NOTES
Finally, since the formation of biphenyl from the reaction of (C6H5),& species (where X is a heteroatom) with alkali metals is a fairly common occurrence and can cause complications in the interpretation of e.s.r. spectra, it was considered of interest to analyze several of the radical-containing solutions for biphenyl. An estimate of the maximum concentration of biphenyl which might be present was obtained. It was in the
2779
range 2-5 X loV5M for the various metal radical solutions. Therefore, the effect of biphenyl was considered to be negligible.
Acknowledgments. We wish to express our appreciation to Professor S. I. Weissman for his help in obtaining several of the spectra. We also thank Mr. T. F. Wulfers and Mr. P. L. Hall for their aid in the vapor phase chromatographicanalysis.
NOTES
On the Chlorophyllide- Sensitized Reduction of Azobenzene and Other Compounds by G. R. Seely Contribution No. 186 from the Charles F. Kettering Research Laboratory, Yellow Spring& Ohw (Received February 8, 1966)
We have found1 that the reduction of phenosafranine by hydrazobenzene in ethanol-pyridine mixtures, photosensitized by ethyl chlorophyllide a, is not retarded by one of its products, azobenzene, a t 1.7 X 10-4 M . This suggested that excited chlorophyllide, Chl*, reacts much faster with phenosafranine than with azobenzene. We have now found that azobenzene at the same concentration almost completely prevents the photochemical reduction of ethyl chlorophyllide by ascorbic acid in ethanol-pyridine mixtures.2pa At a lower azobenzene concentration (4 X 10" M ) , where the reduction of chlorophyllide is still strongly retarded, the loss of azobenzene can be followed by the decline of absorptivity at 335 mp. As the azobenzene disappears, the reduction of chlorophyllide is accelerated. Without ascorbic acid there is no loss of azobenzene; there is no reaction in the dark. Livingston and Pariser reported that chlorophyll sensitized the photoreduction of azobenzene by phenylhydrazine, but not by ascorbic acid, in methanol s ~ l u t i o n . ~ It seemed likely that azobenzene was reduced to hydrazobenzene in this reaction. To see whether this was so, or whether azobenzene was converted to
some other product, we tried hydrazobenzene as a reductant, in 1:6 ethano1:pyridine containing 0.05 M benzoic acid. (Hydrazobenzene is inert as a reductant for Chl* in ethanol-pyridine mixtures, unless acid is present.) As expected, the absorption at 335 mp did not remain constant or decrease but slowly increased by an amount commensurate with the amount of chlorophyllide reduced. We believe that azobenzene is reduced by the radical ChlH. formed in the reduction of chl~rophyllide~ for the following reasons. Azobenzene and ChlH2, the stable photoreduction product of chlorophyllide (absorption maximum at 525 mp),2coexist in the dark without reaction, which rules out ChlH2 as the effective reducing agent. Increasing the ascorbic acid concentration from 2 X to 2 X M accelerates both the reduction of chlorophyllide and the reduction of azobenzene, but the former more than the latter. This is compatible with the mechanism (I) Chl*
+ AH2
4ChlH.
+ AH-
+ ChlH2 ChlH. + D +Chl + DH. 2ChlH. +Chl
(1) (2)
(3)
D and AH2 represent the oxidant and the reductant, in this case azobenzene and ascorbic acid. The product of step 1 is written ChlH. because transfer of a (1) G. R. Seely,J. Phys. Chem., 69,2633 (1965). (2) A. A. Kraanovskii, DON.Akad. Nauk SSSR, 60, 421 (1948). (3) G. R. Seely and A. Folkmanis, J. Am. Chem. SOC.,86, 2763 (1964). (4) R.Livingston and R.Pariser, ibid., 78, 2948 (1956). . (5) V. B. Evstigneev and V. A. Gavrilova, Dokl. Akad. Nauk SSSR, 92, 381 (1953).
Volume 69,Number 8 August 1966
NOTES
2780
proton to chlorophyll appears to be indispensable for its photoreduction by ascorbic acid.3 The quantum yield of reduction of azobenzene is not more than about twice that of reduction of chlorophyllide in the absence of azobenzene. Reduction of azobenzene is slow and incomplete in ethanol without pyridine, as is the reduction of chlorophyllide.a The ethyl chlorophyllide sensitized reduction of the azo dye, methyl red, by ascorbic acid, hydrazobenzene, and mercaptosuccinic acid in ethanol goes by a different mechanism (11)6
Chl*
+ D +Chi*+ + D*- (+ H+ +DH.) Chl*++ AH2 +Chl + AH. + H+
(4) (5)
Other compounds for which there is evidence of photoreduction by this mechanism are phenosafranine,' quinones,7 pyocyanine, and phenazine methosulfate.g The existence of different mechanisms for the sensitized reduction of two such apparently similar compounds as azobenzene and methyl red gives rise to the question of which mechanism operates in other cases. They differ in that mechanism I requires conditions under which the sensitizer is reduced. In practice, this means that hydrazobenzene and mercaptans are much less effective than ascorbic acid in (I). Ascorbic acid in ethanol, without base, is active in (11) but not in (I). Hydrazobenzene is active in (I) only in the presence of acid (e.g., pyridiniwn benzoate). With ascorbic acid in pyridine-ethanol, the bimolecular rate const,antfor step 1is 1.8 X lo5M-I sec.-lla but the rate constant for step 4 is around lo9 M-' in those cases studied.lV6J0 The disparity is so great that (I) can only be seen when (11) is inoperative. With ascorbic acid, quantum yields by ('I)are typically or less, whereas initial quantum yields by (11) are typically -10-' or more; however, exceptions must be expected. We now attempt to decide by which mechanism chlorophyll and related compounds perform some of the sensitized reductions reported in the literature. Livingston and Pariser noted a considerable difference in the kinetics of the sensitized reductions of methyl redll and butter yellow.12 We have found that methyl orange (butter yellow sulfonate) is reduced in much the same way as is ambemene, and we therefore assign (I) it and butter 'In the reduetion Of butter yellow, the Of aniline is probably that of activating basela for the reduction of pheophytin. The description of the reduction of fast red s (2hydroxy-1,lr-azonaphthalene-4'-sulfonicacid, sodium The Journal of Physical Chemistry
salt) by chlorophyllin in water certainly supports the mechanism (I) assigned to it.14 However, we have found that fast red S is reduced by hydrazobenzene in ethanol, ethyl chlorophyllide as sensitizer, with a similar quantum yield (-lo+). Mechanism I is inoperative under these conditions. It is possible that the mechanism depends on the solvent or the sensitizer or that the dye reacts via (11) only in its quinone hydrazone tautomeric form. (The maximum absorption in water was at 480 mp14; in ethanol, 510 mp.) Thionine16and riboflavinI6are probably reduced by ascorbic acid v i a (11) because of the high quantum yield. For riboflavin, this conclusion is supported by e.s.r. measurements.'' Safranine T is reduced via (11), analogously to phenosafranine.16,18 The bacteriochlorophyll-sensitized reduction of ubiquinone by reduced phenazine methosulfatel9probably goes via (11). We found that triphenyltetrazolium chloride (1.6 X M ) was reduced rapidly by both ascorbic acid (4.1 X M ) and hydrazobenzene (3.7 X M) in 15 vol. % aqueous pyridine, with ethyl chlorophyllide as sensitizer. The complications encountered with hydrazine20 were absent in these systems. This salt and probably also tetrazolium blue21 are reduced by
(11). The sensitized reduction of pyridine nucleotides22-26 (6) G. R. Seely, J . Phys. Chem., 69,821 (1965). (7) A.A.Krasnovakii and N. N. Drozdova, Dokl. Akad. Nauk SSSR, 150, 1378 (1963); 158, 730 (1964). (8) G. Tollin and G. Green, Biochim. Biophys. Acta, 60,524 (1962). (9) B. Ke, L. P. Vernon, and E. R. Shaw, Biochemistry,4,137 (1965). (IO) E.Fujimori and R. Livingston, Nature, 180, 1036 (1957). (11) R.Livingston and R. Pariser, J. Am. Chem. SOC.,70,1510 (1948). (12) R. Livingston and R. Pariser, ibid., 78, 2944 (1956). (13) A. A. Kraanovskii, G. P. Brin, and K. K. Voinovskaya, Dokl. A M . Nauk SSSR, 69, 393 (1949). (14) G. Oster, J. S. Bellin, and 5. B. Broyde, J . Am. Chem. Soc., 86, 1313 (1964). (15) K. G. Mathai and E. Rabinowitch, J. Phys. Chem., 66, 954 (1962). (16) A. A. Kraanovakii, DolcE. Akad. Nauk SSSR, 61, 91 (1948). (17) G. T o h and G. Green, Biochim. Biophys. Acta, 66,308 (1963). (18) T.T.Bannister, Photochem. Photobiol., 2 , 519 (1963). (19) W.S. Zaugg, L. P. Vernon, and A. Tirpack, Proc. Natl. Acad. Sd.,51, 232 (1964). (20) E. Fujimori, J . Am. Chem. Soc., 77, 6495 (1955). 15, 1639 (1961). (21) L. P. Vemn, A d a Chem. (22) A. A. Krasnovskii and G. P. Brin, Dokl. Akad. Nauk SSSR, 67, 325 (1949). (23) G. P. Brin and A. A. Krasnovskii, Biokhimiya, 24,1085 (1959). (24) T. T.Bannister and J. E. Bernardini, Biochim. Bwphys. Acta, 59, 188 (1962). (25) L. P. Vernon, A. San Pietro, and D. A. Limbach, Arch. Biochem. Bwphys., 109,92 (1965). (26) A. A* amovskii, G. p* ad Drozdova, A M . Nauk SSSR, 150,1167 (1963).
2781
NOTES
Table I : Conditions of Chlorophyll-Sensitized Reductions of Various Oxidants, and Mechanisms Assigned. Comparison with Oxidation-Reduction Potentials at pH 7 (E,)and Polarographic Half-Wave Potentials (E)i12us. Saturated Calomel Electrode Conditions of sensitized reduction
E?
Phenazine methosulfate Pyocyanine 1,4Benzoquinoneh 1,4Benzoquinone Thionine Methyl red, neutral Methyl red, neutral Safranine T Ubiquinone 6 Ubiquinone 6 9,lO-Phenanthrenequinone Trimethylbenzoquinone Triphenyltetrazolium chloride Triphenyltetrazolium chloride Menadione Riboflavin Riboflavin Phenosafranine Fast red S Fast red S Azobenzene Azobenzene NAD NAD Methyl orange, basic Butter yellow Methyl red, basic
-0.07, -0.32' -0.20~ -0.21
+0.08 -0.03 (f0.30)
-0.29 -0.32'
$0.06
-0.33, -0.59,' -0.80, 0.91 -0.35
-0.29
...
(+0.13)
-0.39 -0.42 -0.43, -O.8Ok
(+0.06) (SO.11)
-0.52 -0.55
(
-0.60 (-0.35),'" -0.80, -1.04 -0.91 -1.01, -1.36'
-1.05" -1.09 -1.10'
...
+-0.19 0.01) -0.25
... ...
-0.31
... ... ...
Sensitizef
Chl Chl Chl Chl Chl Chld Chl Chl Chl Chl Chl Chl Chld Chl Chl Chl Pheo2.HCl Chld Chln Chld Chld Chl Chl Chld Chld Pheo Chld
Reductantd
... ... ... ... asc asc, hzb, m a phen 8sC
... ... ... asc, hzb hydrazine
...
aSC
...
hzb asc hzb asc phen asc asc asc WC
hzb
Solvent*
Mechanism
Ref./
eth eth eth-gly, - 70' EPA, -150" 30% aq. P Y ~ eth meth aq. Pyr
I1 I1 I1 I1 I1 I1 I1 I1
eth EPA, -150" EPA, -150' eth 15% aq. PYr meth eth-gly, -70" PYr eth eth-pyr water
I1 I1 I1 I1
8 8
II2
0
I1 I1 I1
20
eth eth-pyr meth aq. PF, 3" 15% aq. pyr, 3" eth-pyr meth eth
9 9
7 8 15 6 11 16,18 9 9
7 16
I1 17 I1 1 I(?) 14 I1 I I
0 O
I
26
I I I
O O
?"
4
12 6
Measured in deaerated 7:2: 1 ethanol:water:pyridine, containing 0.1 M LiClOA as electrolyte and 0.02% polyvinylpyrrolidone as maximum suppressor; pH 8.2. Taken from W. M. Clark, "Oxidation-Reduction Potentials of Organic Systems," Williams and Wilkins Co., Baltimore, Md., 1960. Quinone potentials are not measurable a t pH 7; values listed parenthetically are EO - 0.414. asc = ascorbicacid, hzb = hydrazoChl = chlorophyll, Chld = ethyl chlorophyllide, Chln = chlorophyllin, Pheo = pheophytin. benzene, m a = mercaptosuccinic acid, phen = phenylhydrazine. Where no reductant is given, the reversible interaction of sensitizer with oxidant w&sfollowed directly by e.8.r. or visible absorption changes. ' eth = ethanol, gly = glycerol, pyr = pyridine, meth = methanol, EPA = ether:isopentane:ethanol, 8:3:5 (by volume). "0" refers to present paper. Appear to be one-electron reductions, from low relative values of diffusion current. Benzoquinone and other quinones may have reacted with the solvent before polarogram could be made. However, all quinones gave single, two-electron reduction waves. The wave ascribed to the neutral form of methyl red is suppressed by addition of KOH, and the wave ascribed to the basic form exalted. Safranine 0 (K & L Laboratories) gave four small, overlapping waves, all together barely equal t o a single, two-electron reduction step. Both appear to be two-electron steps; second probably represents reduction of the formazan. Reduction to the formazan is by mechanism 11, but the formazan was not further reduced. '" The three waves had current ratios of 3: 15:lO. The first, small wave may be from an impurity or from the quinonehydrazoneform. Allied Chemical Corp. sample. " Starting material was the acidic (zwitterionic) form of methyl orange. This showed, in addition to the wave for the basic form, a wave a t -0.55 v., suppressed by added KOH and ascribed to the acidic form. ' We detected reversible reduction of the basic form of methyl red, even with hydrazobenzene as reductant. It is not clear whether chlorophyllide reacts with the basic form itself or with a small amount of neutral form in equilibrium with it.
'
is particularly important because it occurs during photosynthesis. The fact that reduction of chlorophyll often accompanies sensitized reduction of nucleotide by ascorbic acid in ~ i t r o ~supports ~ - ~ ~ the original assignment of mechanism I to this reaction.Z2 We found further support for this assignment. NAD
(p-nicotinamide adenine diphosphate, Sigma, 98% assay) a t 1.2 X lova M in 15% aqueous pyridine, 0.1 M in a ~ - ~ ~ m ~is nreduced i a , ~ smoothly ~ ~ ~ ~ by ascorbic acid, 4 X M , in a chlorophyllidt+sensitized reaction. The generation of NADH was followed by the growth of its band at 340 mp, and after precipitaVolume 69,Number 8 August 1966
2782
tion of reaction productsz3the presence of NADH was verified by comparison of the fluorescence spectrum with that of an authentic sample. However, when hydrazobenzene was used in place of ascorbic acid, the rate of formation of NADH, measured a t 340 mp, was less than 1% of that found with ascorbic acid. The inability of hydrazobenzene to replace ascorbic acid under these conditions is concordant with (I) but not with (XI). There is a close relation between the mechanism by which an oxidant is reduced and the polarographic half-wave potential of the oxidant. Table I summarizes reported conditions of sensitized reduction of a number of oxidants and lists the oxidation-reduction potential (when known) and the polarographic half-wave potentials determined by us for each oxidant. Those oxidants for which mechanism I had been assigned (yellow azo dyes and NAD) are all reduced a t 0.9 v. or more below the s.c.e. Those oxidants for which (11) had been assigned are reduced a t potentials 0.6 v. or less below the s.c.e. All oxidants were polarographed in a 7:2:1 mixture of 95% ethanol :water:pyridine of pH 8.2, containing 0.1 M LiClO4 as electrolyte, with a Sargent Model XXI polarograph, over a 2-v. range below the s.c.e. Good polarograms were obtained for all oxidants except the two safranine dyes, which noticeably adsorbed to surfaces. Oxidant concentrations were around M , or lower for the more surface-active dyes. For riboflavin, phenosafranine, and thionine the difference between E7 and El/,(s.c.e.) is about 0.35 v.; this may be regarded tentatively as the difference between Ell2 values, measured under the present conditions, and potentials on the hydrogen scale (pH 7). Acknowledgment. The technical assistance of Mr. D. Stoltz is gratefully acknowledged. The work was supported in part by National Science Foundation Grant No. GB-2089.
NOTES
useful additions to the literature. In this laboratory, they are needed for the understanding of the details of binary diffusion for simple nonaqueous systems. Encouraged by Longsworth’s success with D20-H20 solutions, it was decided to measure tracer diffusion coefficients for a number of hydrocarbons using the Rayleigh interferometric procedure.
Experimental
Department of Chemistry, Yale University, New Haven, Connecticut (Received March SI, 1966)
The modified Rayleigh-Philpot-Cook interferometer2 used is equivalent to an unfolded Spinco electrophoresis unit. With minor changes we have used the equipment in the manner described by Longsworth. The cost of deuterated compounds limited any series of experiments on a given hydrocarbon to the use of a total of 5 ml. of the deuterated species. Therefore a small Pyrocell electrophoresis cell with channel length 6.302mm. was used for this work. In the particular cell chosen, the optical flats which constitute the faces of the cell extend about 3 mm. beyond the channel. The original purpose for this override was to provide compensation in the reference optical path which passed immediately adjacent to the cell channel through a temperature-controlled bath. By cementing a drilled plate against these overlapping optical flats a small auxiliary chamber was formed which could be filled with a reference liquid (in these experiments the appropriate undeuterated hydrocarbon). “Non-Aq” was used as a cell lubricant precluding the use of a water thermostat. Fortunately, the experiments were performed in an isolated basement laboratory. Using large circulating fans behind controlled electric heaters, it was possible to maintain the room at 25O for long periods within *0.lo after preliminary equilibration. During the course of a given experiment (about 2 hr.) temperature variations were rarely greater than 0.05O. Since the deuterated compound was to be reused, the sharpening procedure used was a bit unconventional. It was performed by manual operation of a screw linked to the piston of a 10-ml. syringe. Sharpening was interrupted for about 10 min. after the boundary had been brought on axis and about 2 ml. had been removed. The deuterated material from all sources remaining a t the end of one experiment was reused in another diffusion run against the pure normal hydrocarbon and this procedure was repeated until the refractive increment became too small for further work. Viscosities for the final accumulated dilute mixtures
Self-diffusion cOefficients for hydrocarbons deriving from fairly Precise, absolute nmsurements might be
L. G. Longsworth, J. Phys. Chem., 58, 771 (1954). (2) J. St. J. Philpot and G. H. Cook, Research, 1, 234 (1948). (3) L. G. Longsworth, J. Am. Chem. SOC., 74,4155 (1952).
Diffusion in Deuterio-Normal Hydrocarbon Mixtures by J. D. Bir‘kett and P. A. Lyons
*
~
The Journal of Physical Chemistry
(1)
~~~~
