These slides involves theory, principle,instrumentation, factors affecting, applicatons, and limitation of fluorimetry or spectrofluorimetry

1. Fluorimetry or
Spectrofluorimetry
PRESENTED BY:
PAYAL KHANNA
M.PHARMACY
(PHARMA COLOGY)

2. Fluorescence Spectroscopy (also know
n as Fluorimetry or Spectrofluorimetry) is
a type of electromagnetic spectroscopy that
analyses fluorescence from a sample .

3. Spectroscopy
ABSORPTION
SPECTROSCOPY
(Absobtion of radiations
is studied)
• UV Spectroscopy
• IR Spectroscopy
• NMR Spectroscopy
EMISSION
SPECTROSCOPY
(Emission of radiation is
studied)
• Fluorimetry
• Flame photometry(ato
mic emission spectros
copy

4. THEORY
• Absorption of UV/Visible radiation causes transition of ele
ctrons from ground state (low energy) to excited state (hig
h energy).
• As excited state isn’t stable, excess energy is lost.
• LUMINESCENCE: There are some substances which can
absorb UV/Visible light but lose only part of this excess en
ergy as heat and emit the remaining energy of electromagn
etic radiation of a wavelength longer than that absorbed. T
his process of emitting radiation is collectively known as lu
minescence

5. LUMINESCENCE
(Process of emission of radiation from electronically excited
state of a substance)
occurs when electron return to ground state from an excited
state and loses its excess energy as a photon
• Photo luminescence
 Fluorescence
 Phosphorescence
• Chemical luminescen
ce
Sample
Luminescence
L
Incident
radiation
0
Transmitted
radiation


6. Fluorescence
• When a beam of light is inciden
t on certain substances they emi
t visible light or radiations. This
is known as fluorescence.
• Phenomenon of fluorescence is
instantaneous and starts immedi
ately after absorption of light an
d stops as soon as incident light
is cut off
• Fluorescence materials gener
ally emit radiation within 10-6
to 10-4 sec of absorption
Phosphorescence
• Delayed type of fluorescence is
called phosphorescence. When l
ight radiation is incident on cer
tain substances they emit light c
ontinuous even after incident li
ght is cut off
• Life time of phosphorescence is
much longer than fluorescenc
e and subs. Emit light continu
ous even after incident light
is cut off
• Phosphorescence material emit
excess radiation within 10-
4 to 20 sec.

7. diagram

8. Principle
• Fluorescence spectroscopy is primarily concerned with electronic and
vibrational states.
• Generally the species being examined has a ground excited state (low e
nergy state) and an excited electronic state of higher energy. Within e
ach of these electronic states there are various vibrational states.
• Fluorescence is generated when a molecule transmits from its ground s
tate (So) to one of vibrational energy levels in first excited electronic s
tate(S1) or the second electronic excited state(S2) both of which are sin
glet states.
• Relaxation to the ground state from these excited state occurs by emiss
ion of energy through heat or photons

10. • The difference between the excitation and emission wavelengths is call
ed the Stokes shift.
• Stokes studies of fluorescent substances led to formulation of Stokes la
w ,which states that the wavelength of fluorescent light is always great
er than that of the exciting radiation. Thus for any fluorescent molecule
, the wavelength of emission is always longer than the wavelength of a
bsorbtion.

11. Classification
(based on the wavelength of emitted radiation when
compared to absorbed radiation)
• Stokes fluorescence: wavelength of emitted radiation i
s longer than absorbed radiation
• Anti stokes fluorescence: wavelength of emitted radiat
ion is shorter than absorbed radiation.
• Resonance fluorescence: wavelength of emitted
radiation is equal to absorbed radiation

12. Beer-Lambert law for Fluorimetry
Fluorimetry can be used as a tool for determination of very small concent
ration of substance which exhibit fluorescence. Beer –lambert law is also
applied in case of fluorimetry as:
Ef= Absorbitivity of fluorescent material
c= concentration of substance
b= path length
Isolvent, Isample = intensities of incident radiation energy and transmitted ener
gy respectively
thus the intensity of fluorescence is given by
f=k(Isolvent – Isample)
logIsolvent /log Isample = Ef .c.b.

13. Factors affecting intensity of fluo
rescence and phosphorescence
• Nature of molecules: Only such molecules show fluorescence and phos
phorescence that are able to absorb UV-Visible radiation.
Greater the absorbancy of molecule More intense its luminescence
Molecules having conjugated double bonds (π-bonds) are particularly s
uitable whereas aliphatic and saturated cyclic organic compounds are n
ot suitable.
• Nature of substituents: electron donating groups like -NH2 and –OH o
ften enhance fluorescence whereas -SO3H,NH4
+ and alkyl groups d
on’t have much effect on both phenomenon. Electron withdrawing gro
ups like –COOH, -N=N- and halides decreases or even destroy fluores
cence.
Electron Donating Groups Enhance fluorescence
Electron withdrawing groups decreases or even destroy fluorescence

14. • Effect of rigidity: More rigid the structure of compound more will
be intensity of fluorescence.
Also chelation of aromatic compounds with metal ions promote rigidity
and reduces internal vibrations . Thus chelation promotes fluoresce
nce.
• Effect of viscosity: If there is an increase in viscosity then collision be
tween molecules decreases which results in increase in intensity of flu
orescence
• Effect of temperature: Increase in temperature leads to increases collisi
on between molecules thereby decreasing fluorescence intensity.
• Presence of oxygen: The presence of oxygen may interfere in two way
s 1) by direct oxidation of the fluorescent substance to non- fluoresce
nt substance.
2) or by quenching / decreasing fluorescence
For e.g. anthracene is susceptible to presence of oxygen

15. QUENCHING
 Decrease in fluorescence intensity due to specific
effects of constituents of the solution.
 Due to concentration, ph, pressure of chemical
substances, temperature, viscosity etc.
Types Of Quenching
• Self quenching
• Chemical quenching
• Static quenching
• Collision quenching

17. Chemical Quenching
 Here decrease in fluorescence intensity due to the factors like change i
n pH, presence of oxygen , halide and heavy metals
 pH – aniline at pH 5-13 gives fluorescence but at pH <5 and > 13 it do
esn’t exhibit fluorescence.
 Halides- like chloride, bromide, iodide, and electron withdrawing grou
ps like NO2 ,COOH etc. leads to quenching.
 Heavy metals- leads to quenching because of collision of triplet groun
d state.

18. Static quenching
Occurs due to complex formation.
E.g. caffeine reduces the fluorescence of riboflavin by
complex formation.
Collisional quenching
It reduces fluorescence by collision, where no. of c
ollision increases hence quenching takes place.

19. Instrumentation for fluorescence spectroscopy
Power
supply
Source Excitation
monochromator
Emission monochromator
Detector
Sample cell
Slit
Data processor
General layout of fluorescence spectrophotometer

20. Schematic diagram of a typical spectrofluorometer.

21. 1) Light sources
a. Gas discharge lamps :
Xenon arc lamp
High pressure mercury vapor lamp
b. Tungsten wire filament lamp
c. Laser : tunable dye laser
d. X-ray source for X-ray fluorescence
2) Wavelength selection devices
a. Filters : used in fluorimeter
Absorption filters ( primary filter)
Interference filters ( secondary filter)
b. Monochromators : used in spectrofluorimeter
Gratings
Prism

22. 3) Sample compartment
Fluorescence cells ---- right angle design or small angle(37o) viewing system
Quartz or fused silica ----200 nm ~ 800 nm
Glass or plastic ---- 300 nm ~
path length ----- 10mm or 1cm
4) Detectors
Photomultiplier tubes
Photovoltaic tube

23. Applications of spectrofluorimetry
• Determinations of organic substances
( plant pigments ,steroids,proteins,naphthols etc can be deter
mined at low concentrations.
Generally used to carry out qualitative as well as quantitative
analysis for a great aromatic compounds present in cigarett
e smoking ,air pollutant and automobile exhausts)
• Determination of inorganic substances
• Extensively used in field of nuclear research for the determ
ination of uranium salts.
• Determination of vitamin B1 (thiamine) in food samples lik
e meat ,cereals etc.
• Determination of vitamin B2 (riboflavin) . this method is
generally used to measure the amount of impurities prese
nt in the sample

24. • Most important applications are found in the
analysis of food products,pharmaceuticals,clinical
samples and natural products.
• To measure the amount of impurities present in
the sample
Disadvantages :
• Not useful for all compounds because not all cm
pds fluorescence.
• Contamination can quench the fluorescence and
hence give false/ no results.

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