Fluoresceins

Fluorescein can be crystallized by dissolving it in hot alcohol or in hot glacial acetic acid, and adding water. 

Dissolve a trace of fluorescein in a dilute solution of sodium hydroxide and examine the solution by reflected and by transmitted light. 

Preparation of Eosin (SECTION 641).—In a small flask cover 5 grams of fluorescein with 20 cc. of alcohol. Add slowly in small portions, taking 10 minutes for the operation, 4 cc. of bromine. Shake the flask frequently. When about one-half of the bromine has been added most of the fluorescein passes into solution as the dibrom-substitution-product as the addition continues the tetrabrom-substitution-produet crystallizes out. (Eq.) Allow the mixture to stand for an hour filter and 

Salts of eosin are used for dyeing. Prepare some of the ammonium salt as follows Place in a desiccator about 25 cc. of con- 

In a related phthalein dye, however, this condition is fully met. The dye fluorescein is resorcinol phthalein and is made from phthalic anhydride or phthalyl chloride and resorcinol just as phenol phthalein is made from phthalic anhydride and phenol (p. 750). These relationships may be expressed as follows writing the final dye salt both as a tri-phenyl methane derivative (B) and as a pyronine (C). The reactions are exactly analogous to those given for the preparation of phenolphthalein and its dye salt (p. 750), some of the intermediate steps being omitted in the present case. 

Uranine.—Phenolphthalein is a yellow crystalline compound, m.p. 250°. It is practically insoluble in water but is readily soluble in alcohol in which form it is used as an indicator. Fluorescein is a dark red crystalline compound, practically insoluble in water but soluble in alcohol. Its sodium salt is red in color but in dilute solution exhibits a remarkable green and yellow fluorescence, hence the name fluorescein. The salt is known as uranine. It is not used as a dye by itself because of its faint character but is used to mix with others in order to impart fluorescence. The rhodamines also possess fluorescent properties mostly blue and red. 

A derivative of fluorescein is important as a dye. It is known as eosine from the Greek word for dawn because its color is a fluorescent rose like the color of the sky at dawn. It is used as a silk dye. It is the tetra-hromine derivative of fluorescein potassium salt. 

The preceding discussion of the tri-phenyl methane and pyronine dyes is by no means exhaustive but enough has been said to give the student some idea of the importance of the dye compounds which are derived from the hydrocarbon tri-phenyl methane also to give the principal facts in connection with their relation to the history of synthetic dyes and to the question of chemical constitution and color of dyestuffs. 

This discussion of the constitution of phenolphthalein and fluorescein involves the work of numerous investigators. Among these we 

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Fajans method The titration of Cl" with Ag using fluorescein as an adsorption indicator. At the end point the precipitate becomes red. 

It is extensively used in the preparation of dyestuffs. Combines with diazonium salts to form oxyazo-colouring matters. Gives rise to fluorescein dyes on fusion with phthalic anhydride. Used for production of plasticizers, resins, adhesives. 

Addition of silver nitrate to a solution of a chloride in dilute nitric acid gives a white precipitate of silver chloride, AgCl, soluble in ammonia solution. This test may be used for gravimetric or volumetric estimation of chloride the silver chloride can be filtered off, dried and weighed, or the chloride titrated with standard silver nitrate using potassium chromate(VI) or fluorescein as indicator. 

Silver nitrate is used volumetrically to estimate chloride, bromide, cyanide and thiocyanate ions. Potassium chromate or fluorescein is used as an indicator. 

I. Fluorescein test. Fuse together carefully in a dry test-tube for about 1 minute a few crystals of resorcinol and an equal quantity of succinic acid or a succinate, moistened with 2 drops of cone. H2SO4. Cool, dissolve in water and add NaOH solution in excess. A red solution is produced which exhibits an intense green fluorescence.-f-... 

Does not form a phthalein or fluorescein (distinction from phthalic acid). 

Fluorescein reaction. Repeat Test i, using however resorcinol instead of phenol. A reddish solution having an intense green fluorescence is produced. 

Fluorescein reaction. Fuse together in a dry test-tube o-i g. of succinimide, O l g. of resorcinol and 2 drops of cone. HjSOi, Cool, add water and then NaOH solution in excess. A green fluorescent solution is obtained. 

Fluorescein reaction. Repeat the above test, but use resorcinol instead of... 

The fluorescein test for succinic acid (p. 349) and the phthalein and fluorescein tests for phthalic acid (p. 351) are obviously given also by succinic anhydride and phthalic anhydride, as these tests depend upon the initial formation of the anhy dride in each case. 

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. 

This solution may also be employed in the test for bromine. If iodine has been found, add small amounts of sodium nitrite solution, warm shghtly and shake with fresh 1 ml. portions of carbon tetrachloride until the last extract is colourless boil the acid solution until no more nitrous fumes are evolved and cool. If iodine is absent, use 1 ml. of the fusion solution which has been strongly acidified with glacial acetic acid. Add a small amount of lead dioxide, place a strip of fluorescein paper across the mouth of the tube, and warm the solution. If bromine is present, it will colour the test paper rose-pink (eosin). 

Fluorescein test paper Is prepared by dipping filter paper into a dilute solution of fluorescein in ethyl alcohol it dries rapidly and is then ready for use. The test paper lias a lemon yellow colour. 

Supplement 1952 2666-3031 Carbonyl compounds Ethylene carbonate, 100. Piperonal, 116. Thioindigo, 177. Fluorescein, 222. Carboxylic acids Piperonylic acid, 269. Amines, 328. Three Cyclic Oxygens, 381. Four Cyclic Oxygens, 433. Fiite Cyclic Oxygens, 459.. . . ... 

Fluorescein, ethyl ester O.lNNaOH 0.99 Quinine sulfate 0.99 POPOP... 

Bromophenol blue 2, 7 -DichIorofluorescein Eosin, tetrabromofluorescein Fluorescein Potassium rhodizonate, C404(0K)2 Rhodamine 6G Sodium 3-aIizarinsuIfonate Thorin Dissolve 0.1 g of the acid in 200 mL 95% ethanol. Dissolve 0.1 g of the acid in 100 mL 70% ethanol. Use 1 mL for 100 mL of initial solution. See Dichlorofluorescein. Dissolve 0.4 g of the acid in 200 mL 70% ethanol. Use 10 drops. Prepare fresh as required by dissolving 15 mg in 5 mL of water. Use 10 drops for each titration. Dissolve 0.1 g in 200 mL 70% ethanol. Prepare a 0.2% aqueous solution. Use 5 drops per 120 mL endpoint volume. Prepare a 0.025% aqueous solution. Use 5 drops. 

Xanthenes date from 1871 when von Bayer synthesized fluorescein (5) by the condensation of two moles of resorcinol with one mole of phthaUc anhydride in the presence of concentrated sulfuric acid (1). 

For uniformity with the stmctures given in the Colourindex the ammonium radical (9) is used for the amino-substituted xanthenes and the keto form for the hydroxy derivatives. The xanthene dyes may be classified into two main groups diphenylmethane derivatives, called pyronines, and triphenylmethane derivatives (eg, (4)), which are mainly phthaleins made from phthaUc anhydride condensations. A third much smaller group of rosamines (9-phenylxanthenes) is prepared from substituted ben2aldehydes. The phthaleins may be further subdivided into the following fluoresceins (hydroxy-substituted) rhodamines (amino-substituted), eg, (6) and mixed hydroxy/amino-substituted. 

Most xanthene dyes are classified as basic dyes by their method of appHcation acid dyes can be produced by introduction of sulfonic acid groups. The fluoresceins, which contain carboxy and hydroxy substituents, are also acid dyes for coloration of silk. Some of the fluoresceins in which the carboxy group has been esterified, are soluble in alcohol or other organic solvents and can be classified as solvent dyes. Mordant dyes can be produced by introducing o-dihydroxy or sahcyhc acid groups (2), which when metallised can have very good lightfastness. 

The physical properties of the xanthene type dye stmcture in general have been considered. For example, the aggregation phenomena of xanthene dyes has been reviewed (3), as has then photochemistry (4), electron transfer (5), triplet absorption spectra (6), and photodegradation (7). For the fluoresceins in particular, spectral properties and photochemistry have been reviewed (8), and the photochemistry of rhodamines has been investigated (9). 

Rhoda.mines, Rhodamines are commercially the most important arninoxanthenes. If phthalic anhydride is used in place of formaldehyde in the above condensation reaction with y -dialkylarninophenol, a triphenyknethane analogue, 9-phenylxanthene, is produced. Historically, these have been called rhodamines. Rhodamine B (Basic Violet 10, Cl45170) (17) is usually manufactured by the condensation of two moles of y -diethylaminophenol with phthahc anhydride (24). An alternative route is the reaction of diethylamine with fluorescein dichloride [630-88-6] (3,6-dichlorofluoran) (18) under pressure. 

Acid rhodamines are made by the iatroduction of the sulfonic acid group to the aminoxanthene base. The preferred route is the reaction fluorescein (2) with phosphorous pentachloride to give 3,6-dichlorofluoran (fluorescein dichloride) (23), which is then condensed with a primary aromatic amine in the presence of 2inc chloride and quicklime. This product is then sulfonated. For example, if compound (23) (fluorescein dichloride) is condensed with aniline and the product is sulfonated. Acid Violet 30 Cl45186) (24) is produced. 

Another group of halogenated fluorescein dyes is prepared by condensing chloro derivatives of phthalic anhydride with resorcinol, followed by bromination or iodination. Thus Phloxine B /78472-87-2] (Acid Red 92, Cl45410) (42) is prepared by condensing tetrachlorophthaUc anhydride with resorcinol followed by tetrabromination. Phloxine B undergoes ethylation to yield the yellowish ted acid dye Cyanosine B [6441-80-1] (43). 

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