Xanthene dyes fluorescein

Walthall, W.K. and Stark, J.D. (1999) The acute and chronic toxicity of two xanthene dyes, fluorescein sodium salt andphloxine B, to Daphniapulex, Environmental Pollution 104 (2), 207-215. 

To date, the most popular fluorescent labels for FIA have been those derived from the long-wavelength, strongly emitting xanthene dyes fluorescein isothiocyanate (FTTC) and lissamine rhodamine B (RB200).F The isothiocyanates or isocyanates of these fluorophores can be used to label primary and secondary aliphatic amines in aqueous solutions by simple procedures. 

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). 

In the present work, the synthesis of two MIEPs is described and their application for sensing and separation two xanthene dyes - rhodamine and fluorescein - has been reported. 

Fig. 6 General structure of xanthene dyes containing fluorescein, eosin, and rhodamine...

Fluorescein (703) (Acid Yellow 73 C.I. 45350) is possibly the best known xanthene dye. The sodium salt is soluble in water to which it imparts an intense yellow-green fluorescence, detectable even at a dilution of 0.02 p.p.m. under UV irradiation. This property leads to the use of fluorescein as a location marker for aircraft lost at sea, as a tracer for the detection of a source of contamination in drinking water, and in a number of related situations. The use of fluorescein to detect abrasions of the cornea is also based on its fluorescence. 

The rhodamines are economically the most important amino-substituted xanthene dyes. The total sales of Rhodamine B in the United States in 1980 were over 107. The total domestic market for fluorescein and uranine was estimated to be over 0.5 x 106 /yr. 

By bonding two phenyl rings via a heteroatom bridge in the 2- and 2 -positions, the yellow acridine dyes (4a), the red to violet xanthene dyes (4b), or similarly colored thioxanthene dyes (4c) in the case of amino-substituted di- or triaiyl-methane dyes are obtained. Because of their rigid molecular skeletons these compounds fluoresce. Hydroxyxanthenes behave similarly (e.g., fluorescein) [2],... 

Synthesis of Xanthene Dyes. Phtbalic anhydride reacts with 3-dietbylaminophe-nol in the molten state to give rhodamine B, and with resorcinol to give fluorescein. For unsymmetrical xanthene dyes, dihydroxybenzoyl- (24) or an aminohy-droxybenzoylbenzoic acid (25) is required. 

Like phenolphthalein, xanthene dyes are prepared in a condensation reaction involving phthalic anhydride. However, resorcinol is employed instead of phenol. The simplest representative of this family is C.I. Acid Yellow 73 (fluorescein), which is made via the sequence of steps shown in Fig. 13.109. Similarly, C.I. Acid Red 92 is made by the condensation of tetrachlorophthalic anhydride and resorcinol followed by bromination. 

By contrast, the radicals formed upon single-electron reductions of xanthene dyes are persistent, at least in alkaline solution, and ESR spectra have been recorded. The early reports of persistent paramagnetism by Bubnov et and by Lagercrantz and Yhland have been followed by work reporting ESR data for fluorescein and various of its derivatives. " The hyperfine splittings indicated in 76 (in gauss) for the fluorescein radical-dianion are those of Niizuma et It had been... 

Pulse Radiolysis of Aqueous Fluorescein Dyes. The xanthene dyes of the fluorescein type were investigated using l-/xsec. pulses of 30 Mev. electrons (4, 5, 8). The transient spectra obtained with deaerated fluorescein solutions show three characteristic sets of bands. A prominent peak that shifts from 355 m/x in neutral solutions to 395 m/x in alkaline solutions corresponds with the semiquinone monoanion (pKa = 9.5) (13). This band is quenched by e q scavengers, such as oxygen or H202, and was attributed to reduction of the dye by eaq. A band at 415-420 m/x which does not change with pH was identified with the semioxidized radical monoanion, a phenoxyl derivative first observed in flash photolysis also (13). This band is quenched by formate and was attributed to the oxidative attack of OH. The remaining transient consists of a diffuse... 

Various reports of the photoreduction of organic dyes have appeared. The photoreduction of methylene blue in the presence of several electron donors,93 thionine in the presence of allylthiourea,94 fluorescein,95 Rhodamine B, and other xanthene dyes have all been investigated by spectrophotometric or e.s.r. techniques. 

A number of dyes are derived from the xanthene structure (xanthene dyes) such as fluorescein 26, eosin 27 and pyronine G 28 ... 

The xanthene dyes can be divided into two main groups viz. the biphenylmethane derivatives (pyronins) and the triphenylmethane derivatives (mainly the phthaleins). Among the phthaleins are fluorescein (hydroxy group) and rhodamin (amino group) and the dual compounds (hydroxy and amino). Both fluorescein and rhodamin, currently used as biological dyes, are also photosensitizers. 

The second family of xanthene dyes is fluorescein and its derivatives. Fluorescein itself is only slightly fluorescent in alcohol solution. In contrast, the alkali salt obtained by addition of alkali exhibits the well-known yellow-green fluorescence characteristic of the fluorescein dianion (uranin). Fluorescein and its derivatives, e.g. eosin Yand erythrosin Y, are known to be very sensitive to pH and can thus be used as pH fluorescent probes (see Chapter 10). 

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