sample cell. To achieve adequate signal-tonoise, we collected the fluorescence until -10 000 counts had been detected in our first fitting channel (typically-1 15 ns into the decay). Fits were performed using PeakFit (Jandel Scientific) and weighted according to Poisson statistics.
Results and Discussion The binding of pyrene by aquatic humic substances was measured by both fluorescence quenching and solubility enhancement. We fitted our solubility enhancement data to the model derived by Chiou and co-workers (7, 8): c *
where S ,* and Sw are the respective solubility of pyrene in the presence and absence of humic substances and [HS] is the humic substance concentration expressed as organic carbon (kglL). Fluorescence quenching data were fitted to the static version of the Stern-Volmer eq (1):
As
Suwanee River humic acid Ref 1. Ref 5.
---+
0
21380
.. A
@Saturated
Ar Saturated
F 120 .-. 0.odooo
0.odOol
O.odo02
O.odo03
O.odOo4
O.(
105
Suwannee River Fulvic Acid (KgC L )
FIGURE 2. Fluorescence lifetimes of pyrane in the presence and absence of Suwannee River fulvic acids in air-equilibrated and deoxygenated samples.
3F = 1 + Koc,hs[HS]
(2)
where Fo and Fare the respective probe fluorescence in the absence and presence of a static quencher (i.e.,the humic substances). In using this approach, it was assumed that all the quenching in our system was caused by noncollisional processes (Le., through a partitioning mechanism). Both eqs 1 and 2 possess the identical mathematical expression, and this would allow us to compare directly our results measured by these two methods (Figure 1). In all cases, the fluorescence quenching technique yielded pyrene-Suwannee fulvic acid and humic acid partition coefficients that were a respective factor of 2.7-1.6 times higher than those determined by the solubility enhancement method (Table 1). Our fluorescence quenching results for pyrene and the Suwannee River humic acid are in good agreement with those found by Schlautman and Morgan (3. Conversely, reported FQ-measured Suwannee River fulvic acid-pyrene binding constants reported in the literature ranged over an order of magnitude (Table 1).Our Suwannee River fulvic acid is unique in that it was extracted from water sampled over a period of 1 day and is not a typical IHSS reference substance. Thus, it is plausible that the observed variability could be due in part to temporal variations in the composition of the humic material. Moreover, if we assume that solubility enhancement measures the true pyrene-humic substance binding constant, then these observations could further suggest that mechanisms in the fluorescence quenching approach other than static quenching could be responsible for the discrepancies in measured Koc,hs. VOL. 29. NO. 8.1995 i ENVIRONMENTAL SCIENCE
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0
A
0
I00
Air Saturated (No SRFA); T = 144 ns Air Saturated 12.95 mg C/L SRFA; T = 132 ns Deoxygenated (No SRFA); T = 190 ns Deoxygenated 1I.69 mg CIL SRFA; T = 194 ns
200
300
400
500
600
700
TIME (ns) FIGURE 3. Sample fluorescence decays for pyrene showing the effects of dissolved 02 and Suwannee River fulvic acid (SRFA).Steeper slopes correspond to shorter lifetimes. Spikes at early times are due to SRFA fluorescence.
The fluorescence of PAHs may also be quenched dynamically through collisions with small molecules, and molecular oxygen is well known as an ubiquitous quencher of fluorescence (10). From our observations of the literature, it appears that most PAH fluorescence quenching experiments involving humic substances have been conducted with air-equilibrated samples (I -@. Presumably,dynamic quenching was assumed to be negligible (due to 02’s low solubility in water), and/or its contributions were believed to have been accounted for in separate control experiments. While these approaches are reasonable, they are valid only if the contribution of oxygen to dynamic quenching is unaffected by the presence of humic materials. Following this assumption, it is reasonable to assume that oxygen’s influence would be constant from sample to sample, rendering deoxygenation unnecessary. However, if the quenching process is influenced by interactions between molecular oxygen and the dissolved humic materials, these assumptions and corrections may prove to be inadequate. When dynamic and static quenching occur concurrently, one expects to observe deviations from the Stern-Volmer equation (Le., nonlinear behavior). In the event that dynamic and static quenching can be attributed to one quencher (in our case the humic substance), boththe static quenching constant (equivalent to &,hs for purposes of this work) and the collisional quenching constant (&I, can be elucidated using the following equation:
The values for & and &,hs can be determined by using the following second order polynomial expression:
(4)
+
where I is equal to (Kd &c,hs) and Sis equivalent to &&c,hs (10). At very high humic substance concentrations (’3 x 2164
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kg/L), we observed deviations from eq 2 (figure not shown). Attempts to elucidate I(o& using eq 4,however, were unsuccessful because values of I and S from our data resulted in a quadratic equation that was unsolvable. These results suggest that collisional quenching by the humic substance is not important and that the presence of another species capable of collisionalquenching may be responsible for the observed deviation from linear behavior. PAH fluorescence lifetime measurements made by other investigators (11, 12) have also concluded that collisional quenching by humic and fulvic acids is not significant since no changes in the fluorescence lifetimes were observed for the PAHs that were studied. In an effort to minimize the effects of dissolved oxygen as a quencher, fluorescence lifetime experiments are generally conducted in the presence of an inert gas (e.g., argon) (11). We conducted lifetime experiments using both air-equilibrated and deoxygenated samples. Pyrene lifetimes measured under anoxic conditions (Le., using argon sparged and sealed samples) were not affected by the presence of humic substances (Figure2). Indeed the decay of pyrene fluorescence (expressed as loglo-normalized photon counts) in the presence of 11.69 mg/L Suwannee River fulvic acid as total organic carbon was indistinguishable from those conducted in Milli-Q water (Figure 3). Our findings corroborate the results presented by others regarding the ability of humicsubstunces to act as dynamic quenchers of PAH fluorescence (11, 12) as well as the analysis of fluorescence quenching data using eqs 3 and 4. When similar experiments were conducted under airequilibrated conditions (i.e., in the absence of argon sparging), a decrease in the pyrene lifetime was observed (Figures 2 and 3). This phenomenon was expected given molecular oxygen’s ability to dynamically quench fluorescent substances. An unexpected result was the synergistic effect of oxygen and the humic materials on the lifetime of pyrene. Loglo-normalized photon counts of pyrene in the
presence of molecular oxygen and 12.95 mg/L Suwannee River fulvic acid (as total organic carbon) were steeper than the decay curve of pyrene in air-equilibrated Milli-Q water (Figure 3). Indeed at higher humic substance concentrations, pyrene lifetimes continue to decrease (Figure 2). Lifetime experiments conducted with Suwannee River humic acid yielded similar results. These findings confirm that pyrene’s fluorescence is dynamically quenched in aqueous solution. Of greater importance for our investigations, it appears that oxygen is the dominant dynamic quencher and that the humic substances themselves are involved only indirectly. In order to explain our observations, we observed (from the chemical literature) that the solubility of oxygen (a nonpolar molecule) in organic solvents is significantly higher than its aqueous solubility (13). Indeed, the solubility of oxygen in neat 2-propanol is 30 times higher than its solubility in water. Thus, it is plausible that both molecular oxygen and PAHs in the aqueous phase are attracted to the humic materials through hydrophobic interactions. In these microenvironments, enriched with both species, any “free”pyrene could be dynamically quenched by oxygen. Given pyrene’s relatively long fluorescence lifetime (-200 ns) inwater, the probabilityfor being dynamicallyquenched is increased over other PAHs. Other probes in aqueous matrices have shorter lifetimes, so dynamic quenching by oxygen may be less pronounced. We are currently investigating the effect of molecular oxygen and humic substances on the fluorescence quenching of PAH probes possessing shorter lifetimes. Finally, if static quenching of pyrene by humic substances is incomplete (i.e., through the existence of fluorescent pyrene-HS complexes) (141, the presence of molecular oxygen could further quench any additional fluorescence from the humic substancepyrene complex that could result in higher than expected &,hs values.
Conclusions Fluorescence quenching of pyrene by two humic substances is profoundly affected by the presence of molecular oxygen. In all cases &,hs values determined by fluorescence quenching in the presence of dissolved oxygen were higher than those measured by solubility enhancement. We believe that dissolved molecular oxygen and pyrene are both attracted to and are enriched near these humic materials in the aqueous phase. Given the nature of this dynamic microenvironment, the probability for enhanced collisional quenching of pyrene by molecular oxygen is greatly increased. While this process is important, our data does show that the absolute differences between solubility enhancement-derived Koc,hsand those values obtained by fluorescence quenching for our probe are relatively small (factorsof 2-3). In situations where the quantities of humic substances or dissolved organic matter are limited, fluorescence quenching can still be used to obtain “ballpark estimates” of &c,hs. Moreover, we believe that accurate &hs values can be measured by fluorescence quenching if such experiments are carried out under anoxic conditions.
Research is underway to elucidate the mechanisms that control this enhanced collisional quenching process by molecular oxygen in the presence of humic materials.
Acknowledgments We thank Deb Backhus and Mark Schlautman for their valuable comments and helpful discussions and J. Andrew Sipp for helping us with many of the experiments. This work is a result of research sponsored in part by the Ohio Sea Grant College Program, Project RIPS-9, under Grant NA90AA-D-SG496 of the National Sea Grant College Program, National Oceanic and Atmospheric Administrations in the U.S. Department of Commerce, and from the State of Ohio, and in part byaU.S. Environmental Protection Agency CooperativeAgreement (CR-819615-02-0)between the Ohio State University and the EPA Environmental ResearchLaboratory,Duluth, MN; Douglas Endicott, project officer. We acknowledge Coherent, Inc., for the loan of some of the equipment used in these experiments. We acknowledge the National Science Foundation for support of portions of the instrumentation used in this work under grant CHE9108384. We also acknowledge The Ohio State University for partial support of this work through the Seed Grant Program and the Center for Materials Research. J.S.B. acknowledges financial support from a Department of Education National Need Fellowship.
Literature Cited (1) Gauthier, T. D.; Shane, E. C.; Guerin, W. F.; Seitz, W. R.; Grant, C. L. Environ. Sci. Technol. 1986, 20, 1162. (2) Gauthier, T. D.; Seitz, W. R.; Grant, C. L. Enuiron. Sci. Technol. 1987, 21, 243. (3) Backhus, D. A.; Gschwend, P. M. Enuiron. Sci. Technol. 1990,25, 1214. (4) Chin, Y. P.; Gschwend, P. M. Enuiron. Sci. Technol. 1992, 26, 1621. (5) Schlautman, M. A.; Morgan, J. J. Enuiron. Sci. Technol. 1993,27, 961. (6) Schlautman, M. A.; Morgan, J. J. Enuiron. Sci. Technol. 1994,28, 2184. (7) Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E. Enuiron. Sci. Technol. 1986, 20, 502. (8) Chiou, C. T.; Kile, D. E.; Brinton, T. I.; Malcolm, R. L.; Leenheer, I. A.; MacCarthy, P. Enuiron. Sci. Technol. 1987, 21, 1231. (9) Amy, G. L.; Conklin, M. H.; Liu, H.; Cawein, C. In Organic Substances and Sediments in Water; Baker, R. A., Ed.; Lewis Publishers: Chelsea, MI, 1991. (10) Lakowicz, J. Principles of Fluorescence Spectroscopy; Plenum: New York. (11) Mora, M. J.; Corapcioglu, M.; Wandruzka, R.; Marshall, D.; Topper, K. Soil Sci. SOC.Am. J. 1992, 54, 1283. (12) Chen, S.; Inskeep, W. P.; Williams, S.; Callis, P. R. Enuiron. Sci. Technol. 1994,28, 1582. (13) Cargill, R.; Young, C.; Balfino, R.; Lang, P.; Clever, H. J. Chem. SOC.Faraday Trans. 1981, 7, 190. (14) Backhus, D. A. Colloids in Groundwater: Laboratory and Field Studies of their Influence on Hydrophobic Organic Contaminants. Ph.D. Dissertation, MIT, 1990.
Received for review February 21, 1995. Revised manuscript received May 15, 1995. Accepted May 24, 1995.
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