Activity 6

WMSU-ISMP-GU-002.
00
Effective Date: 7-DEC-2016
Activity No. 6
ELECTROPHILIC AROMATIC SUBSTITUTION REACTION:
SYNTHESIS OF FLUORESCEIN VIA MODIFIED
FRIEDEL- CRAFTS ACYLATION
Friedel- Crafts reactions are a type of electrophilic aromatic

substitution reaction that involve the reaction of an aromatic ring with an
electrophile that has an electron- poor carbon center. One variation
(alkylation) includes generation of a carbocation from an alkyl chloride while
the other one (acylation) involves an acyl halide or anhydride and a Lewis
acid catalyst to form an aromatic ketone.
In this experiment, a Bronsted acid catalyst (i.e. sulfuric acid) will be

used to produce a xanthene dye from a substituted phenol and phthalic
anhydride. Xanthene dyes are triarylmethane dyes in which the two of the
aromatic rings are also connected by an ether linkage to form a fused ring
system.
Two successive Friedel- Crafts acylations will take place to produce

each xanthene dye:
Prepared by the Organic Chemistry Committee

Chemistry Department, College of Science and Mathematics
Western Mindanao State University
WMSU-ISMP-GU-002.00
Mechanism:
In this part, the acyl cation is generated by protonation of the phthalic

anhydride as shown above. The acyl cation will then react with the substituted
phenol (resorcinol). This then produces a substituted benzophenone.
After which, the substituted benzophenone formed in the first reaction

is protonated to form a cationic intermediate that can participate in a reaction
similar to the Friedel- Crafts alkylation reaction. The resulting cation then
undergoes alkylation to complete the assemble of the triarylmethane carbon
skeleton. The subsequent acid- catalyzed esterification (lactonization) then
yields fluorescein.

WMSU-ISMP-GU-002.00
Fluorescein, due to its strong fluorescence and low toxicity is used to

examine wells, trace stream flow and to label biological compounds, to detect
corneal abrasion and as to dye drugs, cosmetics and markers.

WMSU-ISMP-GU-002.00
Procedure
A. Synthesis of fluorescein using concentrated acid as an alternative catalyst
(Note: Use mask to prevent yourself from inhaling various solvent vapors)
1. To a 50 mL clean and dry test tube, add 153 mg of resorcinol and 100
mg of previously- ground solid phthalic anhydride. From this
mixture, an amount of 6 drops of 2N H2SO4 should be added. Careful
not to exceed 6 drops into sample mixture. Stir the resulting product
carefully and briefly using a stirring rod.
2. Place the test tube containing the sample in the preheated sand bath
deep enough such that its contents are just below (0.5 cm) the surface
of the sand. The reaction temperature should be monitored carefully
between 180° and 200 °C. With this, the heater setting in the sand bath
setup should be set periodically and will be checked using a
thermometer in order to keep the temperature within this range. Not
doing so can cause the resulting product to decompose.
3. Have your resulting setup first checked first by your instructor prior
proceeding to the next step.
4. When the reaction had reached 180 °C, start timing the reaction for 30
minutes. The reaction temperature should carefully be kept within this
temperature range. When the temperature has been reached, the solids
is expected to have been melted to form a solution which should
develop an orange- brown color as the reaction time progresses.
5. At the point where the reaction time had lapsed, the test tube in the
setup should be removed in the bath. Allow it to cool for about 5
minutes.
6. Without placing it in the sand bath, set the test tube containing the
sample in an iron stand and clamp. After which add 5 mL of acetone
into the sample for dissolution. The solution should then be stirred for
5 to 10 minutes using either a magnetic stirring bar or a stirring rod.
The solution is expected to turn yellow as the crude fluorescein
dissolves. If the entire product did not dissolve, further dissolution can
be done by placing an additional 5 mL of acetone until the entire
product dissolves. The total amount of solvent should not exceed than
15 mL.
7. Place the solution in a 50 mL beaker. Then, carefully boil off the

acetone in a steam bath (which is set at 56 °C), leaving a crude orange
residue. Record the weight of the crude product.

WMSU-ISMP-GU-002.00
8. Redissolve the residue using 20 mL of diethyl ether (if diethyl ether is

unavailable, use the alternative procedure as indicated in no.13 for
recrystallization) and 1 mL water. (DO NOT inhale the vapors
produced by the ether solvent). Place a magnetic stirring bar in the
solution and cover the mouth of the beaker with an aluminum foil.
After which, mount the beaker over a magnetic stirring plate for
several minutes until all the solids dissolves.
9. Transfer the solution in a small separatory funnel and add 10 mL

distilled water for washing. Shake the separatory funnel containing the
solution and allow it to create two layers. Drain off and discard the
bottom (i.e. aqueous) layer.
10. Following this, extract the ether layer in the separatory funnel once
using a 10 mL saturated NaCl solution. Again, drain off and discard
the aqueous layer.
11. Dry the organic layer using anhydrous sodium sulfate and filter using a
separate, clean, dry and pre- weighed 50 mL beaker. Remove the
solvent in the filtrate to dryness in a water bath to yield an orange solid
product.
12. Based on the data obtained, determine its percentage yield. Record
your result as well as your calculations in the Data Sheet.
13. Recrystallize the crude product by dissolving in 10 mL hot absolute

ethanol in 20 mL test tube. Stir the contents until the solids are
dissolved. Use water bath maintained at 65°C - 75°C if there were still
undissolved solids left when the contents were mixed for 3 – 4
minutes. If the solids were fully dissolved, place the test tube at room
temperature until it is cool enough to touch. If very few or no crystals
were formed, use ice bath until enough crystals are formed. Separate
the crystals by filtration then wash the residue with 3 -5 mL of cold
absolute ethanol. Dry the product in oven at 110°C and record the
weight then proceed to no.12.
B. Observation of fluorescence from the synthesized fluorescein product
1. Dissolve 5 milligrams (or spatula- tip amount) of the synthesized

product in 50 mL of 0.1 M NaOH solution. Place the resulting
solution it on a black, non- reflective surface, such a black cardboard.
2. Place a bright light source (e.g. flashlight, sunny window) on the

opposite side of the designated dark region from the observer and note
the appearance of the solution. In which, the color observed is

WMSU-ISMP-GU-002.00
primarily due to the absorbance of some wavelengths of visible light

passing through the sample form the light source.
3. In a darkened room, shine a UV lamp at the solution. The vial is

expected to visibly glow from the fluoresced light.
4. Paste an image of your observations (with appropriate captions) after

the presentation of your data/ computations.
Cleaning Up
1. Dilute the acids from the reaction mixture with water then neutralize it
using sodium carbonate before flushing down the drain.
2. Dispose the organic waste solution (which contains an organic solvent)

in an organic solvent container.
Guide Questions
1. The reaction in this experiment involves Friedel- Crafts acylation.

How can sulfuric acid be used as an alternative instead of zinc chloride
as a catalyst?
2. Explain why the synthesized compound exhibited fluorescence

activity.
3. Which layer in the separatory funnel would you expect the synthesized
compound be more prevalent? Justify your answer.
4. How can the saturated salt solution aid the recovery of the synthesized
compound in the chosen layer?
References
1. J.V. McCullagh, K.A. Daggett, J. Chem. Ed. 2007, 84(11), 1799
2. Tremayne, M.; Kariuki, B. M.; Harris, K. D. M. Angew. Chem. Int. Ed.

1997, 36, 770-772
3. Williamson, K.L. & Masters K.M. Macroscale and Microscale

Organic Experiments Sixth Edition. (2011). CA, USA: Brooks/Cole
Cengage Learning.


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WMSU-ISMP-GU-002.

Friedel- Crafts reactions are a type of electrophilic aromatic

In this experiment, a Bronsted acid catalyst (i.e. sulfuric acid) will be

Two successive Friedel- Crafts acylations will take place to produce

Prepared by the Organic Chemistry Committee

In this part, the acyl cation is generated by protonation of the phthalic

After which, the substituted benzophenone formed in the first reaction

Prepared by the Organic Chemistry Committee

Fluorescein, due to its strong fluorescence and low toxicity is used to

Prepared by the Organic Chemistry Committee

A. Synthesis of fluorescein using concentrated acid as an alternative catalyst

7. Place the solution in a 50 mL beaker. Then, carefully boil off the

Prepared by the Organic Chemistry Committee

8. Redissolve the residue using 20 mL of diethyl ether (if diethyl ether is

9. Transfer the solution in a small separatory funnel and add 10 mL

13. Recrystallize the crude product by dissolving in 10 mL hot absolute

B. Observation of fluorescence from the synthesized fluorescein product

1. Dissolve 5 milligrams (or spatula- tip amount) of the synthesized

2. Place a bright light source (e.g. flashlight, sunny window) on the

Prepared by the Organic Chemistry Committee

primarily due to the absorbance of some wavelengths of visible light

3. In a darkened room, shine a UV lamp at the solution. The vial is

4. Paste an image of your observations (with appropriate captions) after

2. Dispose the organic waste solution (which contains an organic solvent)

1. The reaction in this experiment involves Friedel- Crafts acylation.

2. Explain why the synthesized compound exhibited fluorescence

1. J.V. McCullagh, K.A. Daggett, J. Chem. Ed. 2007, 84(11), 1799

2. Tremayne, M.; Kariuki, B. M.; Harris, K. D. M. Angew. Chem. Int. Ed.

3. Williamson, K.L. & Masters K.M. Macroscale and Microscale

Prepared by the Organic Chemistry Committee

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