Flourescence spectroscopy- instrumentation and applications

for pharmacy students...

2. FLUORESCENCE
When a beam of light is incident oncertain
substances they emit visible light or radiations.
This is known asfluorescence.
Fluorescence starts immediately after the
absorption of light and stops as soon asthe
incident light is cutoff.
The substances showing thisphenomenon are
known as fluorescentsubstances.

3. Difference b/w absorption and flourescence

4. PHOSPHORESCENCE
When light radiation is incident on certain
substances they emit light continuouslyeven
after the incident light is cutoff.
This type of delayed fluorescence iscalled
phosphorescence.
Substances showing phosphorescenceare
phosphorescentsubstances

5. FLUORIMETRY:-
It is measurement of
fluorescence intensity at
a particular wavelength
with the help of a filter
fluorimeteror a
spectrofluorimeter.
Ananalyticaltechniqueforidenti
fyingandcharacterizing
minuteamountsofasubstanceby
excitationofthesubstancewith
abeamofultravioletlightandde
tectionandmeasurementofthe
characteristicwavelengthofthe
fluorescentlightemitted.

6. PRINCIPLE:-
Molecule contains-sigma
electrons, pie-electrons
and non bonding (n)
electron.
The electrons may be
present in bonding
molecular orbital. It
is called as highest
occupied molecular
orbital (HOMO).It has
lest energy and more
stable.
When the molecules
absorbs radiant energy
from a light source, the
bonding electrons may
be promoted to anti
bonding molecular
orbital (LUMO). It has
more energy and hence
less stable.

8. PRINCIPAL
Fluorescence is the phenomenon of emission of
radiation when there is transition from singlet
excited state to singlet ground state.
Wavelength of absorbed –Excitation
Wavelength of emitted –Emission
These two are specific for a given substance
under idealcondition.

9. CONCEPT OF ELECTRONIC STATE
TheprocessofpromotionofelectronsfromHOMOtoLUMO
withabsorptionofenergyiscalledasexcitation.
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
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 oppositespin)

10. FACTORS AFFECTING FLUORESENCE
1.Concentration
2. Quantum yield of fluorescence
3. Intensity of incident light
4.Oxygen
5.PH
6.Temperature& viscosity
7. Photodecomposition
8. Quenchers
9. Scatter

11. Concentration:
Fluorescence intensity is praportionalto
concentration of substance only when the
absorbance is less than 0.02
A=log Io\It or A= abc
Io=intensity of incident light
a= absorptivity of constant
b= Pathlength
c= concentration

12. Quantum yield of fluorescence
Ø)=NUMBER OF PHOTONS EMITTED\NUMBER OF
PHOTONS ABSORBED
It Is Always Less Than 1.0

13. INTENSITY OF INCIDENT LIGHT
Increase In the Intensity of Incident Light on the sample
Fluorescence Intensity alsoIncreases.
The intensity of incident light depend on the intensity of
light emitted from the lamp
OXYGEN:-
Bydirectoxidationoffluorescent
speciestoanonfluorescentspecies,
Quenchesfluorescentsubstance.

14. PH
Alterationof PH of a solution will have
significant effect onfluorescence.
For ex Aniline in alkali medium gives
visible fluorescence but in acidic condition
gives fluorescence in visibleregion.

15. Temperature and viscosity:-
Temperature
Increases the
collisional de
activation, and
reduce fluorescent
intensity.
•Viscosity of solution
is more the
frequency of
collisions are
reduced and increase
in fluorescent
intensity.

16. Photochemical decomposition:-
Absorption of
intense radiation
leads to
photochemical
decomposition of a
fluorescent
substance to less
fluorescent or non
fluorescent
substance.

17. Quenchers:-
Quenching is the
reduction of
fluorescence
intensity by the
presence of substance
in the sample other
than the fluorescent
analyte.
Quenching is following
types:-
Self quenching
Chemical quenching
Static quenching
Collision quenching

18. SELF QUENCHING
Fl
uo
r
esce
n
ce
Concentration of
fluorescingspecies
Deviations at higher concentrations can be attributed to self-
quenching orself-absorption.
Fluorescence
Concentration of
fluorescingspecies
CALIBRATIONCURVE
(HIGHCON)

19. CHEMICAL QUENCHING
Here decrease in fluorescence intensity due to the
factors like change in PH, presence of oxygen,
halides &heavymetals.
PH-aniline at PH5-13gives fluorescence but at
PH<5 &>13 it does not exhibit fluorescence.
Halides like chloride, bromide, iodide &
electron withdrawing groups likeNO2,COOH
etc. leads toquenching.
Heavy metals leads to quenching,because of
collisions of triplet groundstate.

20. STATICQUENCHING
This occurs due to
complex formation.
e.g.. caffeine reduces
the fluorescence of
riboflavin by complex
formation.

21. COLLISIONAL QUENCHING
It reduces
fluorescence by
collision.
where no. of
collisions increased
hence quenching
takes place.

22. Scatter is mainly due to colloidal particles in
solution. Scattering of incident light after
passing through the sample leads to decrease
in fluorescence intensity
Scatter:-

23. INSTRUMENTATION

24. INSTRUMENTATION
SOURCE OF LIGHT
FILTERS &
MONOCHROMATORS
SAMPLE CELLS
DETECTROS

25. 1)SOURCE OF LIGHT
Mercury vapourlamp: Mercury vapourat high
pressure give intense lines on continuous
background above 350nm.
low pressure mercury vapourgives an additional
line at 254nm.it is used in filter fluorimeter.

26. SOURCE OF LIGHT
Xenon arc lamp: It give more intense
radiation than mercury vapourlamp. it is
used in spectrofluorimeter
Tungsten lamp:-If excitation has to be done
in visible region this can be used.
It is used in low cost instruments.

27. 2) FILTERS
Filters: these are nothing but optical filters
works on the principle of absorption of
unwanted light and transmitting the required
wavelength of light.
In inexpensive instruments fluorimeter
primary filter and secondary filter are
present.
Primary filter:-absorbs visible radiation and
transmit UV radiation.
Secondary filter:-absorbs UV radiation and
transmit visible radiation.

28. Monochromators:
They convert polychromatic light into
monochromatic light. They can isolate a specific
range of wavelength or a particular wavelength of
radiation from a source.
Excitation monochromators:-Provides suitable
radiation for excitation of molecule .
Emission monochromators: Isolate only the radiation
emitted by the fluorescent molecules.

29. Sample cells:
These are mentfor holding liquid samples.
These are made up of quartz and can have
various shapes ex: cylindrical or rectangular
etc.

30. Detectors:
BARRIER LAYEAR DETECTOR -Thedetectorhasathinfilmmetallic
layercoatedwithsilveror gold and acts as anelectrode.
It also has a metal base plate which acts as anotherelectrode.
Thesetwolayersareseparatedbyasemiconductor
layerof selenium.
Whenlightradiationfallsonseleniumlayer,electronsbecome mobile and
are taken up by transparent metallayer.
Thiscreatesapotentialdifferencebetweentwoelectrodes&causestheflow
ofcurrent.
Whenitisconnectedtogalvanometer,aflowofcurrentobservedwhichis
proportionaltotheintensityandwavelengthoflightfallingonit.

31. Photomultiplier tubes:
These are incorporated in expensive instruments like
spectrofluorimeter. Its sensitivity is high due to measuring weak
intensity of light.
•The principle employed in The principle employed in this
detector is that, multiplication of photoelectrons by secondary
emission ofelectrons.
•Inavacuumtube,aprimaryphoto-cathode is
fixedwhich receives radiation from thesample.
•Some eight to ten dynodes are fixed each with increasingpotential
of 75-100V higher than precedingone.
•Nearthelastdynodeisfixedananodeorelectron
collector electrode.

32. Photomultiplier tubes:

33. INSTRUMENTS

34. Single beam (filter) fluorimeter-

35. DOUBLE BEAM FLOURIMETER

36. DOUBLE BEAM Fluorimeter

37. APPLICATIONS

38. APPLICATIONS
Determination of ruthenium ions in presence
of other platinum metals.
Determination of boron in steel, aluminum in
alloys, manganese in steel.
Determination of boron in steel by complex
formed with benzoin.
Estimation of cadmium with 2-(2
hydroxyphenyl) benzoxazolein presence of
tartarate. Respiratory tract infections