m pharmacy notes

1. by
Ayesha B. Mulani
under the guidance of
Dr. B. V. Udugade
FLUORIMETRY
and
Phosphorimetry

2. CONTENTS
Introduction
Principle
Difference
Factors effecting on fluorescence and phosphorescence
Applications

3. FLUORIMETRY
when a beam of light is incident on
certain substances, they emit visible
light or radiation this phenomenon is
known as fluorescnce.
eg. Quinole,indole,fluorene,8-hydroxy
quinoline

4. Phosphorimetry
when a beam of light is incident on
certain substances, they emit visible
light continuosly even after incident
light is cut off .

5. The process of promotion of electrons from HOMO to LOMO with
absorption of energy is called as excitation.
Singlet state:- a state in which all the electrons in a
molecule are paired  
Doublet state:- a state in which un paired electrons is
present  or 
Triplet state:- a state in which unpaired electrons of
same spin present  
Singlet excited state:- a state in which electrons are
unpaired but of opposite spin like   (un paired
and opposite spin)
Principle :-

6. Difference between Phosphorescence and fluorescence :-
Phosphorescence Fluorescence
Theemission could proceed
either from asinglet ortriplet
state.
Theemission could proceed
only from asinglet state.
longer lifetime of theexcited
state
short-live electrons (<10-5s)
in the excited state of
fluorescence

7. 1. Concentration
2. Oxygen
3. Ph
4. Temperature & viscosity
5. Photodecomposition
6. Nature of substituent
7. Scatter
8. Adsorption

8. Concentration:-
•Fluorescence intensity is proportional to
concentration of substance only when the
absorbance is less than 0.02
Beer-Lambert Law
A = log(I /I 0) = €.c.l
I = transmitted light
intensity
Io = incident light
intensity
c = concentration (M)
l = path length of cell (cm)
€= molar extinction coefficient (usually in the range 0-
105 , with <103 considered to be a week absorption

9. Dissolved oxygen:-
•Oxygen with unpaired electrons dramatically
decrease fluorescence and cause interference in
fluorimetric determinations.
•The paramagnetic nature of molecular oxygen
promotes intersystem crossing from singlet to triplet
states in other molecules.
•The longer lifetimes of the triplet states increases
the opportunity for radiation less deactivation to occur.
PH:-
•Relatively small changes in pH can sometimes cause
substantial changes in the fluorescence intensity and
spectral characteristics of fluorescence.

10. Temperature & viscosity:-
•A rise in temperature is almost always
accompanied by a decrease in fluorescence.
•The change in temperature causes the
viscosity of the medium to change which in
turn changes the number of collisions of the
molecules of the fluorophore with solvent
molecules.
•The increase in the number of collisions
between molecules in turn increases the
probability for deactivation by internal
conversion and vibrational relaxation.

11. Photochemical decomposition:-
Absorption of intense radiation leads to
photochemical decomposition of afluorescent
substance to lessfluorescent or nonfluorescent
substance.
Nature of substituents:-
•Electron donating group enhance
flouroscence
•Electron withdrawing group destroy
flouroscence

12. •Scatter is mainly due to colloidal particles insolution
•Scattering of incident light after passingthroughthe
sample leads to decrease in fluorescenceintensity.
•Ahigher fluorescence is observed when the solvents
do not contain heavy atoms while phosphorescence
increases due to the presence of heavy atoms in the
solvent.
Scatter:-

13. Adsorption :-
•This method requires vary dilute solution, 10-100
times weaker than used in other spectroscopy.
•Adsorption of fluorescent substance on the container
wall may create serious problems ,
•Eg. quinine

14. Applications :-
Applications of fluorimetry-
1. Determination of uranium in salts.
2. Determination of aluminium in alloys.
3. Calcium is also estimated by fluorimetry with calcein
solution.
4. Determination of vitamin B 1-thaimine is non
fluoroscent where its oxidation product thiochrome
gives fluoroscense with blue colour. this property is
used for determination of vit B1 in food samples and
nutrients.
5. In organic analysis –used for both qualitative as well as
quantitative analysis.
6. Analysis of food sample, clinical sample and natural
products.

15. Application of phosphorimetry:-
1. Determination of asprin in blood serum at liquid nitrogen
temperature,by this method 0.02 to 1 mg of asprin per ml
of serum can be analysed .
2. Low concentration of procaine, cocaine, phenobarbital,
chlorpromazine in serum sample can be analysed
3. Phosphorimetry has been emplyoeded in combination
with TLC or paper chromatography
4. Cocaine and atropine in urine sample can be determined
by phosphorimetry.

16. References:-
•Phosphorimetry
R. J. Keirs, R. D. Britt, W. E. Wentworth
•Phosphorimetry : theory, instrumentation, and
applications Robert J. Hurtubise.
•Principles of Fluorescence
Spectroscopy Authors: Lakowicz,
Joseph R.
•Google Scholar and Google images.