Fluorescein: Wikis

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Fluorescein
Identifiers
CAS number 2321-07-5 Yes check.svgY
PubChem 16850
EC number 219-031-8
MeSH Fluorescein
SMILES
Properties
Molecular formula C20H12O5
Molar mass 332.306 g/mol
Melting point

314 - 316 °C

Solubility in water Slightly
Hazards
S-phrases S26 S36
 Yes check.svgY (what is this?)  (verify)
Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)
Infobox references

Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein has an absorption maximum at 494 nm and emission maximum of 521 nm (in water). The major derivative is Fluorescein isothiocyanate (FITC)

Fluorescein also has an isosbestic point (equal absorption for all pH values) at 460 nm. Fluorescein is also known as a color additive (D&C Yellow no. 7). The disodium salt form of fluorescein is known as D&C Yellow no. 8.

Contents

Chemical and physical properties

Fluorescein under UV illumination

The fluorescence of this molecule is very high, and excitation occurs at 494 nm and emission at 521.

Fluorescein has a pKa of 6.4 and its ionization equilibrium leads to pH-dependent absorption and emission over the range of 5 to 9. Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately 3 and 4 ns, which allows for pH determination from non-intensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry.

Derivatives

Fluorescein isothiocyanate

There are many fluorescein derivatives, for example fluorescein isothiocyanate, often abbreviated as FITC. FITC is the original fluorescein molecule functionalized with an isothiocyanate group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure. This derivative is reactive towards amine groups on proteins inside cells. A succinimidyl-ester functional group attached to the fluorescein core, creating NHS-fluorescein, forms another common amine reactive derivative.

Other green dyes include Oregon Green, Tokyo Green, SNAFL, and carboxynaphthofluorescein. These dyes, along with newer fluors such as Alexa 488 and DyLight 488, have been tailored for various chemical and biological applications where higher photostability, different spectral characteristics, or different attachment groups are needed.

Synthesis

Fluorescein was first synthesized by Adolf von Baeyer in 1871. It can be prepared from phthalic anhydride and resorcinol in the presence of zinc chloride via the Friedel-Crafts reaction.

ZnCl2 fluorescein.png

A second method to prepare fluorescein uses methanesulfonic acid as a Brønsted acid catalyst. This route has a high yield under milder conditions. [1] [2]

Applications

Uses in river systems

One of its more recognizable uses was in the Chicago River, where fluorescein was the first substance used to dye the river green on St. Patrick's Day in 1962. In 1966 environmentalists forced a change to a vegetable based dye to protect the thousands of goldfish that populate the river.[citation needed]

Other uses of fluorescein include using it as a water-soluble dye added to rainwater in environmental testing simulations to aid in locating and analyzing any water leaks, and in Australia and New Zealand as a methylated spirit dye.

Biochemical research

In cellular biology, the isothiocyanate derivative of fluorescein is often used to label and track cells in fluorescence microscopy applications (for example, flow cytometry). Additional biologically active molecules (such as antibodies) may also be attached to fluorescein, allowing biologists to target the fluorophore to specific proteins or structures within cells. This application is common in yeast display.

Fluorescein can also be conjugated to nucleoside triphosphates and incorporated into a probe for in situ hybridisation. Fluorescein-labelled probes can be imaged using FISH, or targeted by antibodies using immunohistochemistry. The latter is a common alternative to digoxigenin, and the two are used together for labelling two genes in one sample [3].

Health care applications

Fluorescein drops being inserted for an eye examination.

Fluorescein sodium is used extensively as a diagnostic tool in the field of ophthalmology and optometry, where topical fluorescein is used in the diagnosis of corneal abrasions, corneal ulcers and herpetic corneal infections. It is also used in rigid gas permeable contact lens fitting to evaluate the tear layer under the lens.

Intravenous or oral fluorescein is used in fluorescein angiography in research and to diagnose and categorize vascular disorders in e.g. legs, including retinal disease macular degeneration, diabetic retinopathy, inflammatory intraocular conditions, and intraocular tumors , and increasingly during surgery for brain tumors.

Safety

Topical, oral, and intravenous use of fluorescein can cause adverse reactions including nausea, vomiting, hives, acute hypotension, anaphylaxis and related anaphylactoid reaction,[4] cardiac arrest,[5] and sudden death.[6][7]

The most common adverse reaction is nausea, due to a difference in the pH from the body and the pH of the sodium fluorescein dye, however a number of other factors are considered contributors as well.[citation needed] The nausea usually is transient and subsides quickly. Hives can range from a minor annoyance to severe, and a single dose of antihistamine may give complete relief. Anaphylactic shock and subsequent cardiac arrest and sudden death are very rare but because they occur within minutes, a health care provider who uses fluorescein should be prepared to perform emergency resuscitation.

Intravenous use has the most reported adverse reactions, including sudden death, but this may reflect greater use rather than greater risk. Both oral and topical uses have been reported to cause anaphylaxis,[8][9] including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop.[5] Reported rates of adverse reactions vary from 1% to 6%[10][11][12][13] The higher rates may reflect study populations that include a higher percentage of persons with prior adverse reactions. The risk of an adverse reaction is 25 times higher if the person has had a prior adverse reaction.[12] The risk can be reduced with prior (prophylactic) use of antihistamines[14] and prompt emergency management of any ensuing anaphylaxis.[15] A simple prick test may help to identify persons at greatest risk of adverse reaction.[13]

See also

References

  1. ^ Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P., Synthesis of Fluorinated Fluoresceins. Journal of Organic Chemistry 1997, 62, (19), 6469-6475.
  2. ^ Yuichiro Ueno; Jiao, G.-S.; Burgess, K., Preparation of 5- and 6-Carboxyfluorescein. Practical Synthetic Procedures 2004, 31, (15), 2591-2593.
  3. ^ Noga E. J., Udomkusonsri, P. (2002). "Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish". Vet Pathol 39: 726–731(6). doi:10.1354/vp.39-6-726. PMID 12450204. http://www.vetpathology.org/cgi/reprint/39/6/726.pdf. Retrieved 2007-07-16. 
  4. ^ The Diagnosis and Management of Anaphylaxis - XXI. Anaphylactoid reactions to fluorescein. J Allergy Clin Immunol 1998;101:S465-528. Free full text; see also The diagnosis and management of anaphylaxis: an updated practice parameter. from the National Guideline Clearinghouse.
  5. ^ a b el Harrar N, Idali B, Moutaouakkil S, el Belhadji M, Zaghloul K, Amraoui A, Benaguida M (1996). "[Anaphylactic shock caused by application of fluorescein on the ocular conjunctiva]" (in French). Presse Med 25 (32): 1546–7. PMID 8952662. 
  6. ^ Fineschi V, Monasterolo G, Rosi R, Turillazzi E (1999). "Fatal anaphylactic shock during a fluorescein angiography". Forensic Sci. Int. 100 (1-2): 137–42. doi:10.1016/S0379-0738(98)00205-9. PMID 10356782. 
  7. ^ Hitosugi M, Omura K, Yokoyama T, Kawato H, Motozawa Y, Nagai T, Tokudome S (2004). "An autopsy case of fatal anaphylactic shock following fluorescein angiography: a case report". Med Sci Law 44 (3): 264–5. PMID 15296251. 
  8. ^ Kinsella FP, Mooney DJ (1988). "Anaphylaxis following oral fluorescein angiography". Am. J. Ophthalmol. 106 (6): 745–6. PMID 3195657. 
  9. ^ Gómez-Ulla F, Gutiérrez C, Seoane I (1991). "Severe anaphylactic reaction to orally administered fluorescein". Am. J. Ophthalmol. 112 (1): 94. PMID 1882930. 
  10. ^ Kwan AS, Barry C, McAllister IL, Constable I (2006). "Fluorescein angiography and adverse drug reactions revisited: the Lions Eye experience". Clin. Experiment. Ophthalmol. 34 (1): 33–8. doi:10.1111/j.1442-9071.2006.01136.x. PMID 16451256. 
  11. ^ Jennings BJ, Mathews DE (1994). "Adverse reactions during retinal fluorescein angiography". J Am Optom Assoc 65 (7): 465–71. PMID 7930354. 
  12. ^ a b Kwiterovich KA, Maguire MG, Murphy RP, Schachat AP, Bressler NM, Bressler SB, Fine SL (1991). "Frequency of adverse systemic reactions after fluorescein angiography. Results of a prospective study". Ophthalmology 98 (7): 1139–42. PMID 1891225. 
  13. ^ a b Matsuura M, Ando F, Fukumoto K, Kyogane I, Torii Y, Matsuura M (1996). "[Usefulness of the prick test for anaphylactoid reaction in intravenous fluorescein administration]" (in Japanese). Nippon Ganka Gakkai Zasshi 100 (4): 313–7. PMID 8644545. 
  14. ^ Ellis PP, Schoenberger M, Rendi MA (1980). "Antihistamines as prophylaxis against side reactions to intravenous fluorescein". Trans Am Ophthalmol Soc 78: 190–205. PMID 7257056. 
  15. ^ Yang CS, Sung CS, Lee FL, Hsu WM (2007). "Management of anaphylactic shock during intravenous fluorescein angiography at an outpatient clinic". J Chin Med Assoc 70 (8): 348–9. doi:10.1016/S1726-4901(08)70017-0. PMID 17698436. 

External links


1911 encyclopedia

Up to date as of January 14, 2010

From LoveToKnow 1911

FLUORESCEIN, or Resorcin-Phthalein, C20H1205, in chemistry, a compound discovered in 1876 by A. v. Baeyer by the condensation of phthalic anhydride with resorcin at 195-200° C. (Ann., 1876, 183, p. 1). The two reacting substances are either heated alone or with zinc chloride for some hours, and the melt obtained is boiled out with water, washed by dilute alcohol, extracted by means of sodium hydrate, and the solution so obtained is precipitated by an acid. The precipitate is well washed with water and then dried. By repeating this process two or three times, the fluorescein may be obtained in a very pure condition. It forms a yellow amorphous powder, insoluble in water but soluble in alcohol, and crystallizing from the alcoholic solution in small dark red nodules. It is readily soluble in solutions of the caustic alkalis, the solution being of a dark red colour and showing (especially when largely diluted with water) a brilliant green fluorescence. It was so named on account of this last character. By brominating fluorescein in glacial acetic acid solution, eosin (tetrabromfluorescein) is obtained, the same compound being formed by heating 3.5-dibrom-2.4-dioxybenzoylbenzoic acid above its melting point (R. Meyer, Ber., 1895, 28, p. 1576). It crystallizes from alcohol in yellowish red needles, and dyes silk, wool, and mordanted cotton a fine pink colour. When heated with caustic alkalis it yields dibromresorcin and dibrommonoresorcin-phthalein. The corresponding iodo compound is known as erythrosin. Fluorescein is readily nitrated, yielding a dior tetra-nitro compound according to conditions. The entrance of the negative nitro group into the molecule weakens the central pyrone ring in the fluorescein nucleus and the diand tetra-nitro compounds readily yield hydrates (see J. T. Hewitt and B. W. Perkins, Jour. Chem. Soc., 1900, p. 1326). By the action of ammonia or amines the di-nitro fluoresceins are converted into yellow dyestuffs (F. Reverdin, Ber., 18 97, 3 o, p. 33 2). Other dyestuffs obtained from fluorescein are safrosine or eosin scarlet (dibromdinitrofluorescein) and rose Bengal (tetraiodotetrachlorfluorescein).

On fusion with caustic alkali, fluorescein yields resorcin, C6H 4(OH)2, and monoresorcin phthalein (dioxybenzoylbenzoic acid), (HO) 2 C 6 H 3 CO C H 4 COOH. With zinc dust and caustic soda it yields fluorescin. By warming fluorescein with excess of phosphorus pentachloride it yields fluorescein chloride, C20H1003C12 (A. Baeyer), which crystallizes from alcohol in small prisms, melting at 252° C. When heated with aniline and aniline hydrochloride, fluorescein yields a colourless anilide (0. Fischer and E. Hepp, Ber., 1893, 26, p. 2236), which is readily methylated by methyl iodide and potash to a fluoresceinanilidedimethyl ether, which when heated for six hours to 150° C. with acetic and hydrochloric acids, is hydrolysed and yields a colourless fluoresceindimethyl ether, which melts at 198° C. On the other hand, by heating fluorescein with caustic potash, methyl iodide and methyl alcohol, a coloured (yellow) dimethyl ether, melting at 208° C. is obtained (Fischer and Hepp). By heating the coloured dimethyl ether with caustic soda, the monomethyl ether is obtained (0. Fischer and E. Hepp, Ber., 1895, 28, p. 397); this crystallizes in triclinic tables, and melts at 262° C. It is to be noted that the colourless monomethyl ether fluoresces strongly in alkaline solution, the dimethyl ether of melting point 208° fluoresces only in neutral solution (e.g., in alcoholic solution), and the dimethyl ether of melting point 198° C. only in concentrated hydrochloric or sulphuric acid solution (Fischer and Hepp). Considerable discussion has taken place as to the position held by the hydroxyl groups in the fluorescein molecule, C. Graebe (Ber., 1895, 28, p. 28) asserting that they were in the ortho position to the linking carbon atom of the phthalic anhydride residue. G. Heller (Ber., 1895, 28, p. 312), however, showed that monoresorcin-phthalein when brominated in glacial acetic acid gives a dibrom derivative which, with fuming sulphuric acid, yields dibromxanthopurpurin (I. 3-dioxy-2.4-dibromanthraquinone), a reaction which is only possible if the fluorescein (from which the monoresorcin-phthalein is derived) contains free hydroxyl groups in the para position to the linking carbon atom of the phthalic anhydride residue.


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