Luminol quantum yields

A substantial effort has been appHed to iacreaskig i by stmctural modification (114), eg, the phthalaziQe-l,4-diones (33) and (34) which have chemiluminescence quantum yields substantially higher than luminol (115,116). The fluorescence quantum yield of the dicarboxylate product from (34) is 14%, and the yield of singlet excited state is calculated to be 50% (116). Substitution of the 3-amino group of lumiaol reduces the CL efficiency > 10 — fold, whereas the opposite effect occurs with the 4-amino isomer (117). A series of pyridopyridaziae derivatives (35) have been synthesized and shown to be more efficient than luminol (118). 

Lee, J., and Seliger, H. H. (1972). Quantum yields of the luminol chemiluminescence reaction in aqueous and aprotic solvents. Photochem. Pho-tobiol. 15 227-237. 

The overall reaction scheme of the luminol chemiluminescence in an aqueous medium is shown in Figure 1. The luminol oxidation leads to the formation of an aminophthalate ion in an excited state, which then emits light on return to the ground state. The quantum yield of the reaction is low ( 0.01) compared with bioluminescence reactions and the emission spectrum shows a maximum1 at 425 nm. 

DPA) in dimethylphthalate at about 70°, yields a relatively strong blue Umax =435 nm) chemiluminescence the quantum yield is about 7% that of luminol 64>. The emission spectrum matches that of DPA fluorescence so that the available excitation energy is more than 70 kcal/mole. Energy transfer was observed on other fluorescers, e.g. rubrene and fluorescein. The mechansim of the phthaloyl peroxide/fluorescer chemiluminescence reaction very probably involves radicals. Luminol also chemiluminesces when heated with phthaloyl peroxide but only in the presence of base, which suggests another mechanism. The products of phthaloyl peroxide thermolysis are carbon dioxide, benzoic acid, phthalic anhydride, o-phenyl benzoic acid and some other compounds 65>66>. It is not yet known which of them is the key intermediate which transfers its excitation energy to the fluorescer. 

Hydrazide chemiluminescence has been investigated very intensively during recent years (for reviews, see 1>, p. 63, 2>, 90>). Main topics in this field are synthesis of highly chemiluminescent cyclic diacyl hydrazides derived from aromatic hydrocarbons, relations between chemiluminescence quantum yield and fluorescence efficiency of the dicarboxylates produced in the reaction, studies concerning the mechanism of luminol type chemiluminescence, and energy-transfer problems. 

Another rather striking example demonstrates that the fluorescence efficiency of the respective dicarboxylates is not the most important factor in determining the chemiluminescence efficiencies of the hydrazides 9.10-diphenylanthracene-2.3-dicarbonic acid 25 has a fluorescence efficiency of about 0.9 (as has the parent compound 9.10-di-phenylanthracene) 94>. The corresponding hydrazide 26, however, gives a quantum yield of 48% that of luminol only (in DMSO/t-BuOK/ O2) 95) although 3-aminophtalic acid has a fluorescence efficiency of about 0.3 only. 

As can be seen from Table 2, certain phenanthrene derivatives produce more chemiluminescence than luminol. The diphenyl derivative 33 diluminyl evidently has its benzene rings in a non-coplanar position this is apparent from the fact that this compound does not behave as a benzidine derivative under the oxidative conditions of chemiluminescence reactions. The chemiluminescence quantum yield is only about one third that of luminol 100>. 

In DMS0/tert.Bu0K/02 quantum yields found were 3 x I0 4 for 50 and 5 X 104 for 49. These values are not regarded as maximum values, as a strong influence of the reaction conditions was noted 104). Whereas, in luminol-type compounds, any substitution in the cyclic hydrazide ring system renders the compound non-chemiluminescent 105>, this is not the case with these open-chain hydrazides the methylated compounds 49, 50 (R=N(CH3)NH2) and 50 (R=N(CH3)NH(CH3)) were also found to be chemiluminescent, though less so than the unsubstituted hydrazide. E. H. White and coworkers 104> therefore suggest a mechanism via acyl anions for the non-cyclic hydrazides (see Section VI. C.). 

However, the formation of these products does not appear to play a critical role in the decision as to whether the 425 nm and 480 nm maxima are due to different states of the same molecule or to different compounds. It was reported that special care was taken to ensure the purity of luminol and of 3-aminophthalate 109>. In commercially available 3-amino-phthalic acid a yellowish impurity exhibiting brilliant green fluorescence was detected 109> this substance also formed in neutral solutions of pure 3-amino phthalic acid and crystallized from these solutions in yellow crystals. The structure of this substance was determined to be 53 its absorption spectrum has a maximum at 388 nm the fluorescence maximum is at 475 nm, with a fluorescence quantum yield of about 0.75 in DMF i 9). 

Table 5. Luminol chemiluminescence quantum yields in different solvents with some oxidative systems (after J. Lee and H. H. Seliger 115>)...

Luminol concentration (M) Solvent T[°C] pB Reaction conditions Chemiluminescence quantum yields Qo... 

The photosensitized results are from I.B.C. Matheson and J. Lee 118h It is seen that the quantum yields in photosensitized oxidation depend on the concentrations of luminol and base, and on temperature. At higher temperature (50°) and low luminol concentrations, the quantum yields reached those of hemin-catalyzed hydrogen peroxide oxidation of luminol in aqueous-alkaline solution. Primary products of the photosensitized oxidation are singlet oxygen (1Ag02) or a photoperoxide derived from methylene blue, but neither of these is directly responsible for the luminol chemiluminescence. 

Luminol amidine 132, synthesized from luminol and the Vilsmeier reagent from DMF and thionyl chloride, has been proposed as a suitable luminol derivative for analytical purposes because, unlike luminol, it can be easily purified by recrystallization from water. 132 exhibits a chemiluminescence quantum yield of about 20% of luminol in ferricyanide-catalyzed oxidation by aqueous alkaline hydrogen peroxide Amax of the emission is 452 nm 196>. 

The oxidation of luminol in basic solution is one of the best known and most efficient chemiluminescent reactions, having a quantum yield of CL of about 0.01 in water and 0.05 in DMSO. 

The dismutation (disproportioning) of two free radicals is accompanied by release of a portion of reaction energy as a light quantum. As the quantum yield of such a process is extremely low, the detection of this type of chemiluminescence is technically complicated. Several compounds like lucigenin and luminol have a high quantum yield after reaction with peroxide radicals. Therefore, they are widely used for the detection of these radicals, particularly in the examination of phagocyting cells. 

In chemiluminescence immunoassay the antigen is tagged with a molecule such as luminol or an acridinium ester which emits light with a high quantum yield on oxidation. Alternatively, the antigen may be labelled with a bio-luminescent molecule such as luciferin, which emits light when oxidized by the enzyme luciferase. 

We have recently described a calibration procedure for the determination of excitation quantum yields on commercial fluorimeters, utilizing the luminol standard , and have thereby determined singlet excitation quantum yields for the peroxyoxalate reaction with bis(2,4,6-trichlorophenyl) oxalate (TCPO), hydrogen peroxide and imidazole, using various activators . The same calibration method has been utilized to determine the singlet quantum yields obtained in the induced decomposition of protected phenoxyl-substituted 1,2-dioxetanes 6 and and compared them to the well-investigated... 

Scheme 28) Quantum yields of lucigenin oxidation by hydrogen peroxide in alkaline media are comparable with the values obtained in luminol oxidation (1.24 x 10 E mol ) ° . However, the use of other peroxides, such as tcrt-butyl hydroperoxide, results in a decrease of chemiluminescence quantum yields of two orders of magnitude, confuming the hypothesis that a 1,2-dioxetane is the HEI, since its formation would be impossible with alkyl peroxides . 

TABLE 3. Luminol chemiluminescence quantum yields in different solvents with some oxidative... 

Some chemical reactions give rise to light emission. A well-known example is that of luminol, used for non-electric emergency lighting or the manufacture of luminous colour wands. The reaction that causes emission is an oxidation that takes place in the presence of a catalyst. Its quantum yield is close to 1. 

A review of chemiluminescent and bioluminescent methods in analytical chemistry has been given by Kricka and Thorpe. A two-phase flow cell for chemiluminescence and bioluminescencc has been designed by Mullin and Seitz. The chemiluminescence mechanisms of cyclic hydrazides, such as luminol, have been extensively analysed. " Fluorescence quantum yields of some phenyl and phenylethynyl aromatic compounds in peroxylate systems have been determined in benzene. Excited triplet states from dismutation of geminate alkoxyl radical pairs are involved in chemiluminescence from hyponitrite esters. Ruorophor-labelled compounds can be determined by a method based on peroxyoxalate-induced chemiluminescence. Fluorescence and phosphorescence spectra of firefly have been used to identify the multiplicity of the emitting species. " The chemiluminescence and e.s.r. of plasma-irradiated saccharides and the relationship between lyoluminescence and radical reaction rate constants have also been investigated. Electroluminescence from poly(vinylcarbazole) films has been reported in a series of four... 

---