Fluorescein and eosin

Fluorescein is obtained by condensing phthahc anhydride (1 mol) with resorcinol (2 mols) in the presence of anhydrous zinc chloride. The tetra-bromo derivative, readily prepared by the addition of the calculated quantity of bromine, is eosin. 

Dibromofluorescein is prepared by treating fluorescein in 80 per cent, acetic acid solution with the theoretical quantity of bromine. 

The fluorescein may be purified by dissolving it in dilute sodium hydroxide solution, filtering if necessary, precipitating with dilute hydrochloric acid (1 1), filtering, washing and drying. 

Eosin (Tetrabromofluorescein). Place 16 5 g. of powdered fluorescein and 80 ml. of rectified (or methylated) spirit in a 250 ml. flask. Support a small dropping funnel, containing 36 g. (12 ml.) of bromine, above the flask make sure that the stopcock of the funnel is well lubricated before charging the latter with bromine. Add the bromine diopwise during about 20 minutes. When half the bromine has been introduced, and the fluorescein has been converted into dibromofluor-escein, all the solid material disappears temporarily since the dibromo derivative is soluble in alcohol with further addition of bromine the tetrabromofluorescein (sparingly soluble in alcohol) separates out. Allow the reaction mixture to stand for 2 hours, filter ofiF the eosin at the pump, wash it with alcohol, and dry at 100°. The yield of eosin (orange-coloured powder) is 25 g. 

Sodium salt of eosin. Grind together in a mortar 12 g. of eosin with 2 g. of anhydrous sodium carbonate. Transfer the mixture to a 250 ml. conical flask, moisten it with 10 ml. of rectified spirit, add 10 ml. of water and warm on a water bath, with stirring, until the evolution of carbon dioxide ceases. Add 50 ml. of ethyl alcohol, heat to boiling, and filter the hot solution through a fluted filter paper (supported in a short-stemmed funnel) into a beaker, and allow to stand overnight. Filter ofiF the browiiish-red crystals of sodium eosin, wash with a little alcohol, and dry. The yield is 10 g. 

Fluorescein. Grind together in a mortar 15 g. of phthalic anhydride and 22 g. of resorcinol, and transfer the mixtuie to a 350 or 500 ml. conical flask. Support the flask in an oil bath and heat to 180° (internal temperature), the oil bath is being heated, weigh out rapidly 7 g. 

After standing for two hours the mixture is filtered and the precipitate, after being washed several times with alcohol, is dried on the water bath. The material so obtained still contains alcohol of crystallisation, which is removed by drying at 110°, when the colour becomes lighter. Observe the magnificent fluorescence 

Ammonium Salt of Eosin.-—Place a very stout filter paper on a flat-bottomed crystallising basin which is one-third full of concentrated aqueous ammonia solution, spread eosin to a depth of about 0-5 cm. on the paper, and cover the whole with a funnel. Very soon the light red crystals acquire a darker colour, and after about three hours they are completely converted into the ammonium salt, which forms dark red crystals having a green iridescence. When a sample of the material dissolves wholly in water the reaction is known to be complete. 

Sodium Salt of Eosin.—Grind 6 g. of eosin with 1 g. of anhydrous sodium carbonate, transfer the mixture to a moderate-sized, widenecked, conical flask, moisten with a little alcohol, add 5 c.c. of water, and warm on the water bath until evolution of carbon dioxide ceases. To the aqueous solution of the sodium salt thus obtained now add 20 g. of alcohol, heat to boiling, and filter the hot solution. From the cooled filtrate there separate beautiful brownish-red crystals with metallic lustre, often only after long standing. Collect them at the pump and wash with alcohol. 

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The first sensor proposed for detecting gastric and oesophageal pH24, made use of two fluorophores, fluorescein and eosin, immobilised onto fibrous particles of amino-ethyl cellulose, fixed on polyester foils. Only tested in vitro, the sensor reveals a satisfactory response time of around 20 seconds. 

Rose Bengal differs in reactivity from fluorescein and Eosin because of the iodines at C-2, C-4, C-5, and C-7 which differentiate the reactivity of the phenoxide from the carboxylate and make substitution at C-2 possible in lieu of substitution at C-6. Essentially three different kinds of Rose Bengal derivatives have been synthesized. The most common include derivatives with gegen ions other than sodium at both C-2 and C-6. Many esters of C-2 have been synthesized and compounds with a number of different gegen ions at C-6 have been reported. The rarest derivatives of Rose Bengal are the C-6 ethers, of which only three have been reported. 

Resorcin.—Alefeld and Vaubel,2 by electrolytic oxidation, liave obtained dyes of different shades from resorcin and other hydroxyl derivatives of the aromatic series, such as gallic acid, tannic acid, fluoresceins and eosins. An investigation of the dyes was not made. 

Fluorescein.—It is an important compound in its reaction with phthalic anhydride, yielding beautiful dyes known as fluorescein and eosine. Hydroxy azo compounds formed from it, however, are not valuable as dyes. 

In this case, the activated compound (C) cannot be produced by direct irradiation with light of frequency v. Instead, the activated photosensitizer (Z) transfers energy to tiie monomer or initiator at an appropriate frequency that can be absorbed by C. Typical photosensitizers used in photochemically-initiated polymerizations are benzophenone, fluorescein, and eosin. 

Dye-sensitized photopolymerizations are of interest in that the spectral range of photoinitiation can be extended into the visible region. A variety of dyes such as methylene blue, thionine, fluorescein, and eosin undergo excitation and the excited dye can interact with an appropriate substance to produce radicals which, in turn, can initiate polymerization of the substrate monomer. Dye-sensitized photopolymerizations often comprise redox systems involving electron or hydrogen transfer between the excited dye and the other substance. An example is the methylene blue-p-toluenesulfinate ion system. The p-toluenesulfinate ion is oxidized by the irradiated dye to the corresponding radical which initiates polymerization of the monomer ... 

Table II. Pulse Radiolysis Rate Constants for Fluorescein and Eosin...

The reaction of triplet dye with e (l leading to the excited semiquinone has been observed only with fluorescein and eosin thus far (9) ... 

Commercial production of phthalic anhydride (PA) was taken up by BASFm 1872, by the oxidation of naphthalene with manganese dioxide and hydrochloric acid, to obtain the required base material (PA) for the manufacture of the dyestuffs fluorescein and eosine, and later for phenolphthalein however, the yield was only 5 to 7%. 

Haugland, R. R Fluorescent labeling reagents containing the fluorescein and eosin chromophores. U.S. Patent 4213904,1980. 

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