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Synthesis of Fluorescein

Experimental Problem

To explain utility of given dicarboxylic acid (phthalic acid) / anhydride derivative

Educational Purpose

By performing this lab, the student will:

1. Synthesize fluorescein.2. Identify the wavelength of maximum absorbance ( max)in different pH.Tutorials

Cf. UV-visible notes Fluorescence Phosphorescence

What To Turn In

1. Questionnaire: prelab and postlab2. UV spectra (2 in number)

Theory:

Fluorescein is an organic fluorescent dye used in several areas, ranging from medicine toresearch applications. Structurally, fluorescein is similar to phenolphthalein, a well known dye

indicator used in acid-base titrations. The difference can be seen in Figure 1, illustrating oxygenbridging the aromatic rings on fluorescein. This relatively simple change has a significant impacton the spectral behavior of these two dyes. Fluorescein is fluorescent whereas phenolphthalein is

not.

Fluorescein Phenolphthalein

Figure 1 Neutral structures of fluorescein and phenolphthalein

Fluorescence occurs due to the emission of light from the relaxation of the molecule from theexcited state to the ground state. The electronic transition to the excited state occurs when the

excitation wavelength corresponds to the energy difference between the ground state and the

excited state. In the excited state, some energy is lost to vibrational relaxation. As a result,

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fluorescence energy is less than the absorption energy and the emitted light is observed at a

longer wavelength than the excitation wavelength.

In this experiment, you will synthesize fluorescein by the reaction shown in Figure 2. This test is

given by compounds which have two carboxylic groups and aromatic in nature or it can also beused as test for resorcinol. Dicarboxylic acids or its anhydride (succinic acid, phthalic acid) when

heated with resorcinol in the presence of conc. sulphuric acid, a fluorescent dye calledfluorescein is obtained.Visible absorbance and fluorescence spectroscopy will be best to be used to identify the

wavelength of maximum absorbance ( max) and emission wavelength.

Phthalic anhydride Resorcinol Fluorescein

Figure 2 Synthesis of fluorescein

MATERIALS

Boiling tube, utility clamp, Bunsen burner, 250 mL beaker, Glass rod, UV-vis spectrometer andcuvettes

Resorcinol, phthalic anhydride, 0.5 M sulfuric acid, 0.1 M sodium hydroxide, 0.1 M

Hydrochloric acid

PROCEDURE

Part I Synthesis of Fluorescein

1. Obtain and wear goggles. Protect your arms and hands by wearing a long-sleeve lab coat andgloves. Conduct this reaction in a fume hood.

2. Clamp a boiling tube to a stand.

3. Prepare the reaction mixture.a. Weigh out 0.3 g of resorcinol and transfer to the boiling tube. Record the actual mass to the

nearest 0.0001 g.

b. Weigh out 0.2 g of phthalic anhydride and transfer to the boiling tube. Record the actual mass

to the nearest 0.0001 g.c. Slowly add approximately 1 mL of 0.5 M H2SO4to the mixture of solids in the boiling tube.

CAUTION:Handle the sulfuric acid with care. Can cause painful burns if it comes in contact

with the skin.

4. Heat the reaction at approximately 160180C for 2-3 minutes. Monitor the reaction either bytemperature using a Wide-Range Temperature Probe or thermometer or color change. Note: A

red-brown solid will form as the reaction progresses. If there is no colour change heat it more

(Ask your instructor)5. Let the flask cool for at least 5 minutes.

Part II Spectrophotometric Absorbance

You will analyze your samples by UV vis spectrometer.This technique is also to determine the amount of fluorescein in your synthesized

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Sample (cf. KMnO4experiment). You can also use this information to calculate its purity.

1. Prepare a blankby filling a cuvette 3/4 full with water

2. Record UV vis spectra of given sample in water. Choose New from the File menu of the data-collection program.

3. Determine the optimal wavelength for analysis by collecting a full absorbance spectrum.4. Repeat step 1-3 with 0.1M HCl aq. solution5. Then repeat step 1-3 with 0.1M NaOH aq. solution (only on advice of your i nstructor)

Note: If the absorbance value does not fall within range of your standard solutions, prepare amore dilute or more concentrated sample, depending on the absorbance value from your test.

Pre Lab (20 marks)

1. Explain the difference between an absorbance and fluorescence spectra. (5)2. Explain the difference between phosphorecence and fluorescence spectra. (5)

3. Draw the three charged species of fluorescein (cation, monoanion, dianion). (3)

4. Draw the three neutral species of fluorescein (quinonoid, lactone, zwitterion). (3)5. What is the utility of fluorescein. Give two examples. (2+2)

Post Lab (10 marks)1. Will benzoic acid give fluorescence under same chemical treatment. Support your

answer.

2. What is the colour change in your sample at different pH conditions. Explain it w.r.t. theUV-visible spectra recorded and presence of predominant species at that pH.