Luminol

Chemiluminescence Energy transfer Reduction of a nitro group Amide formation 

In this experiment, the chemiluminescent compound luminol, or 5-amino-phthalhydrazide, will be synthesized from 3-nitrophthalic acid. 

The first step of the synthesis is the simple formation of a cyclic diamide, 5-ni-trophthalhydrazide, by reaction of 3-nitrophthalic acid with hydrazine. Reduction of the nitro group with sodium dithionite affords luminol. 

In neutral solution, luminol exists largely as a dipolar anion (zwitterion). This dipolar ion exhibits a weak blue fluorescence after being exposed to light. However, in alkaline solution, luminol is converted to its dianion, which may be oxidized by molecular oxygen to give an intermediate that is chemiluminescent. The reaction is thought to have the following sequence  

The dianion of luminol undergoes a reaction with molecular oxygen to form a peroxide of unknown structure. This peroxide is unstable and decomposes with the evolution of nitrogen gas, producing the 3-aminophthalate dianion in an electronically excited state. The excited dianion emits a photon that is visible as light. One very attractive hypothesis for the structure of the peroxide postulates a cyclic endoperoxide that decomposes by the following mechanism  

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Detecting the presence of small, even invisible, amounts of blood is routine. Physical characteristics of dried stains give minimal information, however, as dried blood can take on many hues. Many of the chemical tests for the presence of blood rely on the catalytic peroxidase activity of heme (56,57). Minute quantities of blood catalyze oxidation reactions between colorless materials, eg, phenolphthalein, luco malachite green, luminol, etc, to colored or luminescent ones. The oxidant is typically hydrogen peroxide or sodium perborate (see Automated instrumentation,hematology). 

Chemiluminescent Immunoassay. Chemiluminescence is the emission of visible light resulting from a chemical reaction. The majority of such reactions are oxidations, using oxygen or peroxides, and among the first chemicals studied for chemiluminescence were luminol (5-amino-2,3-dihydro-l,4-phthalazinedione [521-31-3]) and its derivatives (see Luminescent materials, chemiluminescence). Luminol or isoluminol can be directly linked to antibodies and used in a system with peroxidase to detect specific antigens. One of the first appHcations of this approach was for the detection of biotin (31). 

Luminol (Phthalhydrazide). Chemiluminescence from luminol [521-31-3] (3-aminophthalhydrazide) (29), isoluminol [3682-14-2] (4-aminophthalhydrazide), and analogues has been studied extensively (104—106). 

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). 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Divalent copper, cobalt, nickel, and vanadyl ions promote chemiluminescence from the luminol—hydrogen peroxide reaction, which can be used to determine these metals to concentrations of 1—10 ppb (272,273). The light intensity is generally linear with metal concentration of 10 to 10 M range (272). Manganese(II) can also be determined when an amine is added to increase its reduction potential by stabili2ing Mn (ITT) (272). Since all of these ions are active, ion exchange must be used for deterrnination of a particular metal in mixtures (274). 

Chromium (ITT) can be analy2ed to a lower limit of 5 x 10 ° M by luminol—hydrogen peroxide without separating from other metals. Ethylenediaminetetraacetic acid (EDTA) is added to deactivate most interferences. Chromium (ITT) itself is deactivated slowly by complexation with EDTA measurement of the sample after Cr(III) deactivation is complete provides a blank which can be subtracted to eliminate interference from such ions as iron(II), inon(III), and cobalt(II), which are not sufficiently deactivated by EDTA (275). 

Iron(II) can be analy2ed by a luminol—air reaction in the absence of hydrogen peroxide (276). Iron in the aqueous sample is reduced to iron(II) by sulfite other metals which might interfere are also reduced to valence states that are inactive in the absence of hydrogen peroxide. The detection limit is 10 ° M. 

Titration Indicators. Concentrations of arsenic(III) as low as 2 x 10 M can be measured (272) by titration with iodine, using the chemiluminescent iodine oxidation of luminol to indicate the end point. Oxidation reactions have been titrated using siloxene the appearance of chemiluminescence indicates excess oxidant. Examples include titration of thallium (277) and lead (278) with dichromate and analysis of iron(II) by titration with cerium(IV) (279). 

Hydrogen Peroxide Analysis. Luminol has been used for hydrogen peroxide analysis at concentrations as low as 10 M using the cobalt(III) triethanolamine complex (280) or ferricyanide (281) as promoter. With the latter, chemiluminescence is linear with peroxide concentration from... 

Luminol-based chemiluminescence methods have also been employed for detection of analytes in flowing stream analytical techniques such capillary electrophoresis (282), flow injection analyses, and hplc (267). AppHcations of the enhanced luminol methodology to replace radioassay methods have been developed for a number of immunological labeling techniques (121,283). 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Luminol chemiluminescence has also been recommended for measuring bacteria populations (304,305). The luminol—hydrogen peroxide reaction is catalyzed by the iron porphyrins contained in bacteria, and the light intensity is proportional to the bacterial concentration. The method is rapid, especially compared to the two-day period required by the microbiological plate-count method, and it correlates weU with the latter when used to determine bacteria... 

Chemiluminescent labels, in which the luminescence is generated by a chemical oxidation step, and bioluminescent labels, where the energy for light emission is produced by an enzyme-substrate reaction, are additional labeling types (39,42). Luminol [521 -31 -3] CgHyN202, and acridine [260-94-6] C H N, derivatives are often used as chemiluminescent labels. 

Peroxymonosulphuric acid (PMSA, H SO ) proved to be a promising oxidizer in reactions with chemiluminescent substances (luminol) with participation of such ions as Mn(II), Cu(II), Ni(II), Cr(IV), V(V). The literature data show the possibility of utilization PMSA in indicating reaction with ferroin ([Fe(l,10-phenanthrolyne) ] ) which is accelerated by Mn(II) compounds. 

Present research is devoted to investigation of application of luminol reactions in heterogeneous systems. Systems of rapid consecutive reactions usable for the determination of biologically active, toxic anions have been studied. Anions were quantitatively converted into chemiluminescing solid or gaseous products detectable on solid / liquid or gas / liquid interface. Methodology developed made it possible to combine concentration of microcomponents with chemiluminescence detection and to achieve high sensitivity of determination. 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

CHEMILUMINESCENT ANALYSIS BASED ON THE LUMINOL OXIDATION REACTION IN SPECIFIC REGIMES... 

Using the luminol photochemiluminescence it is possible to determine not only the nitrates (as reported by us earlier), but also the nitrites. The urotropin is added to the water sample, and the solution obtained is illuminated by the Hg lamp. The chemiluminescence is measured after the addition of basic luminol solution to the illuminated solution. The detection limit is 2-10 M. The nitrates contained in the drinking water do not interfere at tenfold excess. 

We have shown that known reaction of luminol with peroxydisulphate at low luminol concentrations takes place in the regime of controlled generation of SO ion-radicals at spontaneous destruction of peroxydisulphate. The detection limit for various types of antioxidants in water using this reaction is varied from 10 to 10 M. It is possible also to determine some polluting admixtures present in the atmosphere. The reagent used is the mixture of the luminol, base and K S O, which, once prepai ed, could be used during a working day. 

Luminol (5-aminophthalazin-l,4-dione) [521-31-3] M 177.2, m 329-332°, pK] 3.37, pK2 6.35. Dissolved in KOH soln, treated with Norit (charcoal), filtered and ppted with cone HCl. [Hardy, Sietz and Hercules Talanta 24 297 1977.] Stored in the dark in an inert atmosphere, because its structure changes during its luminescence. It has been recrystd from O.IM KOH [Merenyi et al. J Am Chem Soc 108 77716 1986]. 

Assayed by the luminol chemiluminescence method (Puget and Michelson, 1974). One unit corresponds to 0.35 unit of the cytochrome c method (McCord and Fridvich, 1969). 

Calibration with luminol luminescence (Lee et al., 1966). Calibration with luminol can be performed in aqueous solution in the presence of H2O2 and a suitable catalyst (luminescence range 380-550 nm, Amax 430 nm), or in DMSO in the presence of potassium t-butoxide (A.mav 486 nm). The calibration method in aqueous medium requires the following three reagents ... 

Luminol stock solution (approx. 20 mg luminol in 1 liter of 10 mM NaOH, to give an A value of 0.80 at 347 nm). Working solution dilute the stock solution 10 times with pH 11.6 buffer (3 g of Na2HP04 7H2O and 15 g of Na3P04 I2H2O in 1 liter of water). 

Under the conditions described above, 1 ml of luminol solution of A347 1.0 will emit a total of 9.75 0.7 x 1014 photons. 

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. 

Rost, M., Karge, E., and Klinger, W. (1998). What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence 1. Investigations with hydrogen peroxide and sodium hypochlorite. J. Biolumin. Chemilumin. 13 355-363. 

Tawa, R., and Sakurai, H. (1997). Determination of four active oxygen species such as H2O2, OH, OJ and 02 by luminol-and CLA-chemiluminescence methods and evaluation of antioxidative effects of hydroxybenzoic acid. Anal. Lett. 30 2811-2825. 

Eucheuma platycladum (thalli segments) breaking of thalli suspension agitation with magnetic bar luminol dependent chemiluminescence [179]... 

Holt, M.E., Ryall, M.E.T, and Campbell, A.K. (1984). Albumin inhibits human polymorphonuclear leucocyte luminol-dependent chemiluminescence evidence for oxygen radical scavenging. Br. J. Exp. Path. 65, 231-241. 

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