Fluorescein is an organic compound and dye that is available as a dark orange/red powder that is slightly soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications. It is prepared by heating phthalic anhydride and resorcinol over a zinc catalyst, and it crystallizes as a dark red powder. The reagent fluoresces strongly green in the presence of alkaline solutions and is therefore used as a marker when conjugated to antibodies or as a contrast agent for ophthalmic angiography.
The color of its aqueous solution is green by reflection and orange by transmission, which can be noticed at the bubble level, for example, where fluorescein is added as a colorant to the alcohol in the filled tube to increase the visibility of the bubbles contained therein ( Thereby improving the accuracy of the instrument). More concentrated fluorescein solutions may even appear red.
There are two variants of fluorescein: stable red variant B and yellow variant A. The molecular formula is C20H12O5 and the molecular weight is 332.31. B is a red crystalline powder with green fluorescence. Melting point 314~316*C (decomposition). Soluble in hot alcohol, hot aniline, hot acetone, hot formic acid, slightly soluble in water, alcohol, ether, acetic acid, insoluble in petroleum ether. A is a yellow amorphous powder with a melting point of 314-316°C. Soluble in acetone, methanol, formic acid, slightly soluble in water, alcohol, ether, chloroform, benzene, acetic acid, xylene, nitrobenzene, insoluble in petroleum ether. Turns into a red variant when heated. Used as an indicator for chemical analysis and a biological stain. It is obtained by the reaction of resorcinol and phthalic anhydride.
Physicochemical properties of fluorescein:
Appearance: Orange or light yellow-red to red crystalline powder.
Odor: Odorless
Melting point/freezing point (°C): 125-127°C (dec.)
Boiling point, initial boiling point, boiling range (°C): 620.8°C/760mmHg
Solubility: Soluble in acetone, pyridine, hot ethanol, methanol, formic acid, carbonate alkali and hydroxide alkali solution, and show bright green fluorescence; slightly soluble in water, alcohol, ether, benzene, chloroform, acetic acid, xylene, nitro Benzene, insoluble in petroleum ether.
Fluorescein Toxicological Data:
acute toxicity
Rat intraperitoneal LDLo: 600 mg/kg
Mouse intravenous LC50: 300 mg/kg
Mouse LCLo: 600 mg/kg
Rabbit intravenous LDLo: 300 mg/kg
Guinea pig LDLo: 400 mg/kg
Mutagenicity
DNA adduct detection system: Bacteria – Escherichia coli: 15umol/L
Fluorescein numbering system:
CAS number: 2321-07-5
MDL number: MFCD00005050
EINECS Number: 219-031-8
RTECS No.: LM5075000
BRN number: 94324
PubChem number: 24859566
Description of fluorescein properties:
It reacts with strong bases such as sodium carbonate (potassium) and sodium hydroxide (potassium) to produce sodium fluorescein (potassium), which is easily soluble in water and has strong green fluorescence with a fluorescence quantum yield of 0.65 (pH=7 aqueous solution).
Fluorescein Instructions for Use:
1. Adsorption indicator for precipitation titration:
Dubbed into 0.1% ethanol solution, or dubbed with its sodium salt into 1% aqueous solution. Use Ag+ to titrate Cl-, Br-, I-, SCN-, CN-, C2O42-, SO42-, CO32-, etc. In neutral or weakly alkaline medium, the color of the titration end point changes from yellow-green to rose red.
2. Fluorescein is the substrate of luminescent substances. The substance that makes many living things fluorescent. It forms a complex with ATP (fluorescein adenosine), which then binds to luciferase. Fluorescein activated during oxidation emits light. The entire reaction is used for the detection of living organisms or for quantification of very low levels of bacterial contamination. For example, it can be measured with a fluorophotometer.
3. Used as indicator for chemical analysis, biological dye and cosmetic colorant.
4. The important pigment of antifreeze can make the antifreeze show green fluorescence.
5. Fluorescent dyes, chemicals that emit light when excited by light. The emitted light typically has a longer wavelength than the incident light. Fluorescent dyes are substances that cause other substances to fluoresce, i.e. dyes used to label or tag other compounds with fluorescence.
6. Contrast agents, substances used to enhance visualization of tissue.
Synthesis method of fluorescein:
Heat resorcinol to 150°C to melt it completely, add a theoretical amount of phthalic anhydride while stirring, mix and melt, then raise the temperature to 185°C, keep it warm for half an hour, and then slowly add an appropriate amount of freshly roasted Anhydrous zinc chloride, when it is completely dissolved, gradually heat up to 210-215°C, the whole process needs to be stirred continuously, when the reaction liquid starts to thicken, stop stirring, continue to heat at this temperature until it is completely solidified, grind After crushing, a powdery crude product is obtained. Mix the powdered crude product with dilute hydrochloric acid, heat and boil to extract zinc chloride and the remaining resorcinol, filter it with suction, wash it with water, dry it, extract it with ethanol, and get red solid fluorescein after drying.
How to store fluorescein:
Avoid contact with oxides, keep container tightly closed, in a tightly closed container, and store in a cool, dry place.
