J. Phys. Chem. lQ83, 87, 1809-1812
1809
Kinetics and Mechanism for the Oxidation of Ascorbic Acid/Ascorbate by H02/02Radicals. A Pulse Radiolysis and Stopped-Flow Photolysis Study Dlane E. Cabelll and Benon H. J. Blelskl" Department of Chemistry, Brmkhaven National Laboratory, Upton, New York 11973 (Received: October 11, 1982)
The reaction between AH2/AH- and HOz/02-was followed from pH 0.3 to 11, leading to the following rate constants: k8 = 1.6 X lo4 M-ls-l for the reaction between AH2 and HO,, k17 + 0.365k18= 1.22 X lo7 M-' s-l for the reactions between AH- and H02and AH2and Of, respectively, and k19 = 5 X lo4M-' s-l for the reaction between AH- and 0,. The radical-radical reactions of A-- with H02and Of were measured and found to proceed at rates of k9 = 5 X lo9 s-l and kll = 2.6 X lo8 M-' s-l, respectively. An overall mechanism for the oxidation of AH2/AH- by HOz/Oz-consistent with the experimental results is also proposed.
Introduction While ascorbic acid is best known for its antiscorbutic properties and its wide-ranging role in biological processes,l chemists use it extensively as a chelating and reducing agent.24 Its efficiency as an antioxidant is most likely due to a combination of the chemical properties of various components of the ascorbic acid system, namely, ascorbic acid (AH,), ascorbate (AH-), the ascorbate free radical (A-a), and dehydroascorbic acid (A). While AH2/AH- is a very efficient radical scavenger (OH, H02/02-, R.,RO., RO,., etc.), A-. is relatively unreactive toward other chemical compounds and reacts preferentially with itself or other free radicals, thus terminating the propagation of free-radical reaction^.^,^ In view of the wide use of ascorbic acid in aerobic systems, the importance of understanding the chemical reactions which it and its free radical undergo with molecular oxygen or its derivatives (OH, H02/02-, lo2, H202)is apparent. Despite numerous investigations on the autooxidation of AH,/AH-, the mechanism(s) of how electrons are transferred to oxygen or hydrogen peroxide and the subsequent steps in the oxidation process remain obcure.^-^ Studies involving superoxide dismutase,'O the enzyme that catalyzes the disproportionation of H02/02-, suggests that 0,- might be generated as a transient during autooxidation: AH- + 02 A-e + 0 2 - + H+ (1) The absence of superoxide radical in neutral/alkaline oxygenated ascorbate solutions led to the suggestion that autooxidation of ascorbate may involve a single two-electron oxidation step:l1 AH- + 02 + H+ A + H202 (2) The present investigation is a systematic study of the reaction mechanisms by which H02/02-oxidize AH2/AHand its free radical A-.. Although some of these reactions and their corresponding mechanisms had been studied previously by a variety of techniques (60Coy radiolysis,12
-
-
(1)Second Conference on Vitamin C: King, C. G., Burns, J. J., Eds. Ann. N . Y. Acad. Sci. 1975,258. (2)Pelizzetti, E.; Mentastic, E.; Pramauro, E. Inorg. Chem. 1978,17, 1181. (3)Krishnan, C. V.; Sutin, N. J. Am. Chem. SOC.1981, 103, 2141. (4)Creutz, C. Inorg. Chem. 1981,20, 4449 and references therein. (5)Bielski, B. H. J.; Richter, H. W.; Chan, P. C. Ann. N . Y. Acad. Sci. 1975,258,231. (6)Packer, J. E.; Slater, T. F.; Willson, R. L. Nature (London) 1979, 278,737. (7)Weissberger, A.; LuValle, J. E.; Thomas, D. S., Jr. J. Am. Chem. SOC.1943,65,1934. (8)Blaug, S. M.; Hajratwala, J. J. Pharm. Sci. 1972,61, 556. (9)Fedorova, D. S.;Brednikov, V. M. Khim. Vys. Energ. 1978,12,463. (IO) Puget, K.,Michaelson, A. M. Biochimie. 1974,56, 1255. (11)Yamazaki, I.; Piette, L. H. Biochim. Biophys. Acta 1961,50,62. 0022-365418312087-1809$01 .50/0
flash photoly~is,'~ stopped-flow photolysis,14enzymes," pulse radiolysis15J6),unambiguous kinetic parameters over a broad pH range (0.3-11) had never been established.
Experimental Section Chemicals and Solutions. All aqueous solutions were prepared with water which, after distillation, had been passed through a Millipore ultrapurification system. Sodium formate, trisodium phosphate (both Baker analyzed reagents), and EDTA (Fisher Scientific Co.) were purified by repeated recrystallizations as described previ0us1y.l~ Both ethanol (USP 190 proof, US. Industrial Chemical Corp.) and ascorbic acid (Fisher Scientific Co.) were used without further purification. The pH of the solutions was adjusted by the addition of potassium hydroxide (Baker analyzed reagent), sulfuric acid ("Aristar" from BDH Chemicals Ltd.),and/or trisodium phosphate. Oxygenated solutions were prepared by bubbling with UHP oxygen (99.99%, Matheson Co.). Equipment. Pulse radiolysis experiments were carried out by using a 2-MeV Van de Graaff generator with 0.41.8-~spulses and doses varying from 100 to 900 rd. The data were analyzed by a PDP-11 computer interfaced to both the Van de Graaff and the photodetection system. The number of free radicals formed per pulse was computed by using (SCN),- as the calibrant and taking G(-(SCN),-) as 6.13 and the molar absorbance of the radical at 472 nm as 7575 M-' cm-l.l' The spectral path length in these experiments was 6.1 cm. A Durrum Model D-110 stopped-flow spectrophotometer modified by the attachment of a Xe-plasma lamp was used as described previo~sly.'~J~ All routine spectrophotometric measurements were made on a Cary 210 UV/vis spectrophotometer. Results Pulse Radiolysis. The radiolysis of water leads to the formation of the following radical species:20 HzO OH (2.75), ea; (2.65), H (0.651, Hz02(0.70), HZ(0.45) (1)
--
(12) (a) Sadat-Shafai, T.; Ferradini, C.; Julien, R.; Pucheault, J. Radiat. Res. 1979,77,432. (b)Rao, B. S. N. Ibid. 1962,5, 683. (c) Barr, N. F.; King, C. G. J. Am. Chem. SOC.1956,78, 303. (13)Nadezhdin, A. D.;Dunford, H. B. Can. J.Chem. 1979,57,3017. (14)Bielski, B. H. J.; Richter, H. W. J.Am. Chem. Soc. 1977,99,3019. (15)Elstner, E.F.;Kramer, R. Biochim. Biophys. Acta 1973,314,340. (16)Allen, J. F.; Hall, D. 0. Biochem. Biophys. Res. Commun. 1973, 52,856. (17)Schuler, R.H.; Patterson, L. K.; Janata, E. J. Phys. Chem. 1980, 84,2089. (18)Bielski, B. H. J.; Gebicki, J. M. J.Am. Chem. Soc. 1982,104,796. (19)Holroyd, R. A.; Bielski, B. H. J. J. Am. Chem. Soc. 1978,100, 5796.
0 1983 American Chemical Society
1810
The Journal of Physical Chemistry, Vol. 87, No. 10, 1983
Cabelli and Bielski
TABLE I : Observed Rate of Reaction between AHJAH- (Atot) and HO,/O,- ( R )at Variable pH As Determined by Pulse Radiolysis and Stopped-Flow Photolysis kobsd21
PH 0.36 0.56 0.76 0.83 1.09 1.43 1.52 1.66 1.72 2.23 2.98 3.26 3.98 4.22 1.6 4.75 4.9 a
M" s -
Atot, PM
1000, 500
4-6 4-6 4-6 4-6 4-6 4-6 6-11 4 -6 3.1-3.3 3.1-3.3 3.3 3.3 3.3 3.3 5-15 3.3 5-15
500 5 00 500
1000, 500 1000, 500 1000 1000, 5 0 0 25 25 21-107 15-25 25-50 27.4 96.5 27.4 96.5
method'
3.15 x 1 0 4 3.3 x 104 3.2 x 104 3.2 x 104 3.7 x 104 6.2 x 104 4.2 x 104 9.5 x lo4 2.0 x 105 3.4 x 105
1.0 x lo6 1.9 x 1 0 6
5.5 8.8 7.6 8.7 7.3
lo6 X lo6 x
lo6 lo6 x lo6 X X
obsd,
M-I ..-1
PH 5.11 5.27 5.68 6.0 6.2 6.35 6.71 7.25 7.31 7.67 7.94 8.14 8.20 9.43 10.37 11.04
96.5 96.5 27.4 125 125 125 125 200 27.4 200 200
6.5 X l o 6 5.2 X l o 6 3.3 x 106 1.5 X lo6 8.9 X l o 5 7.3 x l o 5 3.6 X l o 5 2.1 x 105 3.1 X l o 5 1.4x 105 1.2 x 105 1.2 x 105 9.9 x 104 9.9 x 104 9.3 x 104 9.5 x 104
5-15 5-15 3-3 4-13 4-13 4-13 4-13 4-6 3-3 4 -6 4-6 4 -6 4 -6 4-6 4 -6 4-6
100 200 200 200 200
methoda pr Pr Pr pr pr pr pr sf pr sf sf
sf sf Sf
Sf
sf
pr = pulse radiolysis; sf = stopped-flow photolysis.
where the values in parentheses are G values, that is, the number of radicals produced per 100 eV of energy dissipated in the system. The addition of sodium formate to an oxygenated aqueous solution leads, upon radiolysis, to the following reactions: eaq-+ O2 H
+ 02
-
+
02-
HOz
HOz + H+ + 02-
+ HCOZ- H2O + C02coz- + 02 coz + 02-
OH
-
-+
(3) (4)
H.J . Chem. Educ. 1981, 58, 101.
AH2
(5-5) (6)
(7)
thus converting the primary radicals to H02, OZ-, or both dependent only upon the pH since reaction 5 is more rapid than any of the other reactions in an aqueous solution. If the concentration of formate is at least 2 orders of magnitude greater than the concentration of AHz/AH-, virtually all of the OH radicals will react according to reaction 6 rather than reacting directly with AH2/AH-. The observed rate of reaction of AHz/AH-with H02/O; was determined under pseudo-first-order conditions (0.1 M formate, 1.2 mM Oz, 0.05-1 mM AH,/AH-, 3-9 pM H02/02-,21.5 "C, variable pH). The molar absorbance of AHz/AH- was measured spectrophotometrically and, since it varies markedly with pH, the rates of reaction were determined by following the disappearance of the absorbance between 250 and 290 nm, wherever the signal-tonoise ratio was largest. Observed rates for this reaction are tabulated in Table I. Pulse radiolysis was a viable technique only from pH 1.5 to 8.0 as at or below pH 1.5 the absorbance of AHz/AH- is sufficiently blue shifted as to be obscured by the presence of formate. Also, above pH 8.0 ascorbate solutions are not sufficiently stable in the presence of oxygen to be used in pulse radiolysis studies. Stopped-Flow Photolysis. The generation of H02/Ozin oxygenated aqueous solutions containing ethanol as a primary radical scavenger has been discussed in great detail elsewhere and will not be repeated.18 The rate of reaction between AH2/AH- and H02/02-,upon mixing in a 1:l volume ratio in the stopped-flow apparatus, was determined under pseudo-first-order conditions (50 mM EtOH, 0.5-1.0 mM AH2/AH-, 0.6 mM 02,10 pM EDTA, 3-10 pM H02/02-,24 "C, pH 0.3-1.5, 8.0-11.0). The rate (20) Schwarz,
of reaction between AH2/AH- and HOz/02- was again determined by following the disappearance of the absorbance of AH2/AH-; observed rates are tabulated in Table I. Radical-Radical Reactions. Although the reaction between AH2and H02has been postulated to proceed by the following mechanism12J3
+ HO2 A-e
-.*
+ HO2
A-. +
+ H+ + HzO2 A
+ Hog-
(8)
(9)
-
no direct observation of reaction 9 has been reported; a value of kg log M-' s-l was obtained by Sadat-Shafai et al. from 6oCostudies.12 Using pulse radiolysis, one can determine values for kg with two alternative experimental procedures. A competition between reactions 8 and 9 can be established by varying the dose (i.e., quantity of H 0 2 formed) and varying the initial AH2 concentration. The quantity of A-. being formed can be determined by measuring the absorbance of A-. at 360 nm since the molar absorbance of A-. is well established.21 Using this method, we determined a lower limit for the rate of reaction 9 to be of the order of log M-' s-l, in agreement with the value reported previously. The rate of reaction 9 can, however, be measured directly in an oxygen-saturated AH2 solution between pH 1 and 3. Upon radiolysis, HOz is formed according to reactions 3-5 but, since this solution no longer contains formate, the OH radicals will now react directly with AH2to yield AH2 + OH
-
A-a:
A-.
+ H++ H20
(10)
When one chooses a suitable concentration of AH2 (0.1-1 mM), tl12of reaction 8 is of the order of 0.5-5 p ~ . ~Since ' reactions 3-5 go to completion within 1-2 ps, within a few microseconds of the pulse the radical mixture in solution consists almost totally of A-. and HOP. The reaction between AH2 and HOz at these concentrations occurs on a seconds time scale and can be neglected. The disproportionation of A-. has been shown previously to occur at lo8 M-'s-' (ref 24) in this pH range and is therefore 1 order of magnitude slower than the lower limit for k , that we found in the competition study. Hence, the rate of disappearance of A-., measured at 360 nm, represents the rate of reaction of A-. and HOP This reaction, measured at pH (21)Schuler, R.H.Radiat. Res. 1977, 69,417.
The Journal of Physical Chemistry, Vol. 87, No. 10, 1983 1811
Oxidation of Ascorbic Acid/Ascorbate
2.72 and 1.42 and variable dose, leads to a rate constant
k, = (5.0 f 0.5) X lo9 M-I s-l. An analogous experiment was carried out at pH 8; here the radicals in solution are A-e and 02-: A-. + 02- product (11) At both pH 7.8 and 8.0 the disappearance of A-. was monitored under experimental conditions similar to those described for measuring reaction 9 and a rate was determined, kll = (2.6 f 0.4) X lo8 M-’ s-l.
-
Discussion The following chemical properties and some well-established reactions of AH2/AH- and A-. are relevant to this study. Ascorbic acid has two pKs, one at 4.25,, which represents the equilibrium AH2+ AH- H+ and another at 11.7922which represents AH- * A2- + H+. The redox potential of ascorbic acid is -0.70 V, pH 6.4.4 AH2/AHcan undergo a reversible Michaelis two-step oxidationreduction process with the formation of a free radical A-. (pK = -0.45 for AH. e A-. + H+)23as an intermediate:
u
+
-
AH-
+le
A-.
A-.
1
+ H+
t
-le
e A +le
‘“‘0
1
< 2
4
6
8
IO
12
PH Although the ascorbate free radical was first discovered during the oxidation of ascorbate by an enzyme,l’ its Figure 1. Plot of k , vs. pH as determined by (0) stopped-flow properties were mainly determined by o p t i ~ a 1 ~and ~ * ~ ~ vphotolysls ~ ~ and (0)pulse radiolysis and a theoretical profile of k, vs. pH (-) as computed by eq I V . ESR-pulse radiolysis e ~ p e r i m e n t s . ~In~ the > ~ ~absence of other reactants, the free radical disproportionates to assince it could not be determined experimentally. In view corbate and dehydroascorbic acid by a proton-dependent of this, we are assuming a 2:l ratio under the pseudosecond-order process which involves a transient dimer:24 first-order conditions with which we measured the rate A-. + A-. A22(14) constants. Under our previously described experimental conditions, A,2- H+ AH- + A (15) reactions 9 and 11occur at rates considerably faster than A22- HzO AH- A OH(16) those for reactions 8,17, and 18. Therefore, as soon as A-. is formed in these reactions, it reacts with the remaining The reaction mechanism for the oxidation of AH2/AH- by H02/02- in the system, giving steady-state conditions. H02/02-over the pH range from 0.3 to 11is described by Hence, the overall stoichiometry of superoxide/perthe following reactions: hydroxyl radicals to ascorbic acid/ascorbate consumed is AH2 HO2 A-* H+ H202 (8) 2:l or AH- HO2 A-* H202 (17) -(dR/dt) = 2k8[AH2][HO2] 2k1,[AH-][HO,] + AH2 02- A-. H202 (18) 2k18[AH2][02-1 + 2kIg[AH-]LO,-] (111)
+
+
+
- +
+ + + - +
-+ + +
AH-
+ 02-
+
transient adduct or product
(19) A-. HOz A + HO; (9) A-* 02- HzO A HO2- + OH(11) The overall change in superoxide/perhydroxyl radical concentration (where R = [HO,] + [Of]) with time in this system is given by -(dR/dt) = ks [HO,][AHz] + k17[HO2][AH-] + ~ 1 8 [ 0 2 - 1[AH21 + k19[0,-I [AH-] + k9[A-.l [I3021 + kii[A-*lIO,-I (11) The observed stoichiometry of 0; to AH- consumed was 2:l when reaction 19 was measured under strictly second-order conditions by mixing equimolar quantities of AH- and 02-at pH 8.6. Whether the same stoichiometry prevails under pseudo-first-order conditions is uncertain +
+
+
+
+
(22) ‘Handbook of Chemistry and Physics”, 56th ed.; CRC Press: Boca Raton, FL, 1976; p D150. (23) Laroff, G. P.; Fessenden, R. W.; Schuler, R. H. J.Am. Chem. SOC. 1972, 94, 9062. (24) Bielski, B. H. J.; Allen, A. 0.; Schwarz, H. A. J. J.Am. Chem. SOC. 1981,103, 3516. (25) Schoneshofer, M. 2.Naturforsch. B 1972,273, 649. (26) Fessenden, R. W.; Verma, N. C. Biophys. J. 1978,24, 93.
In this system both ascorbate and superoxide radicals are in equilibrium with their conjugate acids. Therefore, [AH-] = [AH,](Km,/[H+]) where Km2/[H+]is defined as X and [02-] = [ H 0 2 ] ( K ~ ~ T / [ Hwhere + ] ) KHOz/[H+] is defined as Y. The total radical concentration reduces to R = [H0,](1 + y) and the total ascorbic acid concentration ( A , = [AH,] + [AH-]) reduces to A,, = [AH2](1+ X). Substitution of the above relationships into eq 111, using values of 4.25 for KAHzand 4.7 for KH02,27 yields 2k8 2k17X + 2k18Y 2k1gXY (IV) kobsd = (1 + X)(1 + Y)
+
+
In the very low pH region (plateau, pH 0.3-1.0; see Figure 1)where k&d does not change with pH, the terms involving kl,,k18, and klg are negligible and eq IV reduces to kobad = 2k8 SO k8 = 1.6 X lo4 M-’ s-’. In the very high pH region (plateau, pH 8.2-11.0; see Figure 11,the terms involving he, k17, and k18 are negligible and (1 + X)(1 + Y) N xu,so eq I v reduces to k&sd = 2kI9 and k19 = 5.0 X 104 M-l s-’. As Km, is very similar to KHG, the two cross reactions (reactions 17 and 18) are never present singly but (27) Bielski, B. H. J. Photochem. Photobiol. 1978, 28, 645.
1812
J. Phys. Chem. 1983, 87,1812-1818
always in conjunction with one another. Therefore, since a line-fitting process to obtain the best values for k17 and k,, independently leads to rather arbitrary values, the definition of a composite rate constant appears more practical as well as more exact, k17+ 0.356k18= 1.22 X lo7 M-' This composite rate constant was calculated by using the aforementioned values for k,, k19,X , and Y and the experimentally determined values for kobsdin the pH range 2-7. Although k l , and k,, can be varied, the relationship k,: + 0.356 = 1.22 X 10' M-' s-l was found to be very exact. Using these rate constants, k,, k17+ 0.356k18,and k19, one can calculate a koM vs. pH curve (see Figure 11, in good agreement with the experimental values. As discussed previously, the reaction between AH2/AHand H02/02-(reactions 8,17, and 18) was measured under steady-state conditions. If, however, the AH2/AH- concentration is increased substantially, all of the HOz/Ozis consumed in reaction with AH2/AH- (reactions 8, 17, and 18) and the A-- that is formed subsequently disappears by reactions 14-16. Since the molar absorbance of A-. is well established (+60nm = 3300 M-' cm-' (ref 21)), the quantity of A-. that is formed can be measured. It was found that at pH 6-7 (0.5 M AH2/AH-, 5 X lo4 M 02-/ H02) the formation of A-e was quantitative while at pH 8.5-9.5 (at pH 9.5 02- was added anaerobically in order to eliminate the formation of A-. by an autooxidation process) no A-. was observed. These experiments indicate that, although reactions 8,17, and 18 lead to the formation of A--, reaction 19, the reaction between AH- and 02-,does not yield A-.. As discussed previously, a further probe of reaction 19 involved mixing equimolar amounts of AH- and 02-at pH 8.6. Since the ratio of 02-to AH- consumed was 2:1, the product of reaction 19 either reacts rapidly with another
02-radical or disproportionates in a manner similar to A-. (reactions 14-16). The possibility that AH- reacts with 02-in a single two-electron step as suggested previously'l cannot be ruled out at this point. Unfortunately, none of these possibilities for reaction 19, can, at present, be either verified or eliminated. Conclusion The presented results indicate that the oxidation of AH2/AH- by H02/Oz-proceeds with the following rates: AH2
+ HO2
AH-
ks = 1.6 X lo' M-'8-l
+ 02-
A-.
kls = 5 x 104 M-,
+ H202 + H+
* product
In addition, the radical-radical reactions between A-. and H02/02-proceed by the following rates:
A-* + HO2 A-.
+ 02-
kg = 5
X
lo9 M-l 8-l
+ H02A + HOZ- + OH+
k,, = 2.6 X lo8 M-l e'l +
H2'3
A
Acknowledgment. We thank Dr. H. A. Schwarz for many stimulating discussions on the present work and Mr. D. A. Comstock for excellent technical assistance. This research was carried out at Brookhaven National Laboratory under contract with the U.S.Department of Energy and supported in part by its Office of Basic Energy Sciences. Registry No. AHz, 50-81-7;AH-, 299-36-5;HOz, 3170-83-0; 02-, 11062-77-4;A-., 34481-26-0.
Kinetics of the Reaction of Chlorine Atoms with Vinyl Bromide and Its Use for Measuring Chlorine-Atom Concentrations Jong-Yoon Park, Irene R. Slagie, and David Gutman' Department of Chemistry, Illinois Institute of Technology, Chicago, Illlnois 606 16 (Received: October 27, 1982)
The reaction of chlorine atoms with vinyl bromide, C1+ C2H3Br CzHBCl+ Br, has been tested as a titration reaction for chlorine atoms in gas-phase fast-flow reactors using both continuous and pulsed atom sources. The vinyl chloride produced by this reaction was determined by using photoionization mass spectrometry and was found to be an accurate measure of the in situ chlorine-atomconcentrationdown to 5 X 1OO ' atoms ~ m - The ~. reaction is rapid and stoichiometricand involves the use of easily handled substances. It is particularly well suited for use with photoionization mass spectrometer detectors because of the low ionization potentials and high photoionization cross sections of both vinyl bromide and vinyl chloride. The rate constant of this reaction cm3 molecule-' s-l. was measured at 298 K and determined to be 1.43 (f0.29) x -+
Introduction The rapid reactions of chlorine atoms with organic molecules have been found to be useful sources of polyatomic free radicals in many chemical kinetic studies. In most investigations it has not been necessary to accurately know the concentration of either the chlorine atoms or the free radicals which were produced. These studies include ones conducted under photostationary-state conditions, where rates have been measured and mechanisms established, and others in which the reaction of the free
radical of interest could be isolated for quantitative study under pseudo-first-order conditions. Examples of the first kind of such Studies include the many Smog-chaber experiments, particularly by Niki and co-workers,1*2 which (I) Niki, H.; Maker, P. D.; Savage, C. M.; Breitenbach, L. P. Chem. 1978,59,78;1979, Phys. Lett. 1974,&, 567;1978,55,289;1978,57,596; l9809 739 43; 1980*759 533. 61, (2)Niki, H.; Maker, P. D.; Savage, C. M.; Breitenbach, L. P. J.Phys. 1982,86,3825-9; Int. J.Chem. Kinet. Chem. 1980,84,14;1981,85,877; 1980,12,100, 915.
0022-3654/83/2087-1812$01.50/00 1983 American Chemical Society
