Luminol preparation

D. S. Bersis and J. Nikokavouras 108> came to the conclusion, on the basis of their chromatographic investigations of luminol preparations, that these two emission maxima of luminol chemiluminescence were actually due to impurities in luminol, not to different states of 3-aminophthalate. Recent investigations of 3-aminophthalate in solution 109)... 

On the basis of chromatographic investigations, the results mentioned above were challenged, and it was suggested that the 425 and the 480nm luminol emission were caused by impurities in the luminol preparations, and not by different species of luminol itself [31]. 

Experiment 3 In general, sonoluminescence emission is not discemable with the naked eye. The luminosity of the secondary emission from luminol (oxidised by sonochemically produced OH radicals) however, is several orders of magnitude brighter and is easily seen in a dark room. Prepare a 0.1 mM aqueous luminol solution in 0.1 M NaOH. Sonicate this solution and observe the emission pattern. This will appear as bands of light and dark if a standing wave reactor is used or in more elaborate forms in different reactors. If a 20 kHz horn is used, a cone shaped zone of luminescence will be observed. Explain the emission pattern. 

The luminol reaction has also been used for the CL determination of organic substances such as penicillins [32] and tartrate ion [30] in pharmaceutical preparations by their inhibitory effect on the luminol-iodine and luminol-periodate-manganese(II)-TEA system, respectively. As can be seen from Table 1, the results were quite satisfactory. In the indirect determination of penicillins by their inhibitory effect on the luminol-iodine system, the stopped-flow technique improves the accuracy and precision of the analytical information obtained, and also the sample throughput [32], Thus, in only 2-3 s one can obtain the whole CL signal-versus-time profile and calculate the three measured parameters formation and... 

Some derivatives of luminol and acridine are currently used for the HPLC-CL detection. They are prepared by changing a substituent or introducing a new functional group into the luminol or acridine skeleton to give better CL efficiency or reactivity with analyte(s). 

The RP-HPLC method based on the CL reaction of luminol (124) catalyzed by Co(II) (Section in.B.2.c) can be applied for determination of peroxycarboxylic acids, esters and diacyl peroxides (see examples of LOD in equation 67, Section V.B.2.c) . The biosensor prepared according to equation 70, that is effective in the determination of hydroperoxides (Section V.B.6.b), becomes deactivated after three days of operation when trying to determine t-butyl peracetate. ... 

The attempt to prepare a compound analogous to endoperoxide 30 from the reaction of 1,4-dimethoxyphthalazines with singlet oxygen resulted in the formation of the corresponding phthalate by elimination of N2, albeit no chemiluminescence was observed . Even though no experimental evidence indicates its existence, the endoperoxide intermediate 30 is one of the most well-accepted HEI postulated for the chemiexcitation step in luminol chemiluminescence. 

Qian KJ, Zhang L, Yang ML, He PG, Fang YZ (2004) Preparation of luminol-doped nanoparticle and its application in DNA hybridization analysis. Chinese J Chem 22 702-707... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

As an example, a recent intercomparison (37) included three N02 measurement techniques aTDLAS-based system and two chemical-based systems— the photolysis-ozone chemiluminescence system diagramed in Figure 7 and an instrument based on N02 plus luminol chemiluminescence. Above 2 ppbv the three instruments gave similar results, but at sub-ppbv the results from the three techniques became dissimilar. Tests on the prepared mixtures showed that the luminol results were affected by expected interferences from 03 and PAN. No interferences were found in the TDLAS system, but near the detection limit the data analysis procedures calculated levels of N02 that were too high. The outcome of this intercomparison was close to the ideal the sensitivity, specificity, accuracy, and precision of each instrument were objectively analyzed previous data sets taken by different systems can now be reliably evaluated and each investigator was able to perceive areas in which the technique could be improved. 

In common with all other sensitive detection systems, maintenance of the label enzyme in its active state is important. The precautions detailed in Notes 1—3 should be observed to maximize the sensitivity achieved. Reagents for enhanced chemiluminescence can be prepared in the laboratory or ure available commercially (see Note 4). The purity of the substrate solution is important in achieving maximum sensitivity. Therefore, the precautions detailed in Notes 5-7 should be followed if preparing substrate solutions. The free base form of lummoi undergoes rearrangement ro a mixture of luminol and a series of contaminants. Therefore, luminol should be purified by recrystaliistation as the sodium salt before use (see Note 8). 

Ternaux and Chamoin described an enhanced chemiluminescence assay method for the determination of acetylcholine [48]. Reaction medium was prepared by mixing 250iu/mL of choline oxidase (100 pL), 2mg/mL of horse-radish peroxidase (50 pL) and 10-120 pM luminol in 100 pL of 0.1 M Tris buffer (pH 8.6), or 100 pL of 10-100 pM 7-dimethylamino-naphthalene-l,2-dicarbonic acid hydrazide, for 10min in 5mL of 0.1 M sodium phosphate buffer (pH 8.6). Aqueous 0.325-80 pmol of acetylcho-lineesterase (50 pL) purified on a Sephadex G50 coarse column was mixed with 450 pL of reaction mixture, and the chemiluminescence was measured at 21° C. 

SuperSignal West Pico Chemiluminescent Substrate— Pierce catalog 34080. Prepare the working solution immediately before use by mixing equal volumes of the SuperSignal luminol/enhancer solution and the stable peroxide solution. Each group will require 5 to 10 ml, depending on the size of the membrane. 

This reaction can be run on a scale five times larger. Dissolve the first crop of moist luminol (dry weight about 40-60 mg) in 2 mL of 10% sodium hydroxide solution and 18 mL of water this is stock solution A. Prepare a second stock solution, B, by mixing 4 mL of 3% aqueous potassium ferricyanide, 4 mL of 3% hydrogen peroxide, and 32 mL of water. Now dilute 5 mLofsolution Awith35 mLofwater and, in a dark place, pour this solution and solution B simultaneously into an Erlenmeyer flask. Swirl the... 

Amino-2,3-dihydro-l,4-phthalazinedione, also called luminol and 3-aminophthalhydrazide, has been prepared from 5-nitro-2,3-dihydro-1,4-phthalazinedione by reduction with ammonium sulfide or stannous chloride and by catalytic hydrogenation over palladium on charcoal in alkaline solution and by the reaction of 3-aminophthalimide with hydrazine hydrate. 

For the measurement only two materials were selected rhodamine B, which shows a beautiful orange luminescence, and luminol—i.e., 3-aminophthalhydrazide or 2,3-phthalazdione. Luminol was preferred because the yield seemed to be higher and its emission spectrum corresponded better to the spectral sensitivity curve of the photomultiplier. The rhodamine type of compound—i.e., the compound without any substitution—prepared in the authors laboratory, was not found suitable. 

Preparation of Papers. The solution used contained 100 mg. of Kodak luminol (or... 

Amino-2,3-dihydro-l,4-phthalazdione (3), known as luminol because oxidation of the substance is attended with a striking chemiluminescence, can be prepared in about 25 min. as follows. A mixture of 1 g. of 3-nitrophthalic acid and 2 ml. of an 8% aqueous solution of hydrazine in a 20 x 150-mm. test tube is heated over a free flame until the solid is dissolved, and 3 ml. of triethylene glycol is added. The solution... 

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