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Fluorescein Synthesis Experiment


Chungnam Samsung Academy

Have you ever wondered how highlighters or glow sticks emit fluorescent light? Ignis chemistry club conducted an experiment synthesizing a fluorescent substance called fluorescein.


The experiment was conducted to observe the luminescence of fluorescein and analyze the material by using UV-Vis spectroscopy.



- Chemical formula: C20H12O5.

- It is a yellow acidic dye belonging to the triphenylmethane group of phthalene, appearing as a red powder and being difficult to dissolve in water. It is a fluorescent chemical substance that absorbs light in aqueous solution and emits strong yellow-green fluorescence (530-570nm), hence its name derived from the meaning of fluorescence. Although it is difficult to dissolve in water, it dissolves in alkaline solutions and exhibits green fluorescence, even in small amounts. Currently, it is used as an analytical reagent for sodium salt adsorption indicators, fluorescent indicators, and other analysis indicators.

- Fluorescein absorbs peak absorption and has a blue light at wavelengths of 465 to 490nm. Fluorescent emission occurs at wavelengths of 520 to 530nm, producing a yellow-green color.

Fluorescein synthesis



Ultraviolet-visible spectrophotometer. It can measure the electronic spectrum in the ultraviolet-visible region (190-900nm) and is capable of qualitative and quantitative analysis of samples. It can measure transmittance, reflectance, and color difference.

Transmittance: The ratio (or percentage) of the intensity (or number of particles) of the transmitted wave through a substance layer or interface to the incident particle number, or the reciprocal of the corresponding absorption coefficient. In the case of light, it refers to the ratio or percentage of the transmitted light energy to the incident light energy when parallel rays enter one side of the boundary between two media.

Absorbance: It refers to the extent to which an object absorbs light. The method for determining absorbance is A=log(I0/I) (A=absorbance, I0=intensity of light incident on the object, I=intensity of light transmitted through the object). In some cases, Lambert-Beer's law applies to absorbance, where A=log(I0/I)=εcd (ε=molar absorptivity, c=molar concentration, d=thickness of the absorbing layer). As the equation shows, it is proportional to the concentration and thickness of the substance

How to use uv-vis

1. Turn on the computer and turn on the uv-vis device after running the uv-vis program.

2. Turn on the two light bulbs on the left side of the screen and set the spectrum/peak when the main body is lit.

3. Specify the range of the wavelength to be measured in parentheses of from () to ().

4. Put the blank solution in a cuvette for at least two-thirds and put it in the device with the transparent side facing the device.

5. When yellow light comes in after clicking the blank button, check that the peak value of the graph matches the wavelength investigated in advance.

6. Investigate the wavelength of the sample solution in the same way as the blank solution, but press the sample button instead of the blank button.

7. Turn off the two light bulbs on the left side of the screen and press the switch at the bottom left of the device to power off.

8. Close the uv-vis program on your computer and turn it off.



1. Measure 0.2g of Resorcinol and 1.6g of Phthalic Anhydride on the electronic scale.

2. Put 0.2g of Resorcinol and 1.6g of Phthalic Anhydride in a beaker.

3. Place 5 drops of dilute phosphoric acid in a beaker.

4. Heat the solution at about 110 degrees for 5 minutes using a stirrer.

5. Cool the beaker at room temperature for 1 minute.

6. Put 0.3g of Sodium Hydroxide in a beaker.

7. Observe the changes.



Reasons for using phosphoric acid (sulfuric acid) as a catalyst, and for using sodium hydroxide

The original experiment video we referred to used sulfuric acid, but in our experiment, we used phosphoric acid. Phosphoric acid, like sulfuric acid, can serve as a Brønsted acid catalyst. A Brønsted acid catalyst operates based on the transfer of hydrogen ions (H+), or protons, according to the Brønsted-Lowry acid-base theory. The catalyst's "oxygen vacancies" (vacant oxygen atoms in the substance) alter the structure of the transition metal (in this experiment, Zn), facilitating chemical reactions. It was discovered that the oxygen vacancies change the energy levels of the outermost electron shell (orbital), enhancing overall performance. As the oxygen vacancies increase, both oxygen generation and reduction reactions are promoted.

Concentrated sulfuric acid is known to be an excellent catalyst for alkylation. This is because sulfuric acid, as an acidic catalyst, provides hydrogen ions to promote the reaction. Therefore, the reason for adding sulfuric acid in the synthesis of fluorescein is its role as a catalyst in reacting phthalic acid to synthesize fluorescein.

Additionally, the reason for adding sodium hydroxide is to maintain the reaction solution's pH at a neutral level. The synthesis reaction of fluorescein occurs in a neutral environment, so if the pH is too high or too low, the reaction may not proceed as desired. Therefore, adding sodium hydroxide to maintain a neutral pH ensures that the synthesis reaction of fluorescein proceeds as intended.

How to increase the intensity of chemiluminescence

The intensity of chemiluminescence is influenced by factors such as pH, type of solvent, ion concentration, and temperature. A cooler solution slows down the reaction and prolongs the chemiluminescence. Chemiluminescence lasts only a few minutes and is best observed in very dark environments. Adding nanoparticles such as gold or silver increases the intensity of the emitted light compared to the standard sample (where water is injected instead of nanoparticles). Therefore, to increase the luminescence intensity of the experimented fluorescein, the solution can be cooled or additional nanoparticles can be added.

During the investigation of fluorescein synthesis, it was found that fluorescein can be used as an indicator for neutralization titration.

It appears in two main forms:

1) Adsorption indicator: Used for the titration of chloride ions, bromide ions, and iodide ions in neutral or slightly alkaline solutions. When adsorbed onto silver halide, it turns red.

2) Fluorescent indicator: Exhibits green fluorescence in alkaline solutions and loses fluorescence at pH 4.3-3.8, making it suitable for use as an indicator in neutralization titration.

These forms of indicators are collectively referred to as fluorescent indicators. It is said that when used for silver titration, fluorescein loses its green fluorescence at the endpoint.

Furthermore, through UV-Vis spectroscopy, it is known that fluorescein is a synthetic organic compound that absorbs light at a wavelength of around 490 nm and emits strong fluorescence at around 510 nm. Significant changes were observed in the range of 400-500 on the device. This is due to the fact that a substance that absorbs light (usually ultraviolet or visible light) emits light again when transitioning from an excited electronic state to a lower energy state, typically the ground electronic state.


The experiment synthesized fluorescein using resorcinol and phthalic anhydride. Phosphoric acid was used as a catalyst, and sodium hydroxide was added to dissolve the substances. Upon heating, a small amount of red light emitting yellow substance was synthesized, which exhibited fluorescent light when dropped into water. When illuminated with a UV lamp, the fluorescence was stronger. In the first experiment, due to various reasons noted in the observation, the fluorescein layer showed signs of separation. The UV-Vis spectrum of the unseparated layer showed a wavelength range similar to the expected, but there was an error in the absorbance exceeding 3, possibly due to incomplete dissolution. In the second experiment, although successful in obtaining fluorescent light, the observation of proper fluorescein was hindered during the wastewater treatment process. In the final third experiment, the desired fluorescein was obtained, and the fluorescent reaction was also observed.


∙ Chemical substance information search. (n.d.).

ㆍTuning the Chemiluminescence of a Luminol Flow Using Plasmonic Nanoparticles. (n.d.). Nature.


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