Electrogenerated chemiluminescence. 29. The electrochemistry and chemiluminescence of chlorophyll a in N,N-dimethylformamide solutions.

The electrochemistry and ecl o f chlorophyl l a (Chl a) have been studied in highly pur i f ied D M F solutions. Cyc l ic voltammetric and coulometric experiments demonstrate that both the radical anion, (Chl a)-., and the dianion, (Chl a)2-, are fa i r ly stable even under bulk electrolysis conditions in the absence o f proton donors and oxygen. The protonation o f ( C H I a)-. and ( C h l a)*b y hydroquinone was also studied. Luminescence was observed for the reduction o f Chl a i n the presence o f oxygen, but not for the Chl a anion-cation annihi lat ion i n the absence o f oxygen. Chemiluminescence on mix ing a solution o f (Chi a)-. and oxygen was also observed. Chlorophyll a (Chl a ) plays an important role in photosynthesis in the initial redox events following the absorption of light. Thus the nature and reactions of the oxidized and reduced forms of Chl a and the potentials associated with the electron-transfer processes of this molecule have been the subject of a number of investigations. Several authors have discussed the electrochemical behavior of Chl The oxidation5-? of Chl a in aprotic solvents (e.g., CH2C12) occurs with the reversible formation of the radical cation which is stable even for time scales characteristic of bulk electrolysis. The reduction, which has been studied a t a mercury electrode, shows two one-electron waves, similar to those observed with other metal porphyrin^.^.^ These latter results suggested that the radical anion formed a t the first wave [(Chl a)-.] was not very stable. Thus Berg and KramarCzyk3 observed some reoxidation of (Chl a)-. in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (Me2SO) using the Kalousek commutator technique and estimated the half-life of the radical anion as between 0.01 and 0.1 s. The second reduction wave for Chl a was irreversible. Similarly, Kiselev et al.4 reported, by using cyclic voltammetry, that the first reduction wave of Chl a in D M F was quasireversible and the second wave was irreversible. Felton et al.2 reported that controlled-potential coulometric reduction of Chl a a t a Pt electrode in MezSO a t potentials of the first wave yielded an n a p p value of 1 . I to 1.3. However, the solution produced by reduction could neither be reduced further a t potentials of the second wave nor oxidized at potentials a t the foot of the first wave. Spectroscopic examination of the reduced solution suggested that (Chl a)-. decomposed by loss of a hydrogen atom (at the 10 position) to form the phase test intermediate; the hydrogen atom acceptor was not identified. The instability of (Chl a)-. and (Chl a)2as compared with the relative stability of the reduced forms of other metalloporphyrins and the free porphins8-'I suggested that further investigation of the electrochemical reduction of chlorophyll be undertaken. The question of ecl in chlorophyll systems is also of interest. Radical ion annihilation luminescence has been observed for cy, /3, 7, 6 , tetraphenylporphin (TPP),'O and for PtTPP and PdTPP.' I Fairly extensive experiments in our laboratories failed to show ecl from Chl a in several solveqts with careful purification and deaeration.I2 However, Krasnovskii and Litvini3 reported studies of Chl a in D M F with LiCl supporting electrolyte in which ecl was observed both upon cycling the potential of a Pt electrode between the oxidation and reduction waves (i.e., radical ion annihilation ecl) and upon reduction of Chl a in the presence of oxygen. In this paper we describe experiments which demonstrate that both (Chl a)-and (Chl a)2are quite stable in purified D M F and discuss the conditions for observing chemiluminescence in (Chl a)-containing systems. Experimental Section Chlorophyll a S e p a r a t i ~ n . ' ~ ' ~ Chl a was isolated f rom spinach leaves. After extraction with 80% acetone, the pigments were adsorbed on talc, eluted wi th ethyl ether, and separated by chromatography on a column o f fine powdered sugar. T o remove yellow compounds, the adsorbed pigments were washed w i t h I : I n-pentane:benzene. Chl a and Chl b were further separated chromatographical ly by washing with n-pentane containing 0.5% 1 -propanol. The Chl a zone was eluted wi th ethyl ether and dried on a vacuum l ine for I day, then stored in a drybox under a helium atmosphere. The final separation was carried out under subdued light, and the C h l a was stored i n the dark. Reagents. D M F was first puri f ied by st irr ing for several days over molecular sieves and then using the procedure reported previously, method A.16 For further purification, the solvent containing support ing electrolyte was treated wi th neutral alumina as recommended Saji, Bard / Electrogenerated Chemiluminescence