DaMocles SS 2011 – Jonas von Irmer, Jhilmil Hossain, Sebastian Dewald & Maximilian Gatterdam

Fluorescein
Fluorescein is a fluorescent dye, which is 3. Synthesis 4
mostly used as a chemical indicator in
many applications. Its disodium salt is a
red crystalline (chinoid form) or yellow
(lactoide form) powder called uranine,
soluble in water and alcohol, and belongs
to the group of xanthene dyes.

13

+H+

1. General data 1, 2
-H2O

IUPAC-name: 2-(6-Hydroxy-3-
oxo-3H-xanthen-9-
yl)-benzoic-acid
CAS-No.: 2321-07-5
Molecular Formula: C20H12O5
Molecular Weight: 332,32 g/mol
Melting point: 314 – 316 °C
-H+
Excitation max.: 494 nm
Emission max.: 521 nm
Lifetime: 3-4 ns

2. History 3

Fluoresceine was discovered by Adolf von

Baeyer in 1871 while experimenting with
the analogous phenolphthalein which is
synthesized from Phenol and phthalic
-H2O
anhydride. In comparison fluorescein
(earlier called Resorcinphthalein) is
synthesized by condensation of Resorcinol
and phthalic anhydride as shown in the
mechanism.
 DaMocles SS 2011 – Jonas von Irmer, Jhilmil Hossain, Sebastian Dewald & Maximilian Gatterdam

4. Fluorescence 5 5. Derivatives

Fluorescence is a special type of Not only fluorescein, but also its

luminescence. It describes the absorption derivatives are needed in different fields.
of light or other electromagnetic radiation Just to mention a few of them:
and following emission of light of a
5.1 Eosin B
different wavelength.

As shown in the Jablonski diagram the

absorbed electromagnetic radiation
excites an electron from the ground state
S0 to the excited state S1. This electron can
relax to its ground state by “non-radiative
relaxation” in which the excitation energy
is transformed into vibration (heat) or by 5.2 Eosin Y
emitting radiation (fluorescence). The
absorption and emission wavelength are
specific for every fluorescent substance.
Commonly the lifetime of the excited state
is a few nanoseconds.

An explanation of phosphorescence will

not be given here because it is not
relevant for fluorescein.
Both forms of eosin are used for
14
histological compounds, indicators, to
dye tissues and cells, and to dye paper
and textiles 6.

5.3 Fluorescein isothiocyanate (FITC)

Carboxyfluorescein*

*uses in chapter 6.4

 DaMocles SS 2011 – Jonas von Irmer, Jhilmil Hossain, Sebastian Dewald & Maximilian Gatterdam

6. Applications

6.1 Uses in Water systems 1, 7: 6.3 Uses in analytic chemistry:

Fluorescein is used for dyeing A method to detect bromides is to
rivers and springs to trace their oxidize them with K2Cr2O7 and
stream course also the concentrated sulfuric acid so
subterranean ones. gaseous bromine is produced. The
It can also be applied to visualize bromine reacts with a filter paper
water leaks. drenched in a solution of 0,2g
It was also used for dyeing the uranine in 100ml ethanol and a
Chicago River green on St. Patrick`s drop of ammoniac solution (10%)
Day from 1962-1966. coloring it pink 2.
Shipwrecked crews can deploy Fluorescein (or eosin) can also be
fluorescein packages to color the used as indicator in the
sea to augment the chances of argentometry (Fajans method)9, 10.
being rescued. 500g of uranine can
dye up to 4000 m² 6.4 Fluorescence labeling:
Using this technique antibodies,
6.2 Uses in Angiography 1, 8: proteins, amino acids and peptides
Fluorescein angiography is a are labeled attaching a reactive
visualization of vasculature by derivate of a fluorophore (in this
injection of an uranine solution. case fluorescein). These can then
The now fluorescing veins can be be detected by a fluorescence
detected with a specific camera to microscope.
observe retinal diseases (macular
FITC conjugates primary amines
degeneration, inflammatory
reacting to thiourea. This method
intraocular conditions, diabetic
is applied to visualize antibodies 11.
retinopathy, and intraocular
Carboxyfluorescein can be
tumors). A new application is to
incorporated into liposomes which
show brain tumors during brain
can then be tracked passing the
surgeries.
body 12.

15

Fluorescing tissue (angiography)

 DaMocles SS 2011 – Jonas von Irmer, Jhilmil Hossain, Sebastian Dewald & Maximilian Gatterdam

7. Sources

1
http://en.wikipedia.org/wiki/Fluorescein
(7.7.2011 11:24)
2
http://www.omikron-
online.de/cyberchem/cheminfo/0221-
lex.htm (8.7.2011 14:32)
3
http://de.wikipedia.org/wiki/Fluorescein
(8.7.2011 14:26)
4
http://www.mgh.schulnetz.hamm.de/fae
cher/chemie/farbstoffe/Fluorescein.doc
(8.7.2011 14:29)
5
http://www.zoologie-
skript.de/methoden/fluo/fluo1l.htm
(8.7.2011 14:35)
6
http://en.wikipedia.org/wiki/Eosin
(8.7.2011 14:25)
7
http://en.wikipedia.org/wiki/Dye_tracing
(8.7.2011 14:17)
8
http://en.wikipedia.org/wiki/Fluorescent_
angiography (8.7.2011 14:21)
9
http://de.wikipedia.org/wiki/Titration_na
ch_Fajans (8.7.2011 13:39)
10
http://en.wikipedia.org/wiki/Argentometr
y#Fajans_method (8.7.2011 13:40)
11
http://en.wikipedia.org/wiki/Fluorescein_
isothiocyanate (8.7.2011 14:10)
12
http://en.wikipedia.org/wiki/Carboxyfluor
escein (8.7.2011 14:08)
13
http://upload.wikimedia.org/wikipedia/co
mmons/d/d3/Fluorescein-sample.jpg
(8.7.2011 14:43)
14
http://www.zoologie-
skript.de/methoden/fluo/jablonl.jpg
(8.7.2011 14:37)
15
http://www.optos.com/Global/images/pr
of_genophth_case_retdetach_large.jpg
(8.7.2011 14:39)

All Lewis structures are self-made with

ChemSketch.