Environ. Sci. Technol. 1988, 22, 77-82
Electron Paramagnetic Resonance Measurements of Photochemical Radical Production in Humic Substances. 1. Effects of O2 and Charge on Radical Scavenging by Nitroxided Nell V. Blough
Department of Chemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 ~
rn Electron paramagnetic resonance spectroscopy (EPR) was employed to measure the ability of Aldrich humic acid (HA) to photosensitize the consumption of a series of cationic, neutral, and anionic nitroxides. These stable organic radicals react rapidly with a suite of inorganic and organic radicals to form diamagnetic products. In the presence of 250 pg/mL HA and under near-natural light conditions, easily detectable rates of nitroxide consumption are observed in both air- and Ar-equilibrated samples. However, at a given nitroxide concentration, significantly lower rates of nitroxide loss are observed in the presence of air, consistent with the view that O2and nitroxides compete for a substantial portion of the total radical pool. The initial rates of loss decrease in the order cationic > neutral >> anionic, indicating that there is an electrostatic effect on the rate constants for scavenging. These findings suggest that negative charge on the humic influences the efficiency with which nitroxides can react with radicals on the humic structure. The use of EPR to detect radical scavenging by nitroxides represents a sensitive means for estimating free radical photoinitiation rates in mixtures of chemically ill-defined organic material such as HA. Introduction Numerous studies over the past 10 years have provided evidence that photooxidative processes play an important role in the transformation and degradation of organic materials in natural waters (for reviews, see ref 1-3). Several groups have shown that irradiation of humic or fulvic acid solutions (4, 5 ) , or natural waters containing via energy these species (4-7), generates singlet O2(IO2) transfer from triplet excited states of the humics to ground-state oxygen (302). lo2,in competition with relaxation to 302, can react selectively with electron-rich organics to form peroxides and other oxidized products (Scheme I). This mechanism, commonly known as a type I1 photooxygenation process, may be important in the transformation of certain xenobiotics and biological substances in some waters ( 6 ) . Although a number of significant studies relating to free radical or type I photooxygenation processes have been published (8-1 l ) , less attention has been focused on this mechanism, presumably owing to the difficulty of quantifying the rates of production and yields of radicals capable of reacting rapidly to form superoxide (02-) or chain-propagating with 302 peroxyl radicals (RO,') (Scheme I). This paper presents the results of a study in which stable nitroxyl radicals (Figure 1)were employed to estimate free radical photoinitiation rates in humic acid (HA). The basis of the method is the ability of nitroxides to scavenge efficiently reactive free radicals as well as the hydrated electron (12-15);as the hydrated electron is "radical-like'' by nature, it is defined to be part of radical production. Nitroxides have been used in this capacity in the fields of biology (16-231, polymer chemistry (24-26), and mecha-
Scheme I. Comparison between the Reactions of O2with Triplet Excited States and Radicals Leading to Photooxidation and Reactions of Nitroxides Leading to Excited-State Quenching and Radical Chain Termination via Scavenging TYPE I PHOToOXyGNATION
E =
Oxd%edRc&k
"tn
t.
E~~MIW
h .
yN-O+
nistic photochemistry (27-30). Because nitroxides contain an unpaired electron, but the products of scavenging are diamagnetic (Scheme I, vide infra), the rate of nitroxide consumption upon irradiation can be monitored by electron paramagnetic resonance spectrometry (EPR) (16-21, 24, 25, 28, 29, 31, 32). Nitroxides are particularly well suited for examining the rates of production of radicals involved in photooxidation (Scheme I). They, like 02,couple rapidly to aryl and alkyl radicals but, in contrast to 02, form stable diamagnetic 0-substituted hydroxylamines (14,15,27,33). Also similarly to 02, they can act as one-electron acceptors in type I processes and be converted to 0-unsubstituted hydroxylamines (15-19,24,25). However, aliphatic nitroxides do not react with peroxyl radicals (26),superoxide (34),or singlet oxygen (35). Hydroxylamines react slowly with peroxyl radicals and superoxide to regenerate nitroxides (24, 26, 34). These radicals also exhibit photophysical quenching properties similar to 0,; triplets having energies 118000 cm-l (A < 555 nm) are quenched by nitroxides at rates comparable to O2(36, 37). A comparison between the reactions of nitroxides and O2is summarized in Scheme I. Nitroxides also react with a suite of organic and inorganic radicals such as Br2-, COS-, 12-, and OH (12, 13), which do not react with 02. A simple kinetic model constructed on the basis of these known reactions and employed to interpret the data is presented below. Experimental Rationale and Kinetic Model Nitroxide scavenging of radicals can be understood within the context of the following simplified reaction scheme (see also Scheme I):
+This is Contribution No. 6401 from the Woods Hole Oceanographic Institution. 0013-936X/88/0922-0077$01.50/0
OxidzedProdwk
Z PHOTOOXYGENAATION
0 1987 American Chemical Society
-c + lo2-
C* + 302
k,l
30,
(2)
Environ. Sci. Technol., Vol. 22, No. 1, 1988 77
C*
+ R2N-0 -!% C + R2N*-0 C*
kl
-
R2N-0
2R'
kz'
+ R2N-0
k3
II
0
0
4-Hydroxy-Tempo
4-Amino-Tempo
$ 4
(5)
diamagnetic products
RO,
I
(4)
R' --+ '12R-R R'
cmpo Derivatives
(3)
(6)
Proxy1 DerlLaftLes
m
termination reactions
k
R' t 0,--.%
or ROX
t -0,
+
or
(7)
propagation
Here C* represents an excited state, either singlet or triplet, populated as a result of light absorption by the chromophore C (reaction 1). Subsequently, C* decays either by relaxation to the ground state (reaction l), O2 quenching (reaction 2), nitroxide quenching (reaction 3), or photochemistry to produce radicals R' (reaction 4). Although both singlet and triplet states are capable of being quenched by O2 and nitroxides (38,391,because of the short lifetimes of singlet states and the relatively low concentrations of O2 and nitroxide involved, quenching is limited to triplet states as represented by reactions 2 and 3. Radicals formed from the excited states can undergo recombination (or back-electron transfer) (reaction 5), react with O2 to form peroxyl radicals or superoxide (reaction 7), or undergo termination reactions with nitroxides (R2N-0) to form diamagnetic products (reaction 6). While radical recombination (reaction 5) will be governed by second-order kinetics for simple free radicals in solution, the recombination of radicals generated in close proximity within a macromolecular structure is better approximated as an intramolecular, first-order process. As the charge effect suggests that radicals are indeed formed on the HA framework (40,41), it is assumed that first-order kinetics prevail for reaction 5. By examining the initial rates of nitroxide loss, hydroxylamine to nitroxide recycling can be ignored (24,25) while O2 consumption and changes in the radical formation rate can be minimized. Invoking the steady-state assumption for species C* and R' and solving the rate equations for reactions 1-7 give for the initial rate of nitroxide consumption N
where kz = k:/2 is given by
and F is the rate of radical formation and
If the excited states producing radicals are short-lived with respect to photophysical quenching by O2 and nitroxides (k, + k d >> kql[02]o+ kq2[R2NO]o), F will be independent of the O2and nitroxide concentration, and N will exhibit a hyperbolic dependence on the initial nitroxide concentration [R2NO], (eq 8). In the absence of O2 and at sufficiently high [R2NOl0(k3[R2NOIo>> k 2 ) ,the initial rate of nitroxide consumption will become equal to the rate of radical formation ( N = F),while the rate constant ratio of recombination to scavenging will be given by the initial nitroxide concentration at which N is half of its maximal value: [RZNO]F / 2 = 2 / lZ 3 (10) Because of competition for photochemicalequivalents, the 78
P
R
m
Environ. Sci. Technol., Vol. 22, No. 1, 1988
0 3 - CarboxyProxyl
0 3-CarbamoylProxyl
0 3 - AminomelhylProxyl
Figure 1. Structures of the nitroxides employed in this study.
presence of O2 will shift the dependence of N to higher [R2NOlO;in this case (11) [R2N01~/2= (k2 + k4[021o)/k3 In contrast, if the excited state producing radicals is sufficiently long-lived so that it can be quenched by nitroxide or 02,F and thus N will decrease with increasing [R2NOlOor [O2lO,and a hyperbolic profile will no longer be obtained. The above model represents the simplest possible situation. For HA, the system is likely to be more complicated and involve the heterogeneous populations of chromophores and radicals. In the absence of cross-recombination reactions between radicals formed from the excited states of different chromophores, this simple model can be expanded to include multiple processes by restating eq 8 and 9 as
where n equals the number of independent radical pools. The scavenging rate constant of a given nitroxide (k3*, where x = I-V; Figure 1) is considered to be equal for all pools. Although this assumption may not apply universally, it is consistent with the present data (vide infra). Equation 12 was employed to interpret the data quantitatively.
Materials and Methods Stock solutions of HA (250 pg/mL; Aldrich lot no. 1204PE) were prepared in pH 8.0, 10 mM phosphate buffer. Lower concentrations of HA were obtained by serial dilution of the stock with phosphate buffer. The stock was stored in the dark and used within 1 week of preparation. Nitroxides I-V (Figure 1)were obtained from Aldrich and used without further purification. Stock solutions (10 or 100 mM) were prepared in phosphate buffer and, when necessary, were adjusted to pH 8.0 by the addition of small volumes of concentrated HCI or NaOH. Carefully prepared solutions (100 mM) of 4-hydroxyTempo (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) (I) and 3-carboxy-Proxy1 (3-carboxy-2,2,5,5-tetramethyl-lpyrrolidinyloxy) (111)or 3-carbamoyl-Proxy1(3-carbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyloxy) (IV) were employed as spin concentration standards for Tempo and Proxyl derivatives, respectively. Appropriate volumes of the nitroxide stock solutions were added to 1-10 mL of the HA and control (phosphate buffer) solutions to give ni-
troxide concentrations ranging from -8 to 400 pM. Aerobic samples were drawn into 50-pL calibrated capillaries (Corning), which were then sealed at the bottom with a small amount of high-vacuum silicone grease (Dow Corning) and placed inside 3 mm diameter quartz EPR tubes. Anaerobic samples were prepared by rapidly bubbling Ar through sample solutions contained within l-mL Kimax microvials, which were vented through the 50-pL capillaries via rubber septa. After at least 20 min of flushing, the Ar flow was reduced, and the capillary filled by the positive pressure of the Ar flow; the capillary was then carefully sealed top and bottom and placed within a quartz EPR tube. EPR spectra were recorded on a Bruker 220D spectrometer with a Bruker ER/4102ST rectangular cavity of TE,,, mode. Standard instrument settings were as follows: microwave frequency, 9.77 GHz; power, 10 mW; modulation amplitude, 1.6 G (peak to peak); modulation frequency, 100 KHz; time constant, 0.5 s. For the photochemical experiments, the output of a 300-W xenon lamp (Varian Model PS300-1) operating at 15-18 A was filtered through three Pyrex glass plates (-1 cm; transmitting X > 310 nm) and directed into the EPR cavity through a 50% transmittance irradiation grid. Unless otherwise noted, the light intensity at the exterior of the grid was 175 mW/cm2 as measured with a YSI Model 65A radiometer. The time course of nitroxide consumption was followed at the field position corresponding to the maximum of the low-field hyperfine line, and initial rates of nitroxide consumption were calculated from the amplitude changes measured in these time courses when the consumption of nitroxide was 510%.
Results For nitroxides I-V, the line widths, nitrogen hyperfine splittings, and g values were identical in the presence and absence of HA, indicating that immobilization of these nitroxides by binding to HA does not occur under the conditions of these experiments (32). Also, at the instrument settings used, nitroxide-nitroxide or nitroxide-02 spin-exchange interactions did not visibly alter the line widths. That an exchange mechanism did not significantly broaden the lines was also indicated by the linear response of the signal amplitude of the low-field hyperfine line to nitroxide concentrations over the range of 8-400 pM in the presence and absence of air. Thus, nitroxide concentrations were calculated by comparison of the amplitude of the low-field line of a sample to curves of amplitude versus concentration determined from the spin standards. Incubation of HA with nitroxides I-V in the absence of light did not produce a detectable loss of nitroxide spin, thus showing that these species do not react thermally under the experimental conditions. In the absence of HA, irradiation of nitroxides I-V for 10 min in the presence or absence of air caused no signal loss. However, substantial losses were detected in solutions containing HA. Figure 2 shows typical kinetic traces of 4-hydroxy-Tempo consumption obtained upon irradiation of 250 bg/mL solutions of HA equilibrated with Ar or air. At low concentrations, the loss of 4-hydroxy-Tempo did not follow first-order kinetics (Figure 2A), which suggests that nitroxide recycling may be significant, thus requiring that initial rates of consumption be measured (24,25). At a constant initial concentration of 4-hydroxy-Tempo, the initial loss rate under air or Ar decreased linearly with decreasing HA concentration (Figure 3) and light intensity (Figure 4). The dependence of the initial rates of loss on initial nitroxide concentration was influenced significantly by the Under charge on the nitroxide and by the presence of 02.
'
hv0Ni
hvOFF
AIR
AR AIR
8 D 42
-
46 -
20 24 28 32 36 0
200
600
400
Time l s l Figure 2. Typical kinetic traces of 4-hydroxy-Tempo consumption photosensitized by 250 pg/mL HA in pH 8.0, 10 mM phosphate buffer equilibrated with air or Ar, temperature 23 f 2 OC. Initial nitroxide concentration: (A) 10 KM; (B) 25 pM; (C) 50 HM; (D) 100 wM. Light intensity was 175 mW/cm2 (A > 310 nm) as measured at the exterior of the 50% optical transmittance grid of the EPR cavity.
0
Humic Acid Conceniraiion h g d , Flgure 3. Dependence of the initial rate of 4-hydroxy-Tempo loss on the concentration of HA equilibrated with Ar (W) or air (0). Initial nitroxide concentration was 100 pM; light intensity and other conditions were as in Figure 2.
Ar, increasing the concentration of the neutral 4hydroxy-Tempo from 10 to 100 pM increased the initial rate of its consumption from 4 to -7 pM min-l (Figures 2 and 5), while a further increase to 300 pM produced only a small additional increase in the rate to 8 pM min-' (Figure 5). In contrast, the initial rates of loss for the cationic 4-amino-Tempo (4-amino-2,2,6,6-tetramethylpiperidinyloxy) rose from -8 pM min-l to a plateau value of 11-12 pM min-l over the same concentration regime. In the presence of -250 pM 0, (air), the loss of 4hydroxy-Tempo decreased to 1 pM min-' and became nearly independent of concentration, while the loss rate of 4-amino-Tempo increased dramatically with concentration, rising from a low of 1pM min-l at 10 pM to 10 pM min-' at 375 pM (Figure 5). Identical trends were observed in the initial rate profiles of the Proxy1 nitroxides (Figures 1and 6). Under Ar, the
-
Environ. Sci. Technol., Voi. 22, No. 1, 1988 79
8 I
Figure 4. Dependence of the initial rate of 4-hydroxy-Tempo loss on light intensity for solutions equilibrated with Ar) . ( or air (0). Initial nitroxide concentration was 100 pM; HA concentration was 250 pg/mL. Light intenstty was reduced by decreasing the current to the lamp. Light Intensity was measured as described under Materials and Methods.
0
“0
100 200 300 /nitia/ Mtroxide Concentration(uM)
400
Figure 5. Dependence of the initial rate of loss of 4-amino-Tempo (open symbols) and 4-hydroxy-Tempo (solid symbols) on initial concentration under Ar).( or air (0).HA concentration and light intenstty were as in Figure 2. The solid lines represent the predictions of a three-pool model (eq 12);the parameters are given in Table I.
rate of consumption decreased in the order cationic (V) > neutral (IV) >> anionic (111),and for each nitroxide, the presence of 0, reduced the rate of loss. Compared to the corresponding Tempo nitroxides, the initial rates of loss of the cationic and neutral Proxyl nitroxides were approximately 30% lower. Differences of this magnitude are close to the uncertainty estimated for the calibration of absolute concentration for Proxyl and Tempo derivatives in this study and, thus, do not appear to reflect a meaningful difference in loss rates. While the estimated uncertainty in the absolute rate measurements is -3O%, the uncertainty (reproducibility) in the rate measurements for nitroxides of a given derivative type is closer to 10% (Figure 5).
Discussion That the consumption observed in this study could arise predominately from the quenching of long-lived HA triplets via electron transfer to the nitroxides appears highly unlikely, given the low electron affinity of nitroxides (42). Hydrogen atom abstraction by excited-state nitroxides is also unlikely, as this process is very inefficient (24; N. V. Blough, unpublished observations). The protective effect of O2must then result either from quenching of the excited states giving rise to radicals (h,
