Environ. Sci. Technol. 1992, 26, 1787-1792
Fluorescence Quenching of Synthetic Organic Compounds by Humic Materials Mlchele M. Puchalskl and Matthew J. Morra"
Division of Soil Science, University of Idaho, Moscow, Idaho 83843 Ray von Wandruszka
Department of Chemistry, University of Idaho, Moscow, Idaho 83843 quenching, the slope of the Stern-Volmer plot, K, becomes the binding constant. termine equilibrium constants between synthetic organic When only one type of quenching (static or dynamic) fluorophores and water-soluble humic materials, limitais present and the Stern-Volmer plot is linear, theoretical tions of the technique have not been thoroughly evaluated. constraints of the equation are satisfied and data interDifenzoquat (1,2-dimethyl-3,5-diphenyl-1H-pyrazolium pretation is straightforward. However, in the case of humic methylsulfate) and l-naphthol were used as fluorophores materials, data interpretation is hindered by the strong in quenching experiments with three humic acids. Curabsorption of exciting and emitted radiation by the humic vature in the Stern-Volmer plots toward the Y-axis was quencher. This results in another type of emission not caused by inner filter effects or, in the case of difenabatement termed the inner filter effect (IFE), which must zoquat, its tendency to form dimers. Mathematical in some fashion be separated from true static or dynamic methods to separate a possible combination of static and quenching. Many methods have been proposed to correct dynamic quenching components, which may have confor IFE, but the accuracy and applicability to systems tributed to the curvature, were unsuccessful. The Sterncontaining quenchers which absorb at both the excitation Volmer plots resembled those obtained by other reand emission wavelengths have only recently been evalusearchers working with microheterogeneous micellar systems. This investigation shows that the quenching beated (5). havior of humic acid does not conform to simple SternA second unique feature of humic materials which comVolmer theory, which assumes single-exponential decay. plicates the application of fluorescence quenching to these The use of fluorescence quenching to determine equilibsubstances is their proposed micellar character (6). Mirium constants of interactions occurring between humic cellar systems have been shown to alter quenching by materials and synthetic organic compounds should creating microenvironments in which the physical orientherefore be approached with caution. tation of the quencher and fluorophore are altered (7).The relationship of micelle-like behavior to humic quenchers has been proposed ( 4 ) , and the difficulty in applying Introduction Stern-Volmer theory to microheterogeneous micellar Fluorescence quenching is an attractive method to desystems which do not exhibit single-exponential decay has termine equilibrium constants, because in contrast to other been discussed (8). methods the constants are measured directly without the Because of the unique nature of humic materials, a more need to disrupt system equilibria by separating bound and thorough analysis of their quenching behavior is required unbound fractions. Gauthier et al. ( I ) first introduced the before fluorescence quenching techniques can be applied use of fluorescence quenching as a nondestructive method with confidence. The objective of this study was to to determine equilibrium constants of interactions between evaluate the use of fluorescence quenching as a technique dissolved humic materials and synthetic aromatic hydroto determine binding constants of humic quenchers with carbons. Others have since employed similar techniques fluorescent synthetic organic compounds. to determine binding constants of naphthalene (2) and phenanthrene (3) with water-soluble organic carbon and Materials and Methods to investigate the type of interaction occurring between Difenzoquat (1,2-dimethyl-3,5-diphenyl-1H-pyrazolium naphthalene or l-naphthol and humic acid ( 4 ) . methylsulfate; 98.1% ) was obtained from American Any process which decreases the fluorescence yield of Cyanamid Corp., under the trade name Avenge. Difena compound is termed fluorescence quenching. Dynamic zoquat and l-naphthol (Fisher Scientific, Pittsburgh, PA) quenching refers to a collisional type of quenching in which were used without further purification. Three types of the excited fluorophore is nonradiatively deactivated humic acids were used in this study. The first, HA-1, was during the lifetime of the excited state. No actual binding extracted from a Latahco silt loam soil (Argiaquic Xeric occurs between the fluorophore and quencher. Static Argialboll) maintained in pasture for 20 years. The top quenching resulta from a ground-state association between 30 cm was collected, air dried, and crushed to pass a 2.0the fluorophore and quencher such that the bound fluomm sieve. The soil contained 41.5 g of organic C, 3.9 g of rophore no longer fluoresces. When present separately, total N, 159 g of clay, and 121 g of silt/kg of soil. Organic both types of quenching are described by the linear relaC was determined by the Walkley-Black method (9), tionship originally proposed by Stern and Volmer: particle size distribution by the hydrometer method (lo), and total N by Dumas combustion (LECO CHN-600 de(1) F,/F = 1 + kq7,[Q] = 1 + K[Q] terminator). Humic acid was extracted using a procedure where F, is the fluorescence intensity in the absence and similar to that recommended by Schnitzer (11). A detailed F is the fluorescence intensity in the presence of quencher, description of the modified method can be found in Morra k is the bimolecular quenching constant, 7, is the lifetime et al. (4). Carbonates were first removed from the soil by o! the fluorophore in the absence of quencher, [Q]is the acidification with HC1, and humic materials were extracted concentration of the quencher, and K = kq7, is the with 0.1 M NaOH under N2 for 24 h at room temperature Stern-Volmer quenching constant. In the case of static followed by centrifugation. The supernatant was decanted
rn Although fluorescence quenching has been used to de-
0013-936X/92/0926-1787$03.00/0
0 1992 American Chemical Society
Environ. Sci. Technol., Vol. 26, No. 9, 1992
1787
and centrifuged twice followed by filtration using Whatman 42 filter paper. The filtrate was acidified to pH 2 with 1M HC1 and allowed to stand for 24 h at room temperature. The precipitated humic acid was then separated from the fulvic acid by centrifugation. The humic acid precipitate was redissolved in 0.1 M NaOH, and the pH was brought to 7.0. Soluble salts were removed from the dissolved humic acid using a method similar to that reported by Kwak et al. (12). Distilled-deionized water (18 Ma cm resistivity) contained in a pressurized reservoir was passed through a 50-mL ultrafiltration cell in a continuous-flow mode using N2. The cell containing the humic acid was fitted with an ultrafiltration membrane having a MW cutoff of 10000 (PM10 Amicon, Danvers, MA). The extracts were then freeze-dried and stored in sealed containers at room temperature. HA-1 contained on a moisture-free basis, 337 g of total C, 26.6 g of total N, 34.5 g of H, 25.3 g of Fe, 30.8 g of Al, and 115 g of Si/kg of humic acid. Carbon, N, and H were determined by Dumas combustion (LECO CHN-600 determinator) of solid samples, and Fe, Al, and Si were determined by inductively coupled plasma emission spectroscopy of aqueous solutions. The second humic acid, HA-ref, was purchased from the International Humic Substances Society (soil humic acid reference 1R102H). This humic acid was extracted from a mollisol and contained on a dry, ash-free basis 604 g of C, 49.2 g of H, 25.2 g of N, 5.5 g of S, and 5.0 g of P/kg of humic acid. The third type of humic acid used, HA-2, was prepared from HA-1 following the deashing procedure recommended by Schnitzer (11). After treatment with a HC1-HF wash followed by a distilled-deionized water wash, the humic acid was freeze-dried and stored in a desiccator at room temperature. This deashed humic acid contained, on a moisture-free basis, 462 g of C, 35 g of N, and 1.9 g of Fe/kg of humic acid. Fresh stock solutions (1 mg/mL) of HA-1 and HA-2 were prepared weekly in glass containers using distilleddeionized water. To completely dissolve the humic acid, the solutions were sonified in a 40 O C water bath for 15-20 min. The solution pH WM approximately 6.0. Fresh stock solutions (1mg/mL) of HA-ref were prepared similarly, with the exception that 1M NaOH was added to solubilize the H+-saturated humic acid and adjust the pH to that of HA-1 solutions. Stock solutions of difenzoquat (6.9 X M) were prepared as needed using distilled-deionized water. Stock solutions of l-naphthol (1 X M) were prepared by first dissolving l-naphthol in a small amount of methanol (Omnisolve,EM Science, Cherry Hill, NJ) and then diluting with distilled-deionized water. All stock solutions were stored in the dark in glass containers at 4 M) were prepared "C. Stock solutions of KI (1.0 X fresh for each experiment using distilled-deionized water. Triplicate sample solutions of the fluorophore and humic acid were made from stock solutions and allowed to equilibrate before measurements were taken. The samples were protected from light to prevent decomposition. Triplicate sample solutions of difenzoquat and KI were made from stock solutions and used immediately to avoid decomposition of the KI. Fluorescence spectra were recorded with a Perkin-Elmer MPF 66 spectrofluorometerequipped with a thermostated cell holder. The instrument gives corrected spectra through the use of a built-in rhodamine 101 quantum counter. Fluorescence-free quartz cells were used, and blank subtraction was performed by instrument software. The excitation wavelength for difenzoquat was set at 256 1788
Environ. Sci. Technoi., Vol. 26, No. 9, 1992
6
4
G
\ LL 2
-b
6
ti
Humic acid (mg C
18
/
i4
L)
Figure 1. Fluorescence quenching of difenzoquat by HA-1, HA-2, and HA-ref at 22 "C expressed in the form of Stern-Volmer plots. F, and F a r e the fluorescence intensities of the fluorophore In the absence and presence of humic acid, respectively. The ratio of the IFE-corrected to uncorrected fluorescence intensities exceeded 3.0.
nm. The emission wavelength was read for maximum intensity and varied between 366 and 372 nm. Excitation and emission slit widths were set at 5.0 and 1.8 nm band pass, respectively. The concentration of difenzoquat used was 6.9 X low5M unless otherwise noted. Fluorescence measurements of samples containing HA-1 and HA-2 were taken at 22 and 42 "C. Fluorescence measurements of samples containing HA-ref were taken at 22, 32, and 42 "C. The excitation wavelength for 1-naphthol was set at 293 nm. The emission wavelength was read at 479 nm. Excitation and emission slit widths were set at 5.0 and 1.5 nm band pass, respectively. The concentration of 1naphthol was 7 X loT5M, and measurements were taken at 22 "C. Absorbance measurements were taken with a PerkinElmer Lambda 4C spectrometer on the same samples used for fluorescence measurements. Corrections for inner filter effects were made using these absorbances following the method of Gauthier et al. (1). Time-resolved fluorescence lifetimes were measured using a pulsed laser fluorometer (4). The concentration of difenzoquat used was 5 X lo3 M. Oxygen was removed by freeze-pump thawing the samples.
Results and Discussion Curved Stern-Volmer plots were obtained when difenzoquat was quenched by HA-1, HA-ref, and HA-2 at 22 "C (Figure 1). The plots were also curved when quenching experiments were conducted at 32 and 42 "C (data not shown). Curvature toward the Y-axis may indicate incomplete correction for inner filter effects (IFE). We have shown that the IFE correction of Gauthier et al. ( I ) ,as used here, corrects for curvature and removes IFE at both the excitation and emission wavelengths when both are considered separately (5). However, Parker (13)reported that inaccurate corrections for IFE may occur if the ratio of the corrected to the uncorrected fluorescence intensity of the fluorophore exceeds 3.0. Since this was the case for all concentrations of the three humic acids (Table I) and a portion of the IFE was caused by difenzoquat itself, the experiment was repeated with a lower concentration of difenzoquat (1 X M). The plot retained its curvature
Table I. Ratio of Corrected F (F,,,,) to Observed F (Fob.) Values for 6.9 X lo-&M Difenzoquat and HA-1, HA-2, and HA-ref at 22 OC
HA-1
HA-2
LL
1
3.37 3.60 4.01 4.54 5.12 7.95 7.95 9.64 11.17 3.25 3.48 3.75 3.95 4.43 5.15 5.18 5.71 3.20 3.60 4.20 4.90 5.60 6.10 7.20 8.50
0.00 1.69 3.37 5.06 6.74 10.11 13.48 16.85 20.22 0.00 3.02 6.04 9.06 12.08 15.01 18.12 21.14 0.00 2.31 4.62 6.93 9.23 11.54 13.85 16.15
HA-ref
3.5
Fcarr/Fahs
+
Y = 1.02
0.082X
+
0.01 1X2
P
r2 = 0.99
2.5 -
hLI
1.5
-
0.5
I
0
4
I
b
Humic Acid ( m g C
12
/
L)
Flgure 2. Fluorescence quenching of difenzoquat by HA-1 at 22 O C expressed in the form of a Stern-Volmer plot. F, and F a r e the fluorescence Intenstties of the fiuorophore In the absence and presence of humic acid, respectively. The ratio of the IFE-corrected to uncorrected fluorescence intensttles was less than 3.0.
(Figure 2) even though the ratio of the corrected to the uncorrected fluorescence intensity was less than 3.0. A second possibility for origin of the curvature relates to the tendency of difenzoquat to form both monomers and dimers (14).The presence of two different fluorophores in solution could result in irregularities in the SternVolmer plot which have no relationship to the nature of the humic acid quencher. Instead, peculiarities in quenching behavior and the respective Stern-Volmer plot would result solely from the unique characteristics of the fluorophore, difenzoquat. A simple quencher, KI, was substituted for humic acid to test this possibility. The Stern-Volmer plot of KI quenching of difenzoquat (1 X M)was linear (data not shown). We concluded that humic acid must in some unknown fashion cause the observed curvature.
0
A HA- 2
n
concn, mg of C/L
humic acid
0 HA- 1 v)
0 7
X
2 I
c
(I
CL
a
2 0.0
1.5
3.0
4.5
6.0
Humic acid (M x Flgure 3. Fluorescence quenching of dlfenzoquat by HA-1 and HA-2 expressed in the form of Stern-Volmer plots modified according to Vaughan and Weber (75) and Lakowicz (76).
Curved Stern-Volmer plots are usually attributed to a combination of static and dynamic quenching mechanisms which can be separated graphically (7,15,16). In order to use these separation methods, it is necessary to express the quencher (humic acid) concentration on a molar basis. Molarities of HA-1 and HA-2 were obtained by assuming a molecular weight of 10000, since this was the molecular weight cutoff of the membrane used in the extraction procedure. Molarities of HA-ref were also obtained by assuming a molecular weight of 10000. The exact molecular weight of humic acid used in the mathematical separation of static and dynamic quenching components is unimportant. The calculated quenching constants are not altered by the selection of a smaller or larger molecular weight because of the relationships detailed in the following equations. In the separation method described by Vaughan and Weber (15)and summarized by Lakowicz (161,a modified Stern-Volmer equation is used to separate the quenching constants: (2) Fo/F = 1 + ( K D+ Ks)[Q1+ K&s[Ql2 where KD is the dynamic quenching constant and Ks is the static quenching constant. A parameter Kappis defined which is equal to (Fo/F- l)/[Q]. Thus, a plot of Kappvs [Q]should result in a straight line with an intercept ( I ) equal to KD + KS and slope (S) equal to K & . The terms for I and S can be rearranged into a single quadratic ex-
pression. By substituting the values for the slope and intercept into Ks2 - KSI + S = 0 (3) K Dand Ks can be determined. Using this method, modified Stern-Volmer plots were constructed (Figure 3). The modified plots were linear for HA-1 and HA-2 (P= 0.OOOl); however, substitution of the slopes and intercepts into the above equation resulted in an equation which had no real solutions for KD and Ks. The plots were not linear (P= 0.0001) for HA-ref. In the method described by Eftink and Ghiron (7), graphical separation is based on another modified SternVolmer equation: Fo/(Fev[Ql)= 1 + KD[Q] (4) where Vis the static quenching constant. In this method Environ. Sci. Technol., Vol. 26,
No. 9, 1992 1789
2.0
a
A
1.5
1.B
u
> e,
e, 1.0
'
C 0
0
LL
f0
1.5
0
Ll-
v)
n
4
0.5
1.3
1.o
/
I
I
2
4
0.0
I
6
1
Humic acid (M x loe6)
Table 11. Eftink-Ghiron V Values, Dynamic Constants ( K D )and , Bimolecular Quenching Constants ( K , ) for Difenzoquat and HA-1, HA-ref, and HA-2
k,, M-l HA-1 HA-1 HA-ref HA-ref HA-ref HA-2 HA-2
V, M-I
KD,h4-l
22
171 000
42
140 000
155 000 229 450 279 610 205 269 261 694 295 166 174 238
temp, "C
22
100 000
32 42 22 42
120 000 90 000 141 500 213 500
X lOI3
1.55
2.30 2.80 2.00 2.60 3.00 1.70
the Eftink Y-axis value or Fo/(Fev[Ql) is plotted vs [Q] for varying values of V. The V value which results in the most linear plot is the static quenching constant for that system. The dynamic constant, KD,is the slope of this plot. This method is based on the "dark complex" model which describes curved Stern-Volmer plots as mixtures of static and dynamic quenching, with the dominant mechanism being dynamic. This type of static quenching is described as instantaneous quenching resulting from the quencher and fluorophore being in close proximity to each other, but not necessarily in physical contact. Eftink and Ghiron (7) have provided a summary of this model as well as other models used to explain quenching which occurs spontaneously. Modified Stern-Volmer plots were constructed using this method (Figure 4). The most linear plots for difenzoquat and the three humic acids resulted from V values and dynamic constants listed in Table 11. Bimolecular quenching constants (k,) were calculated to determine if the dynamic constants were appropriate. Bimolecular quenching constants are determined from the SternVolmer constants and the fluorescence lifetime of the fluorophore in the absence of quencher (TJ: k , = KD/70 (5) Although a specific lifetime could not be determined for difenzoquat because of wide variation in measurements, an upper limit of 10 ns was obtained. Assuming a value of T~ equal to 10 ns gave the smallest estimate of k, possible in this system. The calculated bimolecular quenching constants for the dynamic portions of quenching (Table 11) were much larger than the maximum value (1X 1O1O M-' s-l) that k, can assume for diffusional processes in 1790
Environ. Scl. Technoi., Vol. 26, No. 9, 1992
I
I
I
260
275
290
Wavelength (nm)
Flgure 4. Fluorescence quenching of difenzoquat by HA-1 and HA-ref expressed in the form of Stern-Volmer plots modified according to Eftink and Ghiron (7).
humic acid
I
245
Flgure 5. Absorption spectra of difenzoquat and (A) 0.0, (B) 20.2, and (C) 40.4 mg of C/L of HA-1. Isosbestic point Is representedby arrow.
aqueous solutions (16) and must be inaccurate. Other evidence suggests that the correction of Eftink and Ghiron (7) is inappropriate for the present system. Humic acid is considered to be a large macromolecule with varying amounts of negative charge. The total acidity of humic acid is between 500 and 870 cmol kg-' (17). Since difenzoquat is positively charged, significant interaction or binding with humic acid is expected. This would suggest that difenzoquat should complex with humic acid in such a way as to produce a much larger static than dynamic quenching component. Evidence for complex formation was obtained from the UV-visible spectra of difenzoquat with the humic acids. Although the spectral structure of difenzoquat did not change with the addition of any of the humic acids, the spectra show an isosbestic point (Figure 5), an indication of complexation (18). Application of the methods of Kubota et al. (18) to calculate binding constants for difenzoquat and humic acid using the absorptivities of the fluorophore and quencher was unsuccessful. This was expected since calculation requires complete complexation with a ratio of humic acid to difenzoquat of 1:l. Because each humic acid molecule is large and contains many negatively charged sites, electrostatic attraction is expected to result in a ratio other than 1:l. Other researchers have represented the quenching of nonionic synthetic organic fluorophores by dissolved humic materials in the form of linear Stern-Volmer plots (1-4). In some cases linearity of the Stern-Volmer plot was used to infer the presence of only one type of quenching (1-3). The quenching was considered static, and equilibrium constants were, therefore, derived from the slope of the line. Our previous work using fluorescence lifetime changes has shown that static quenching dominates when 1naphthol or naphthalene interacts with humic acid in aqueous solution (4). However, we described the quenching as apparent static quenching similar to the dark complex model in which a true ground-state association or binding does not occur. One possibility for linearity in the case of these systems, and not in the case of difenzoquat, is the cationic nature of difenzoquat. Thus, although humic acid may cause curvature in Stern-Volmer plots, such curvature may be specific to the cationic interaction of difenzoquat with negatively charged humic acid molecules. This seems unlikely given that difenzoquat should interact in a static
Table 111. Comparison of Linear and Quadratic Stern-Volmer Plots for the Quenching of Humic Materials by Synthetic Organic Fluorophores
data sourceo Morra et al. ( 4 )
quencherb
fluorophore l-naphthol
HA
Morra et al. ( 4 )
HA
naphthalene
experimental
HA
l-naphthol
Traina et al. (2)
WSOC (Na)
naphthalene
Traina et al. (2)
WSOC (Ca)
naphthalene
Traina et al. (2)
WSOC (Al)
naphthalene
modelc
a
b
1 q 1
0.98 1.00 0.99 1.00 0.98
0.022 0.016 0.028 0.027 0.040 0.029 0.078 0.023 0.072 0.039 0.035 0.026
9
1 q 1 q 1 q 1 q
1.00 0.84
1.06 0.84 0.99 0.94 0.98
C
0.00025 0.000042 0.00086 0.0020
0.0012 0.00029
rz 0.98 0.99 0.99 0.99 0.99 1.00 0.95 0.98 0.98 0.99 0.96 0.96
probabilityd x > It1 x2 > It1 0.0001 0.013 0.0001 0.003
0.0001 0.0001
0.18 0.82 0.0002
0.0002 0.27 0.0001
0.035
0.0001
0.0001
0.0001 0.0039
0.28
DDatafrom Morra et al. ( 4 ) taken from Figure 1 and data from Traina et al. (2) taken from Figure 4 of the respective publications. bHumic acid designated as HA and water-soluble organic carbon as WSOC. Cation in parentheses was present in system during quenching. cLinear designated as 1 and Quadraticas a. dProbabilities less than 0.05 were considered Significant.
1
O=CH
HCCC
COOH HO
HO
COOH
R--tH
I
CCOH
Flgure 8. Interaction of a synthetic organic compound (striped circle) with a micellar microenvironment of humic acid. The humic acid molecules were modeled after the structure proposed by Stevenson ( 77).
fashion and produce a linear plot, whereas nonionic compounds would be more likely to interact both statically and dynamically and produce a curve. We propose an alternative explanation which requires careful reexamination of our data as well as that of other researchers. In a previous publication, we described the interaction between l-naphthol or naphthalene and humic acid as a mixture of static and dynamic quenching (4). We described humic acid as forming a pseudomicelle which cages the fluorophore; restraining, but not rigidly binding it in a similar fashion as shown in Figure 6. Likewise, the hydrophobic portion of the difenzoquat molecule is buried in a hydrophobic microenvironment of a localized area of the humic acid molecule. In such situations, curved Stern-Volmer plots are possible (7). In our previous work, Stern-Volmer plots for l-naphthol and naphthalene possessed a linear relationship in the humic acid concentration range used (Table 111). The addition of a quadratic term did not result in a more appropriate model. We speculated that curvature in the plot would materialize if higher quencher concentrations were used. In our current work, we repeated the experiment using the same concentration of l-naphthol but higher concentrations of humic acid. The Stern-Volmer plot of these data was curved (Table 111). The curvature is slight
and a linear relationship is significant, but a curved model provides a better fit for the data. Similarly, the data of Traina et al. (2)produce significantly linear Stern-Volmer plots in two of three cases, but curved models produce a better fit (Table 111). The quadratic term is significant for Stern-Volmer plots of the systems containing watersoluble organic C and either Na+ or Ca2+. Data shown by Gauthier et al. (I) produce a linear Stern-Volmer plot which cannot be better fit by a curved model. However, only quenching data for fulvic acid are available for reevaluation, and fulvic acid would be expected to have much less micellar character than humic acid. Magee et al. (3) do not provide the actual data but only note that a significantly linear Stern-Volmer plot exists. Ziemiecki and Cherry (19) and Miola et al. (20) described experiments with microheterogeneous micellar systems in which the application of Stern-Volmer theory results in plots curving toward the Y-axis. In such systems decay kinetics are often not the result of single-component exponential decay but the sums of exponentials or a distribution function of exponentials (8). This seems likely in the case of humic acid because of its heterogeneous structure. In a previous publication we provided evidence that humic acid has domains with a micelle-like cage structure (4). The heterogeneity introduced by such a configuration would account for the presence of different fluorophore populations and, hence, multiple exponential decay. This, in turn, would lead to a loss of linearity in the Stern-Volmer plots. The relevant equations (8)require a more thorough knowledge of the microenvironment of the fluorophore than is presently available for humic acid. Their application in such systems is therefore premature.
Conclusions Inner filter effects and difenzoquat’s tendency to form both monomers and dimers were eliminated as possible causes for curvature in the Stern-Volmer plots toward the Y-axis. Curvature of the Stern-Volmer plots of difenzoquat and l-naphthol with humic acid suggests that both static and dynamic quenching mechanism are operative. However, it was not possible to separate static and dynamic quenching constants for these curved plots using graphical methods. In addition, the UV-visible spectra of difenzoquat and the three humic acids indicate complex formation. The curved Stern-Volmer plots of difenzoquat and l-naphthol with humic acid are similar to plots obtained from quenching experiments with micelles. Thus, we have provided additional evidence that humic acid Envlron. Sci. Technol., Voi. 26, No. 9, 1992
1781
Environ. Sci. Technol. 1992, 26, 1792-1798
forms a micelle-like structure which quenches the fluorescence of these compounds. Because of the microheterogeneous nature of humic materials, the interpretation of humic acid binding with synthetic organic fluorophores according to single-exponential decay and SternVolmer theory should be avoided. Equilibrium constants derived from such interpretations should be approached with caution.
Acknowledgments Appreciation is expressed to Karl Topper and David B. Marshall, Department of Chemistry and Biochemistry, Utah State University, Logan, UT, for fluorescence lifetime measurements of difenzoquat, Registry No. Avenge, 43222-48-6; 1-naphthol, 90-15-3.
Literature Cited Gauthier, T. D.; Shane, E. C.; Guerin, W. F.; Seitz, W. R.; Grant, C. L. Environ. Sci. Technol. 1986,20, 1162-1166. Traina, S. J.; Spontak, D. A.; Logan, T. J. J. Enuiron. Qual. 1989, 18, 221-227. Magee, B. R.; Lion, L. W.; Lemley, A. T. Enuiron. Sci. Technol. 1991, 25, 323-331. Morra, M. J.; Corapcioglu, M. 0.;von Wandruszka, R. M. A.; Marshall, D. B.; Topper, K. Soil Sci. Soc. Am. J. 1990, 54, 1283-1289. Puchalski, M. M.; Morra, M. J.; von Wandruszka, R. Fresenius’ Z. Anal. Chem. 1991, 340, 341-344. Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E. Enuiron. Sci. Technol. 1986, 20, 502-508. Eftink, M. R.; Ghiron, C. A. J. Phys. Chem. 1976, 80, 486-493. Carraway, E. R.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1991,63, 332-336.
(9) Nelson, D. W.; Sommers, L. E. In Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties, 2nd ed.; Page, A. L., Miller, R., Keeney, D. R., Eds.; Agronomy Monograph No. 9; ASA and SSSA: Madison, WI, 1982; Chapter 29, pp 539-579. (10) Gee, G. W.; Bauder, J. W. In Methods of Soil Analysis,Part 1. Physical and Mineralogical Methods, 2nd ed.; Klute, A., Ed.; Agronomy Monograph No. 9; ASA and SSSA: Madison, WI, 1986; Chapter 15, pp 383-411. (11) Schnitzer, M. In Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties, 2nd ed.; Page, A. L., MiUer, R., Keeney, D. R., Eds.; Agronomy Monograph No. 9; ASA and S S S A Madison, WI, 1982; Chapter 30, pp 581-594. (12) Kwak, J. C. T.; Nelson, R. W. P.; Gamble, D. S. Geochim. Cosmochim. Acta 1977, 41, 993-996. (13) Parker, C. A. Photoluminescence of Solutions; Elsevier: Amsterdam, 1968; p 222. (14) von Wandruszka, R.; Edwards, W. D.; Puchalski, M. M.; Morra, M. J. Spectrochim. Acta 1990, 46A, 1313-1318. (15) Vaughan, W. M.; Weber, G. Biochemistry 1970,9,464-473. (16) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; pp 257-295. (17) Stevenson, F. J. Humus Chemistry; John Wiley & Sons: New York, 1982; p 45. (18) Kubota, Y.; Motoda, Y.; Shigemune, Y.; Fujisaki, Y. Photochem. Photobiol. 1979,29, 1099-1106. (19) Ziemiecki, H.; Cherry, W. R. J. Am. Chem. Soc. 1981,103, 4479-4483. (20) Miola, L.; Abakerli, R. B.; Ginani, M. F.; Filho, P. B.; Toscano, V. G.; Quina, F. H. J. Phys. Chem. 1983, 87, 4417-4425.
Received for review July 12,1991. Revised manuscript received May 19,1992. Accepted May 27,1992. Funding for this research was provided by the Idaho State Board of Education and the Western Region Pesticide Impact Assessment Program.
Riboflavin Tetraacetate: A Potentially Useful Photosensitizing Agent for the Treatment of Contaminated Waters Richard A. Larson,’ Penney L. Stackhouse, and Thomas 0. Crowley Institute for Environmental Studies and Department of Civil Engineering, University of Illinois, 1101 West Peabody Drive, Urbana, Illinois 6 1801
rn The photosensitizing ability of 2’,3’,4’,5’-tetraacetylriboflavin (RTA) was compared to that of riboflavin in the degradation of various organic compounds and contaminated water samples. RTA was found to be superior to riboflavin under the test conditions. The inductive effects of ring substituents on the photolysis rates between riboflavin and RTA and the anilines (aniline, bromochloroaniline, nitroaniline, p-chloroaniline, p-toluidine, p anisidine, and 4-aminobenzotrifluoride) were studied; electron-donating substituents on the aniline ring enhanced the degradation rate for the RTA-mediated reactions but had little effect on riboflavin-promoted photolyses. Work with actual contaminated water samples further demonstrated RTA’s superior ability to photosensitize the disappearance of a mixture of compounds. The esults suggest that RTA may be a promising agent for t e cleanup of some polluted waters.
fi
Introduction Photochemical reactions, whether initiated by sunlight or by alternative sources of actinic radiation such as artificial lamps, may rapidly degrade potentially hazardous contaminants in wastewater and groundwater. Hexa1792 Environ. Sci. Technoi., Vol. 26, No. 9, 1992
chlorocyclopentadiene, which has a half-life of 4 min when exposed to sunlight, is one such compound ( I ) . However, many pollutants, .because of insufficient absorption of actinic radiation or low quantum yields of photoreaction, are not susceptible to such reactions. In these cases, photosensitizers can be deliberately added to facilitate their degradation. A photosensitizer (S) is a substance that absorbs light energy (hv),transforms it into chemical energy, and transfers that energy under favorable conditions to otherwise photochemically unreactive substrates. Redox processes are possible mechanisms for photoinduced energy transfer. (S + hv S*)
+ - + s*+ x - s++ xs*+ z - s- + z+ S*
M
S+
e-
(A)
(B) (C) A photochemically excited molecule may donate an electron to the medium (M, reaction A) or another molecule acting as an acceptor (X, reaction B), or it may act as an electron acceptor when a suitable electron donor is present (Z, reaction C) (2).
0013-936X/92/0926-1792$03.00/0
0 1992 American Chemical Society
