The Journal of Physical Chemistry, Vol. 82, No. 1, 1978 63
Photodegredation of Chlorophyll
Reactions in Microemulsions. 3. Photodegredation of Chlorophyll C. E. Jonest and R. A. Mackay" Department of Chemistty, Drexel University, Philadelphia, Pennsylvania 19 104 (Received August 5, 1977) Publication costs assisted by Drexel University
The anionic oil in water microemulsion system composed of sodium cetyl sulfate/ 1-pentanol/mineraloil/water has been employed as a medium for the investigation of the photochemical behavior of chlorophyll and added oxidants and reductants at a microscopic oil-water interface. The chlorophyll, located in the surface region, undergoes photodegredation and slow dark pheophytinization. The photoreaction is accelerated by added oxidants and reductants, probably by different mechanisms. A complete redox system prevents degredation, and the azobenzene/hydrazobenzene couple can be cycled in the presence of oxygen to protect the chlorophyll.
Introduction We have been interested for some time in the use of microemulsions as media for the examination of reactions and interactions at microscopic oil/water interfa~es.l-~ These systems are clear, stable fluids, containing monodisperse droplets with diameters of 10-60 nma4v5 The volume fraction occupied by the droplets is typically in the range 20-80%, with droplet concentrations on the order of M. This results in a very large oil/water interfacial area of about lo9 cm2/L. The charge and intermediate polarity of the surface(interphase) region of the droplets, as well as the presence of cofactors, can lead to the stabilization of otherwise transient speciesa2Molecules which are quite soluble in the micellar emulsion, but which are not very soluble in any of its components, are expected to be located in the interphase. Chlorophyll is such a molecule, having a somewhat polar porphyrin (chlorin) head group and a long, nonpolar phytol tail. Chlorophyll in a chloroplast membrane would also be expected to be found at the protein-lipid interface,6 analogous to the aqueous-oil interface in the microemulsion. As a prelude to the investigation of chlorophyll sensitized photoreactions in these media, it is necessary to examine the behavior of chlorophyll itself. We report here a study of the spectra and photodegredation of chlorophyll a in a mineral oil in water microemulsion stabilized by an anionic surfactant and pentanol as cosurfactant. Experimental Section Microemulsion Preparation. The oil in water (o/w) microemulsion was prepared by mixing sodium cetyl sulfate (SCS) (12.4% w/w), 1-pentanol (19.2%), mineral oil (8.8%),and water (59.6%). Agitation was not required, but the mixture was stirred to speed formation of the micellar solution. A pseudo three-component phase map of this system may be found in ref 3. Unless otherwise specified, a 0.05 M pH 7.00 phosphate buffer was employed in place of the water. Materials. The phosphate buffer, 1-pentanol, and mineral oil were obtained from Fisher (Certified). The monosodium salt of L-ascorbic acid was obtained from Biochemical Laboratories, Inc., and the azobenzene and hydrazobenzene from Eastman. The SCS was prepared by adding 100 g (0.41 mol) of cetyl alcohol with stirring to 400 mL of a 1:l v/v mixture of chloroform and carbon tetrachloride until the alcohol was dissolved. The mixture was placed in an ice bath and 33 mL (0.49 mol) of chlorosulfonic acid was added over a Part of a Ph.D. Thesis. 0022-365417812082-0063$01.OO/O
TABLE I: Extinction Coefficients of Chlorophyll a in Microemulsion Extinction Extinction Wavecoefr" WavecoefP length, (M-' cm-') length, (M-' cm-*) nm x 10-4 nm x 10-4
668 619 587 537 510 a
7.94 1.68 0.95 0.42 0.29
434 417 382 336
Obeys Beer's law for concentrations
8.02 7.36 5.17 4.09 M.
30-min period. After stirring for an additional 0.5 h, the precipitate which had formed was dissolved by adding 200 mL of 95% ethanol and the solution immediately neutralized with NaOH. After refrigerating overnight, the solid which formed was filtered on a Buchner, washed with ether, and recrystallized from methanol. The procedure followed for the separation of chlorophyll a was similar to that of Anderson and C a l ~ i n .Solutions ~ of chlorophyll in anhydrous ether were made prior to each use. The extinction coefficients of Falk8 were used to determine the concentration. The ether, in a volumetric flask, was carefully evaporated and microemulsion added to the mark. Instruments. A Gary 14 spectrophotometer was used for all of the visible absorption measurements. Electron spin resonance (ESR) measurements were performed on a Varian E-12 spectrometer system operating at 9.5 GHz with 100-kHz field modulation. White light from a Bausch and Lomb tensor lamp was employed for the irradiations. The sample holder from a Beckman DU spectrophotometer was used to position a 1-cm quartz cuvet. The samples were magnetically stirred and the holder was thermostated at 25.0 "C. The sample was irradiated for timed periods and a spectrum taken to determine the extent of reaction.
Results and Discussion ChlorophyllSpectra. As mentioned above, the structure of chlorophyll a, with a long hydrophillic chain and somewhat polar head group, is similar to that of a natural surfactant. It might be expected that the chlorophyll would be preferentially located in the interphase region of the microdroplet. This region is composed primarily of surfactant and alcohol, with some interpenetrating oil and water. The spectrum of chlorophyll at low concentration (5W5M) in the microemulsion is qualitatively similar to that obtained in various solvents. The main differences are small changes in absorption maxima and 0 1978 American Chemical Society
64
The Journal of Physical Chemistry, Vol. 82, No. 1, 1978
C. E. Jones and R. A. Mackay
TABLE 11: Spectral Parameters of Chlorophyll in Various Solvents Solvent Ether Cyclohexanee Benzenee Methanole 1-Pentanole Mineral oil Microemulsion (pH 7 )
h,,a
nm 661 662 666 666 667 664 668
hb nm
es/erC
429 429 433 432 433 431 434
1.31f 1.38 1.29 0.98 1.03 1.27 1.01
A -2
~ ~ / e ~ ' d
1.57 1.41 1.54
-3
1.05 1.18 1.36 1.09
-5
-G -7
a A, is the absorption maximum of the red band. A, is the absorption maximum of the Soret band. eS/er is the ratio of extinction coefficients of the Soret band (blue) t o the red band. e s l e s i is the ratio of extinction coefficients of the Soret band t o the vibrational satellite band near 4 1 0 nm. e From ref 9. f 1.11 in wet ether.
extinction coefficients, which are listed in Table I. In order to gain some insight into the location of the chlorophyll in the microemulsion, several spectral parameters in a number of solvents are compared in Table 11. Generally speaking, the red and blue (Soret) band maxima tend to red shift with increasing solvent polarity. However, the shifts are small and benzene is certainly less polar than methanol. A better indication of the medium which the chlorophyll sees is the ratio of the extinction coefficients of the Soret to red band and the Soret band to the Soret satellite band, which is a shoulder or peak on the high energy side of the Soret near 410 nm. These ratios are lower in more polar solvents, and for the microemulsion lie between those of methanol and pentanol. This strongly suggests that the average location of chlorophyll is in the interphase region. This is consistent with the solubility of chlorophyll in the microemulsion, which is over an order of magnitude greater than that in any of the components, even uncorrected for volume fraction. It should be noted that for the SCS microemulsion composition employed here the droplet diameter is about 10 nm, corresponding to a droplet concentration on the Ma3Thus for a chlorophyll concentration of order of M there is only one pigment molecule per hundred drops on the average, and the probability of a droplet containing more than one is negligible. Therefore, each chlorophyll molecule is isolated and there should be no effects due to any form of aggregation or other interaction. Pheophytinization. The loss of magnesium to form the free base chlorin is accelerated by acid, and is also promoted by light (quantum yield -0.02) in some solvents.1° After 1 week a chlorophyll a solution in unbuffered microemulsion kept in a refrigerator converted to 40-50% pheophytin a. In the buffered (pH 7.00) microemulsion, the rate of pheophytinization was decreased by approximately a factor of 2. There was no evidence for any significant photopheophytinization (vide infra). In general, solutions of chlorophyll a in buffered microemulsion were used within 6 h after preparation. Photodegradation. Chlorophyll a in the buffered microemulsion does react irreversibly when irradiated with white light in the absence of any added oxidants or reductants. A single initial reaction is involved as evidenced by the presence of isosbestic points (Figure 1). The product is not pheophytin a because of the absence of a strong red band at 666 nm and the characteristic bands around 505 nm. Also, pheophytin a has a larger extinction coefficient at the Soret (413 nm) than chlorophyll a, and an increase in absorption would be expected in this region. With the Soret seeming to shift further into the UV and the absence of any distinctive visible absorptions, other than for chlorophyll a, it appears that the product being
-a
I 400
ih1&%8riSth
(nm)
6bo
Figure 1. Photodegradation of chlorophyll a in pH 7.00 microemulsion.
a
I
T h e c f I r r a d i a t i o n (miv.)
20
45
1. D u r o q u i r . o : x and C!:l & 2 . Asc ar,d Chl 3 C h l a ( > : z g e d . %;:it?. l )
2..
Ck.1 a ( 3 J r g e c i wit,: G;j C h l a (,.inpLrgcd)
Chl = C h l G r o F ' f > g l l
AsC
I
60
a0
1'50
5. 6.
Chl g. ani X z o h r x e n e C h l g. ar,d LGraq,4L30r.s
7.
and Asc. Ch1 a , ;zober.aera
So1'J:ior.s : , Z , j , 6 p u r g e d .witn ?i 2 = Asaor?ate
ar.d
a n d Azr'.
7 slso
Figure 2. Absorbance of chlorophyll a at 668 nm with irradiation and added reductant and oxidants (pH 7.00 microemulsion).
formed is not a porphyrin. Rather, a type of degredation is likely taking place such as cleavage of the porphyrin ring at one of the methine bridges to give some sort of tetrapyrrole. The photodegredation is first order in chlorophyll as shown in Figure 2 (curve 4). It may be noted that the degredation of a solution deoxygenated by purging with nitrogen (curve 3) is faster than that of unpurged or oxygenated solutions (curve 4). This is consistent with a ring opening reaction which is retarded by the presence of oxygen. Added Oxidants and Reductants. The addition of the oil soluble duroquinone (DQ) greatly accelerates the rate of chlorophyll (Chl) photodegredation as shown in Figure 2 (curve 1). When the Chl/DQ solution is irradiated in the cavity of the ESR spectrometer, a weak spectrum of about five discernible lines is observed with a g value of 2.005 and a hyperfine splitting of about 1.85 G. That this is the spectrum of the corresponding semiquinone radical anion (DQ-) is demonstrated by the irradiation of a chlorophyll solution containing both duroquinone and hydrazobenzene as shown in Figure 3. No signals were
The Journal of Physical Chemistry, Vol. 82, No. 1, 1978 85
Photodegredation of Chlorophyll
\
S o l v e n t 18 Microeiliulaion
PH
-
?.QQ
I
1
Centered a t 3 3 9 3 . 6 Gauss
U I U
'1.83' 1.87' 1.8 1.90'1.87'1.89'
Flgure 3. Irradiation of chlorophylla, duroquinone, and hydrazobenzene in ESR cavity (no signal without light).
observed in the dark, or under irradiation in the absence of chlorophyll. The presence of semiquinone radicals in the reversible photoreaction of chlorophyll and quinone in ethanol has been reported,l' the source of electrons likely being the solvent. In that case, a triplet exciplex between chlorophyll, quinone, and solvent was invoked to explain the results. Any chlorophyll radical cation (Chl') was presumbly produced independently from excited singlet. Here, however, since the pigment reacts irreversibly, the initial photoreaction is likely electron transfer from excited chlorophyll (Chl*) to duroquinone (eq 1). It Chl* t DQ- Chl' t DQ(1) seems likely that this leads to a net loss of chlorophyll because of a slow back reaction between Chl+ and DQ-, quite probably due to the negative surface charge on the microdroplet. The addition of ascorbate to the Chl-DQ solution prevents the photodegredation of chlorophyll (Figure 2, curve 6). After about 60 min the ascorbate is consumed and the chlorophyll commences to degrade at a rate approaching that for duroquinone alone (curve 1). Thus, as long as a redox system (oxidant plus reductant) is present, there is no net change in the sensitizer (chlorophyll) concentration, presumbly as a result of rapid reduction of Chl+ back to Chl. This has been confirmed with other systems employing various dyes as oxidants and ascorbate or phenylhydrazine as reductants. When only ascorbate (AscH-) is present, the chlorophyll reacts at an intermediate rate (curve 2), which would seem to implicate an initial reductive step, schematially shown by eq 2. If the oxidant azobenzene is added to the solution Chl* t AscH--+ ChlH t Asc-
(2)
containing ascorbate, essentially no photodegredation of
the chlorophyll occurs (curve 7). The only observable change is due to some cis-trans isomerization of the azobenzene. The chlorophyll-sensitized cis-trans isomerization of other compounds has been observed.12 This can be explained in terms of the initial chlorophyllmediated photoreduction of azobenzene to hydrazobenzene by ascorbate. After the ascorbate is depleted, the (reversible) azobenzene-hydrazobenzene redox couple remains. Since there is now no net reaction, the couple can "cycle" indefinitely protecting the chlorophyll from degredation. It has previously been reported that azobenzene prevents the photochemical reduction of ethyl chlorophyllide by ascorbic acid in ethanol-pyridine mixtures,13although the postulated mechanism was based on eq 2. The behavior of a chlorophyll-azobenzene solution (curve 5 ) may also be explained in terms of the formation of a redox couple. The reaction of chlorophyll with azobenzene produces hydrazobenzene which retards the degredation as its concentration increases.
Summary The chlorophyll molecule is located in the surface region of the microdroplet, and undergoes slow pheophytinization. The chlorophyll undergoes a photodegradation in white light which is first order in the pigment, proceeds via a single initial reaction, and is slightly retarded by oxygen. Added oxidants and reductants cause a faster reaction, although likely not by the same mechanism. The negative surface charge on the droplet may contribute to the very rapid reaction with duroquinone. As long as a complete redox system is present, the chlorophyll remains intact. The reversible azobenzene/ hydrazobenzene redox couple can be employed in a cyclic fashion to protect the chlorophyll. References and Notes (1) K. Letts and R. A. Mackay, Inorg. Chem., 14, 2990 (1975). (2) K. Letts and R. A. Mackay, Inorg. Chem., 14, 2993 (1975).
(3) R. A. Mackay, K. Letts, and C. Jones in "Mlcellizatlon, Solubilization and Microemulslons", Vol. 2,K. L. Mittal, Ed., Plenum Press, New York, N.Y., 1977,pp 801-816. (4) J. H. Schulman, W. Stoeckenuis, and L. M. Prince, J. Phys. Chem., 63, 1677 (1959). (5) W. Stoeckenius, J. H. Shulman, and L. M. Prince, Kollold Z.,169,
170 (1959). (6) E. Rabinowitch and Govindjee, "Photosynthesis", Wiley, New York, N.Y., 1969. (7) A. F. H. Anderson and M. Calvin, Nafure(London),194, 285 (1962). (8) J. E. Falk, "Porphyrins and Metalloporphyrins", Elsevier, Amsterdam,
1964. (9) G.R. Seely and R. G. Jensen, Specfrochim. Acta, 21, 1835 (1965). (10) L. P. Vernon, and G. R. Seely, "The Chlorophylls", Academic Press, New York, N.Y., 1966. (11) G. Tollin, Bioenergetics, 6, 69 (1974). (12) H. Claes and T. 0. M. Nakayama, Nafure(Lon&n), 183, 1053 (1959); H. Claes, Z. Naturforsch. B , 16, 445 (1961). (13) G.R. Seely, J . Phys. Chem., 69, 2779 (1965).
