Luminol

940

COMMUNICATIONS TO THE EDITOR

Vol. 86

The infinity absorbance (per cent acyl-enzyme a t equilibrium) and the rate of approach to infinity [kg k5/(l K m / ( S ) ) ] identical for reactions starting are with both the acid and the ester (Fig. 1). Thus an acyl-enzyme is formed from a specific acid substrate of a-chymotrypsin . An excellent preparative method for specific acyl-enzymes maybe based on the data of Table I.

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DEPARTMENT CHEMISTRY OF FERENC. KEZDY J L. NORTHWESTERN UNIVERSITY MYRON BENDER EVANSTON, ILLINOIS 30, RECEIVED NOVEMBER 1963

Chemiluminescence of Luminol : The Chemical Reaction’

Sir:
Chemiluminescence, the production of light in excess of black body radiation by chemical reactions, is not well understood for systenis in the condensed phase. Alarge number of chemiluminescent reactions have been discovered,2 but virtually nothing is known about the chemistry involved. We report here an investigation of the chemiluminescence of lumino13 (5-amino2,3-dihydro-1,4-phthalazinedione,, one of the most I) efficient and probably the best known of the chemiluminescent compounds. Light is produced when basic aqueous solutions of luminol containingoxygen are treated with an oxidizing agent such as potassium ferricyanide, although an increased amount of light is obtained if hydrogen peroxide is included in the system. The oxidizing agents used cause extensive degradation of the reaction products, however, and consequently the only product identified prior to our work was nitrogen. The chemiluminescence in aprotic solvents, such as dimethylsulfoxide and dimethylformamide, is considerably simpler,2eand in these solvents only oxygen and a base are required. 0

that the aminophthalate ion is a product of the chemiluminescence proper, and that it does not stem from some accompanying “dark” reaction. Oxygen (1.04 moles) and 2.00 moles of NaOH were consumed in the reaction, and 0.99 mole of nitrogen was formed per mole of luminol, thusindicating the stoichiometry shown in eq. 1. The reaction of 1 mole of a base with luminol (pKa = 6)4 yields a mononegative ion6 ( H I ) , which does not react with oxygen a t an appreciable rate. The addition of slightly more than 1 mole of base to solutions of luminol containing oxygen leads to chemiluminescence. The dinegative ion (IV) is undoubtedly the critical intermediate formed by theaction of this second molecule of base; the amino group is not directly involved, since the same dependence of chemiluminescence on base was noted for phthalic hydrazide. In the absence of oxygen, basic solutions of luminol are stable indefinitely. The chemiluminescence of luminol, therefore, involves simply the reaction of oxygen with the dinegative ion of luminol (eq. 2). 0

&\

N-

+

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+ products (eq. 1)

H2N

I

d
IV

O K

1
0

H2N

0

I

COz- Na’

“Z

1 1

The products of the reaction are nitrogen and sodium aminophthalate (11) (plus traces of the aminobenzoate) ; these compounds were identified by gas-liquid and paper chromatography and by their spectra. The sodium aminophthalate was converted, furthermore, into acetamidophthalic anhydride andinto dimethyl aminophthalate; yields of up to 90yo of these compounds were obtained. As will be shown elsewhere,j a recovery of this magnitude, coupled with the quantum yields (which require that about 50% or more of the luminol molecules lead to an excited state of 11) ensures
(1) Abstracts, 134th Nacional Meeting of the American Chemical Society,

O I11 T o confirm this sequence of events,and also to show that the two oxygen atoms introduced into the aminophthalic acid framework came from the oxygen gas and not from hydroxide ion or from adventitious water (e.g., in “anhydrous” dimethyl sulfoxide), the chemiluminescence was carried out with OI8 enriched oxygen gas in a system of 70 mole yodimethyl sulfoxide, 30 mole yo water, and an excess of sodium hydroxide. I t was found that...