Fluorescence

Fluorescence
It is a phenomenon of emission of radiation when the molecules are exited by

radiation at certain wavelength.
FLUORIMETRY
It is measurement of fluorescence intensity at a particular wavelength wit the help
of a filter fluorimeter or a spectrofluorimeter.
PRINCIPLE:-
Molecule contains  electrons,  electrons and non bonding (n) electron.
o 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.
o 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.
o The process of promotion of electrons from HOMO to LUMO 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)
When light of appropriate wavelength is absorbed by a molecule the electrons
are promoted from singlet ground state to singlet excited state. once the
molecule is in this excited state relaxation can occur via several process. For
ex by emission of radiation . The process can be the following :-
1) Collisional deactivation
2) Fluorescence
3) Phosphorescence.
1. Collisional deactivation :- In which entire energy lost due to collision de

activation and no radiation emitted.
2. Fluorescence:-excited singlet state is highly unstable. Relaxation of

electrons from excited singlet to singlet ground state with emission of light.
3. Phosphorescence:-At favorable condition like low temperature and absence

of oxygen there is transition from excited singlet state to triplet state which
is called as inner system crossing. The emission of radiation when electrons
undergo transition from triplet state to singlet ground state is called as
phosphorescence.
Factors effecting fluorescence intensity:-
1. Concentration
2. Quantum yield of fluorescence
3. Intensity of incident light
4. Adsorption
5. Oxygen
6. Ph
7. Temperature& viscosity
8. Photodecomposition
9. Quenchers
10.Scatter
1. Concentration
Fluorescence intensity is praportnal to 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
2. QUANTUM YIELD OF FLUORESCENCE:-()
()=NUMBER OF PHOTONS EMITTED\NUMBER OF PHOTONS

ABSORBEDS
It Is Always Less Than 1.0 Since Some Energy Is Lost By Radiation
less Pathways (Collisional, Intersystem Crossing, Vibrational Relaxation)
3. INTENSITY OF INCIDENT LIGHT:-

Increase In The Intensity Of Incident Light On The Sample
Fluorescence Intensity Also Increases.
4. ADSORPTION:-
Adsorption Of Sample Solution In The Container May Leads To A
Serious Problem.
5. OXYGEN:-
Oxidation of fluorescent species to a non fluorescent species,

quenches fluorescent substance.
6. Ph:-
Alteration of ph of a solution will have significant effect on

fluorescence
For ex Aniline in alkali medium gives visible fluorescence but in
acidic condition gives fluorescence in visible region.
7. Temperature and viscosity:-
Temperature Increases Can Increase the collisional de activation, and reduce

fluorescent intensity.
If viscosity of solution is more the frequency of collisions are reduced and
increase in fluorescent intensity.
8. Photochemical decomposition:-
Absorption of intense radiation leads to photochemical decomposition of a

fluorescent substance to less fluorescent or non fluorescent substance.
9. 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:-
 INNER FLUORESCENT EFFECT
 CONCENTRATION QUENCHING/SELF QUNCHING
 COLLISIONAL QUENCHING
 STATIC QUENCHING
 INNER FLUORESCENT EFFECT:- Absorption Of Incident (uv) Light
Or Emitted (fluorescent) Light By Primary And Secondary Filters Leads
To Decrease In Fluorescence intensity.
 SELF QUENCHING:-At Low Concentration Linearity Is Observed, At

High Concentration Of The Same Substance Increase In Fluorescent
Intensity Is Observed. This phenomena is called self quenching.
 COLLISONAL QUENCHING:- Collisions between the fluorescent

substance and halide ions leads to reduction in fluorescence intensity.
 STATIC QUENCHING:- This occurs because of complex formation

between the fluorescent molecule and other molecules.
Ex: caffeine reduces fluorescence of riboflavin.
SCATTER:- Scatter is mainly due to colloidal particles in solution. Scattering of

incident light after passing through the sample leads to decrease in fluorescence
intensity.
INSTRUMENTATION
Instrumentation
 Source of light
 Filters and monochromators
 Sample cells
 Detectors
1. SOURCE OF LIGHT:-
Mercury vapour lamp: Mercury vapour at high pressure give intense lines
on continuous background above 350nm.low pressure mercury vapour gives
an additional line at 254nm.it is used in filter fluorimeter.
Xenon arc lamp: It give more intense radiation than mercury vapour lamp.
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
2) FILTERS AND MONOCHROMATORS:-
 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.
 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.
3) Sample cells: These are ment for holding liquid samples. These are made up
of quartz and can have various shapes ex: cylindrical or rectangular etc.
4) Detectors: Photometric detectors are used they are

 Barrier layer cell/Photo voltaic cells
 Photomultiplier cells
1. Barrier layer /photovoltaic cell:
 It is employed in inexpensive instruments. For ex: Filter Fluorimeter.
 It consists of a copper plate coated with a thin layer of cuprous oxide (Cu2o).
A semi transparent film of silver is laid on this plate to provide good contact.
 When external light falls on the oxide layer, the electrons emitted from the
oxide layer move into the copper plate. Then oxide layer becomes positive
and copper plate becomes negative.
 Hence an emf develops between the oxide layer and copper plate and
behaves like a voltaic cell. So it is called photovoltaic cell..
 A galvanometer is connected externally between silver film and copper plate

and the deflection in the galvanometer shows the current flow through it.
The amount of current is found to be proportional to the intensity of incident
light
BARRIER LAYER CELL

2. 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 this detector Is that, multiplication of
photoelectrons by secondary emission of electrons. This is achieved by using
a photo cathode and a series of anodes (Dyanodes). Up to 10 dyanodes are
used. Each dyanode is maintained at 75-100Vhigher than the preceding one.
 At each stage, the electron emission is multiplied by a factor of 4 to 5 due to
secondary emission of electrons and hence an overall factor of 106 is
achieved.
 PMT can detect very weak signals, even 200 times weaker than that could be
done using photovoltaic cell. Hence it is useful in fluorescence
measurements.
 PMT should be shielded from stray light in order to have accurate results.
INSTRUMENTS
The most common types are:-
 Single beam (filter) fluorimeter
 Double beam (filter )fluorimeter
 Spectrofluorimeter(double beam)
1. Single beam filter fluorimeter
 It contains tungsten lamp as a source of light and has an optical system

consists of primary filter.
 The emitted radiations is measured at 900 by using a secondary filter and
detector. Primary filter absorbs visible radiation and transmit uv radiation
which excites the molecule present in sample cell.
 Instead of 90 if we use 180 geometry as in colorimetry secondary filter has
to be highly efficient other wise both the unabsorbed uv radiation and
fluorescent radiation will produce detector response and give false result.
2. In double beam fluorimeter:-it is similar to single beam except that
the two incident beams from a single light source pass through primary
filters separately and fall on the another reference solution. Then the
emitted radiations from the sample or reference sample pass separately
through secondary filter and produce response combinly on a detector.
3. In spectrofluorimeter:-
In this primary filter in double beam fluorimeter is replaced by excitation

monochromator and the secondary filter is replaced by emission
monochromator.
Incident beam is split into sample and reference beam by using beam
splitter.
Applications
 Determination of inorganic substances.

Al3+,Li+,ZN2+
 Determination of thiamine Hcl.
 Detemination of phenytoin.
 Determination of indoles, phenols, & phenothiazines
 Determination of napthols, proteins, plant pigments and steroids.

 Fluorimetry ,nowadays can be used in detection of impurities in
nanogram level better than absorbance spectrophotometer with special
emphasis in determining components of sample at the end of
chromatographic or capillary column.
 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) benzoxazole in

presence of tartarate .
 Respiratory tract infections.

Fluorescence

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Fluorescence

It is a phenomenon of emission of radiation when the molecules are exited by

o The process of promotion of electrons from HOMO to LUMO with

1. Collisional deactivation :- In which entire energy lost due to collision de

2. Fluorescence:-excited singlet state is highly unstable. Relaxation of

3. Phosphorescence:-At favorable condition like low temperature and absence

Fluorescence intensity is praportnal to concentration of substance only when the

2. QUANTUM YIELD OF FLUORESCENCE:-()

()=NUMBER OF PHOTONS EMITTED\NUMBER OF PHOTONS

3. INTENSITY OF INCIDENT LIGHT:-

Oxidation of fluorescent species to a non fluorescent species,

Alteration of ph of a solution will have significant effect on

Temperature Increases Can Increase the collisional de activation, and reduce

Absorption of intense radiation leads to photochemical decomposition of a

Quenching is the reduction of fluorescence intensity by the presence of

 SELF QUENCHING:-At Low Concentration Linearity Is Observed, At

 COLLISONAL QUENCHING:- Collisions between the fluorescent

 STATIC QUENCHING:- This occurs because of complex formation

SCATTER:- Scatter is mainly due to colloidal particles in solution. Scattering of

Tungsten lamp:- If excitation has to be done in visible region this can be

Primary filter:-absorbs visible radiation and transmit UV radiation.

 Monochromators: they convert polychromatic light into monochromatic

4) Detectors: Photometric detectors are used they are

 It is employed in inexpensive instruments. For ex: Filter Fluorimeter.

 A galvanometer is connected externally between silver film and copper plate

BARRIER LAYER CELL

 These are incorporated in expensive instruments like spectrofluorimeter. Its

1. Single beam filter fluorimeter

 It contains tungsten lamp as a source of light and has an optical system

In this primary filter in double beam fluorimeter is replaced by excitation

 Determination of inorganic substances.

 Determination of thiamine Hcl.

 Determination of indoles, phenols, & phenothiazines

 Determination of napthols, proteins, plant pigments and steroids.

 Determination of ruthenium ions in presence of other platinum metals.

 Determination of boron in steel, aluminum in alloys, manganese in

 Determination of boron in steel by complex formed with benzoin.

 Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in

 Respiratory tract infections.

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