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Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is used as a fluorescent Flow tracer in many applications.
The color of its aqueous solutions is green by reflection and orange by transmission (its spectral properties are dependent on pH of the solution), as can be noticed in spirit level, for example, in which fluorescein is added as a colorant to the alcohol filling the tube in order to increase the visibility of the air bubble contained within. More concentrated solutions of fluorescein can even appear red (because under these conditions nearly all incident emission is re-absorbed by the solution).
It is on the World Health Organization's List of Essential Medicines.
The thyroxine ester of fluorescein is used to quantify the thyroxine concentration in blood.
Fluorescein is also known as a color additive (D&C Yellow no. 7). The disodium salt form of fluorescein is known as uranine or D&C Yellow no. 8.
Fluorescein is a precursor to the red dye eosin Y by bromination.
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, including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop. Reported rates of adverse reactions vary from 1% to 6%. 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. The risk can be reduced with prior (prophylaxis) use of antihistamines
and prompt emergency management of any ensuing anaphylaxis. A simple prick test may help to identify persons at greatest risk of adverse reaction.
Fluorescein has a p Ka of 6.4, and its ionization equilibrium leads to pH-dependent absorption and Fluorescence 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 nonintensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry. Upon exhaustive irradiation with visible light fluorescein decomposes to release Phthalic acid and Formic acid acids and carbon monoxide, effectively acting as a photoCORM. The remaining resorcinol rings react with singlet oxygen formed in situ to give oxidized, ring opening products.
Fluorescein has an isosbestic point (equal absorption for all pH values) at 460 nm.
In oligonucleotide synthesis, several phosphoramidite reagents containing protected fluorescein, e.g. 6-FAM phosphoramidite 2,Brush, C. K. "Fluorescein Labelled Phosphoramidites". . Priority date July 19, 1991. are used for the preparation of fluorescein-labeled .
The extent to which fluorescein dilaurate is broken down to yield lauric acid can be detected as a measure of pancreatic esterase activity.
Fluorescein can also be conjugated to nucleoside triphosphates and incorporated into a probe enzymatically for in situ hybridisation. The use of fluorescein amidite, shown below right, allows one to synthesize labeled for the same purpose. Yet another technique termed makes use of synthetic fluorescein-labeled oligonucleotides. 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.
Intravenous or oral fluorescein is used in fluorescein angiography in research and to diagnose and categorize vascular disorders including retinal disease, macular degeneration, diabetic retinopathy, inflammatory intraocular conditions, and intraocular . It is also being used increasingly during surgery for Brain tumor and spine tumors.
Diluted fluorescein dye has been used to localise multiple muscular ventricular septal defects during open heart surgery and confirm the presence of any residual defects.
As fluorescein solution changes its color depending on concentration, it has been used as a tracer in evaporation experiments.
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 local wildlife.. Greenchicagoriver.com. Retrieved on 2014-08-28.
Fluorescein dye solutions, typically 15% active, are commonly used as an aid to leak detection during of subsea petroleum pipelines and other subsea infrastructure. Leaks can be detected by divers or ROVs carrying an ultraviolet light.
Safety
Oral and intravenous use of fluorescein can cause adverse reactions, including nausea, vomiting, urticaria, acute hypotension, anaphylaxis and related anaphylaxis, The diagnosis and management of anaphylaxis: an updated practice parameter. National Guideline Clearinghouse. causing cardiac arrest and sudden death due to anaphylactic shock.
Chemistry
The fluorescence of this molecule is very intense; peak excitation occurs at 495 Nanometre and peak emission at 520 nm. Values for the deprotonated form in basic solution.
Derivatives
Many derivatives of fluorescein are known. Examples are:
Synthesis
Approximately 250 tons were produced in the year 2000. The method involves the fusion of phthalic anhydride and resorcinol, similar to the route described by Adolf von Baeyer in 1871.Baeyer, Adolf (1871) "Uber ein neue Klasse von Farbstoffen" (On a new class of dyes), Berichte der Deutschen chemischen Gesellschaft zu Berlin, 4 : 555-558; see p. 558. In some cases, acids such as zinc chloride and methanesulfonic acid are employed to accelerate the Friedel-Crafts reaction.
mechanism of the reaction when undertaken with a strong acid (for ex.
Research
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 Fluorescence maximum of 512 nm (in water). The major derivatives are fluorescein isothiocyanate (FITC) and, in oligonucleotide synthesis, 6-FAM phosphoramidite.
Biosciences
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 antibody) 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.
Earth sciences
Fluorescein is used as a rather conservative flow tracer in hydrological Dye tracing to help in understanding of water flow of both and groundwater. The dye can also be added to in environmental testing simulations to aid in locating and analyzing any water leaks, and in Australia and New Zealand as a methylated spirit dye.
Plant science
Fluorescein has often been used to track water movement in groundwater to study water flow and observe areas of contamination or obstruction in these systems. The fluorescence that is created by the dye makes problem areas more visible and easily identified. A similar concept can be applied to plants because the dye can make problems in plant vasculature more visible. In Botany, fluorescein, and other fluorescent dyes, have been used to monitor and study Vascular tissue, particularly the xylem, which is the main water transportation pathway in plants. This is because fluorescein is xylem-mobile and unable to cross Cell membrane, making it particularly useful in tracking water movement through the xylem. Fluorescein can be introduced to a plant's veins through the roots or a cut stem. The dye is able to be taken up into the plant the same way as water and moves from the roots to the top of the plant due to a transpirational pull. The fluorescein that has been taken up into the plant can be visualized under a fluorescent microscope.
See also
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