FLUORIMETRY BASIC IDEA

FLUORIMETRY TECHNIQUE OF DETECTION OF FLUORESCENCE,

1. BY
SUBHASISH DAS
M.PHARM 1
ST
YR.

2. Luminescence is the term applied to
the remission of previously absorbed
radiation .
FLuORescence is instantaneous
,immediate phenomena of emitting
visible light or radiation by a substance
after absorbing visible or UV light.

3. 1. Both the absorption and emission
wavelengths are available .
2. Interferences may be avoided.
3. Data are not subjected to the relative
error curve .
4. Accuracy is maintained at 99.999%
transmittance range.

4. Fluorescence emission spectra arise due
to transition from the free excited state to
the ground singlet state .
Three states involves in luminescence
1. Ground singlet state.
2. Excited single state.(responsible for
fluorescence)
3. Excited triple state.( responsible for
phosphorescence).

5. MODIFIED DIAGRAMME
SIMPLIFIED DIAGRAMME

6. Nature of molecule.
Nature of substituents.
Concentration.
Adsorption.
Light.
Method of illumination.
Oxygen.
pH
Photodecomposition.
Temperature and viscosity.

7. F = 2.303 K I
0
abc

Where
,
F = intensity of fluorescent radiation.
K = proportionality constant.
I
0
= intensity of incident radiation.

a
= absorptivity.
b = length of cell path.
C = concentration of absorbing
substances.

8. SINGLE BEAM FLUORIMETER DOUBLE BEAM FLUORIMETER

9. Determination of URANIUM,RUTHENIUM,
CADMIUM.
Fluorescent indicators – Eosin,
Fluorescein etc.
Fluorometric agent.
Organic analysis.
Pharmaceutical chemistry –
determination of Aneurine.

10. DETERMINATION OF VITAMIN B
1
–
Thiamine is non fluorescent but its
oxidation product thiochrome
fluorescence with blue colour.
DETERMINATION OF VITAMIN B
2
– The
solution is oxidised and fluorescence is
measured by residual technique.

11. FLUORESCENCE MICROSCOPY.
FLUORESCENCE LIFETIME IMAGING
MICROSCOPY.
STUDY OF PROTIENS.
BIOSENSOR.
CELL SORTING.
FORENSIC USE.

12. VALID FOR CONCENTRATION <10
-5
M.- At high
concentration self quenching takes place.
SUSCEPTIBLE TO INNERFILTER EFFECTS.- If emitted light
is absorbed in the sample it cannot reach the detector and
again the apparent fluorescence will decrease.

13. Fluorescence methods have greater
selectivity and specificity than
absorptiometric methods.
Not only in field of pharmaceutical also in
field of animation , electronics , biologics
fluorescence technique as well as
fluorimetry is used .

14. 1. INSTRUMENTA METHODS OF CHEMICAL ANALYSIS
– Gurudeep R. chatwaal , Sham K. Anand ,Ch – 16, Pg .
no. – 2.399 – 2.416.
2. A TEXT BOOK OF PHARMACEUTICAL ANALYSIS –
Kenneth A. Connors , 3
rd
ed. , Ch – 9, Pg . no . – 235 -247.
3. INSTRUMENTAL METHODS OF ANALYSIS – Hobart H.
Williard , Lynne L . Merritt, jr. , John A. Dean , Frank A.
Settle, jr. Ch – 8 , Pg. no. – 197 -223 .
4. PRACTICAL PHARMACEUTICAL CHEMISTRY – A. H.
beckett , J. B. Stenlake, 4
th
ed. , part two, Ch – 9 , Pg. no. –
358 – 378.
5 . ANALYTICAL BIOCHEMISTRY , Vol – 366 , issue 2 ,15
th july 2007 , pg no. – 149 – 155.

15. THANK YOU