INDEX

1. Introduction 3
2. Aim 4
3. Materials required 4
4. Theory 5
5. Procedure 10
6. Observations 12
7. Precautions 17
8. Bibliography 18
ACKNOWLEDGEMENT
I would like to express my special thanks to my Chemistry teachers Ms. Mukta Gupta
and Ms. Amina and our Chemistry lab assistant Mr. Anil Dubey, who gave me the
opportunity to do this project and helped me throughout the process.
Under their guidance, I have learnt a lot while doing this project, be it better conceptual
understanding of the underlying principles or from their timely suggestions
regarding the practical implementation of the project.
Kartikeya Badola
XII-D
CBSE roll no.
qz,octD9
 AIM
To synthesize fluorescein using resorcinol and phthalic anhydride via Fischer
esterification, to test its effects on blood stains and to observe its colour in strongly acidic
and strongly basic medium.

CHEMICALS REQUIRED
1. 20gm Resorcinol
2. 13.3gm Phthalic anhydride
3. 15 drops of cone. Sulphuric acid
4. 40ml Ethanol
5. 1ml glacial Acetic acid
6. 3g Zinc dust
7. 10ml dilute Sodium hydroxide solution
8. 1ml cone. Nitric acid
9. 2ml blood sample
10. 1/4th test tube of both cone. Sulphuric acid and cone. Sodium hydroxide solution.

MATERIALS REQUIRED
1. Hot water bath
2. Bunsen burner
3. Beaker
4. Weighing machine
5. Test tubes
6. Surgical gloves and mask
7. Glass rod
8. Watch glass
9. Uv light

4
INTRODUCTION
Fluorescein is an organic compound which is commercially produced and used as a dye
in fluorescent markers. It is also used as a tracer and is injected in the body of patients to
help map the vascular system and diagnose diseases. It is also used in forensics to reveal
latent blood stains at the crime scene. This compound shows fluorescence i.e it emits
light when irradiated with a beam of suitable wavelength.
 a} Synthesis

Fluorescein is made by the dehydration reaction of Resorcinol and Phthalic Anhydride. It

undergoes Fischer esterification. Resorcinol and Phthalic Anhydride are reacted in 3:2
ratio by mass in presence of a dehydrating agent like cone. Sulphuric Acid. The reaction
mechanism is as follows-:

OH +
o

Q
OH OH
0

:o f :OH
Hv?:(±)
0 H --
#
0

t :OH

- eJ

Q
Acylium ion
OH 0

cc
0 OH

(±)
+
CO2H OH OH

The first reaction is a Friedel-Craft's acylation reaction. Acy[ cation is generated by

protonation of phthalic anhydride which then reacts with resorcinol to produce
a substituted benzophenone.

6
THEORY
Fluorescein isa compound which shows fluorescence. Fluorescence is the emission of
light bya substance that has absorbed light or other electromagnetic radiation. In most
cases the emitted light is of lower frequency hence lower energy than the light which
it absorbs. Fluorescein is excited by a light of wavelength 494 nm and emits the light
of
521 nm.

--Emission I "I
' ''
----Excitation '
'
I
I
I

I '
I
I
'' I
I

I '' II
I

I ' I
I
I I

--
I
#
,
I '' \

300 400 500 600

Fluorescein excitation and emis · specrr--i·,... ..-_.--_.....---------
Fluorescein is a red coloured powaer but its dilute solution in S ct· h . .
O 1um ydroxide 1S
fl11nrocr-ont rrroon 1n f'nlr.11y, 'T'h,r- ;,. h
n ....,_,,,..""' ,...'"'V'\,...,..., ., ,,....,.,.....J Cl
 b) Action on blood stains
Fluorescein is a presumptive blood test for latent bloodstain detection. It has been used
in forensic applications to reveal trace amounts of blood. Traces of latent blood can be
detected even after repeated cleansing of the crime scene. Fluorescein is highly sensitive
to the hemoglobin associated molecules (enzymes and iron) in the red blood cells,
(1:105,000 depending upon the dilution rate of the working solution). Traces of these
hemoglobin associated molecules will embed themselves on the substrate, even after
multiple cleanings. Fluorescein reacts to the proteins and iron ions found in the
hemoglobin and will fluoresce when exposed/excited to light at the 420 nm to 485 nm
wavelengths peaking at about 460 to 480 nm range (blue light, near Ultraviolet).

Fluorescein is reduced by zinc powder in aqueous or alcoholic medium to prepare a

reduced fluorescein solution. This is sprayed over the spot where there may be chance of
blood stains to be present. Then after letting the surface absorb the spray for a while, the
reduced fluorescein solution is oxidised using hydrogen peroxide overspray (or any
other oxidiser). The oxidised solution shows fluorescence whereas the reduced one does
not. If blood is present on the surface then positive test suggests the possibility that
fluorescein was absorbed by the blood molecules adsorbed on the surface it is sprayed
on.

8
 Second Friedel-Crafts:
OH
H
OH 0,.... C02H

,D
I OH

HO

HO # HO

OH
j OH

Proton transfer
-H20

HO HO
OH

Intramolecular
Fischer esterification

- --
...

HO Fluoresce in

Under the reaction conditions used, the substituted benzophenone formed in the first
reaction is protonated to farm 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 assembly of the triarylmethane carbon
skeleton.

7
r

PROCEDURE
Synthesis of Fluorescein

1. In a beaker 20 g of solid Resorcinol was added to 13.3 g of solid

Phthalic Anhydride.
2. 15-20 drops of concentrated sulphuric acid was added to the above beaker.
3. The mixture was heated in a boiling water bath for SO minutes.
4. The mixture was stirred while heating using a glass rod to make sure that flakes of
Resorcinol are completely reacted.
5. After SO minutes the beaker was taken out and cooled. A red semi solid was
formed.
6. The fluorescein formed was transferred into a plastic bag.
7. The final mass of fluorescein formed was 25.33g
8. A sample of fluorescein was added to a test tube containing dilute NaOH soln.
This solution was drop by drop added to a beaker containing water under UV light.
Fluorescence was observed.
 Action on blood stains
1. A very small scoop of Fluorescein was added in a test tube containing 15ml
Ethanol and 1ml glacial Acetic acid. To this test tube 2g of Zinc Dust was added.
The colour of solution changed to bluish green due to reduction of Fluorescein.
2. The above solution was further diluted in another test tube in ethanol. The spray
solution was now ready.
3. The blood sample was added to a watch glass. It was left to dry. Carefully the
blood sample was gently cleaned by filter paper from the middle.
4. The reduced solution was added on the site. It was left to dry.
5. Concentrated HN03 was added on the site. The reduced fluorescein adsorbed
on the surface is now oxidised.
6. The watch glass glowed under uv light due to Fluorescence of Fluorescein.

pH dependant colour change

1. Took aqueous solution of fluorescein (dilute) in a test tube.

2. To one part added cone. Sulphuric acid (about 1/4th test tube).
3. To other added cone. NaOH solution (about 1/4th test tube).
4. Colour of the two solutions was observed.
PRECAUTIONS
1. Phthalic anhydride is a mild irritant. It can cause irritation in eyes and skin
hence gloves and safety glasses are required.
2. Resorcinol is a very dangerous compound. Inhalation of vapors or dust of
resorcinol can causes irritation of respiratory tract. Hence masks are a must
and the container should be emptied away from other people in the lab. It is
also a severe eye irritant and ingestion causes burning of mucous membranes.
Hence after the synthesis it is mandatory to wash our hands.
3. Resorcinol is combustible hence its heating must be done very carefully.
4. Resorcinol should be kept in a place away from disturbance. Too much
shaking may lead to aggregation of charges which would result in higher
reactivity of resorcinol with its surroundings.
BIBLIOGRAPHY
Nile Red: Making Fluorescein https: I /youtu.bel xXZcRwh28TE

Fluorescein detection of latent bloodstains http:l lwww.latent-prints.com/fluorescein2.htm

SYNTHESIS OF XANTHENE DYES USING ELECTROPHILIC AROMATIC SUBSTITUTION

http: I I sites.uci.edul chem52labsl files/ 2014/031M52LC Experiment 1S13.pdf

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