The document discusses the principles of fluorescence and phosphorescence, detailing the processes of singlet, doublet, and triplet electronic states, as well as factors affecting fluorescence intensity and quenching. It explains the instrumentation used for measuring fluorescence, highlighting the importance of concentration, structural and non-structural factors, and types of quenching. Applications of fluorescence measurement include the determination of various inorganic substances and organic compounds.

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Introduction: Fluorescence,
phosphorescence
Concepts of singlet, doublet
and triplet electronic states
Internal and external
conversions
Factors affecting fluorescence,
Quenching
Instrumentation
Applications
CONTENT

3.  It is the measurement of fluorescence intensity at particular wavelength with the
help of fluorimeter.
OR
 Fluorimetry is a process in which concentration of sample is determined by
measuring the intensity of fluorescence or phosphorescence.
GS
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4.  It is a process in which molecules emit a light and get back to ground state from
singlet excited state.
 When a beam of light is incident on certain substance, they emit visible light or
radiation known as fluorescence.
 Fluorescence stops as soon as the incident light is cut off.
 In short, emission of light is called fluorescence.
 Fluorescence ∝ Concentration of sample.
 Time for fluorescence: 10-2 second
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5.  It is same as that of fluorescence, but in this light is emitted continuously for some
time even after incident light is cut off.
Also called as Delayed fluorescence.
 Phosphorescence vary from compound to compound.
 Time for phosphorescence : 10-2 to 100 second
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6.  These are electronic state which describe the amount of unshared pair of electrons
present in a molecules.
 Electrons are present in pair.
GS
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Singlet Doublet Triplet
Formula: 2S + 1
All the electrons
are paired
At least one
Unpaired electron
Two Unpaired
electron, but in the
same spin
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7.  When a beam of light is passed through a sample, molecule of sample absorb it and
get converted into singlet ground state to singlet excited state.
 When molecules get backing to their ground state they emit the light known as
fluorescence.
 By calculating the intensity of fluorescence we can calculate concentration of a
molecule in a sample, because more the concentration more the fluorescence.
 Phosphorescence is a delayed fluorescence.
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Intersystem crossing
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9.  Molecules comes from singlet excited to triplet state, by changing the system called
inter system crossing. (10-9)
 Internal conversion:
 It is intermolecular process which brings down a molecule to a lower energy
electronic state without emitting the light.
 It involves vibration relaxation in singlet excited state to triplet excited state.
 Time: 10-11 to 10-9
 External conversion:
It is a process in which molecules bring down its energy electronic state by emitting
light.
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10. Fluorescence intensity is affected by two factors i.e. structural factors and non structural
factors.
1. Structural factors
 Conjugation
 Nature of substitute group
 Rigidity of structure
2. Non-structural factors
 Temperature
 Viscosity
 oxygen
 concentration
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11. Conjugation:
 Molecule should be unsaturated for fluorescence.
 Conjugation is directly proportional to fluorescence. (=bond, lone pair - increase)
Nature of substituent group
 Electron withdrawing group decreases fluorescence intensity, (e.g. NO2, COOH)
 Shortening of conjugation.
 Electron donating group increases the fluorescence intensity (e.g. NH2, OH group)
Rigidity of the structure
 Rigid structure have more fluorescence intensity. (e.g. fluorine)
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12. Temperature:
 Increase in temperature increases molecular collision and decreases fluorescence
intensity.
Viscosity: inversely proportional to the fluorescence.
Oxygen: inversely proportional to the fluorescence. Oxygen oxidize the molecules and
convert it into non-fluorescent substances.
Concentration:
 It is directly proportional to the fluorescence intensity, but not in every case.
 It depends on intensity of incident light.
pH:
 It affect the fluorescence intensity but vary from compound to compound.
e.g. Imidazole, pH is inversely proportional
Quenching:
 It reduces intensity of fluorescence.
 Inversely proportional 12

13.  It is process by which intensity of fluorescence decreases.
 It is due to effect of sample.
Types of Quenching
1. Concentration Quenching
 When solution absorb excess amount of primary or fluorescent radiation.
 Also called as inner filter effect (Collision)
2. Chemical Quenching
 In this intensity of fluorescence decreases due to changes in chemical nature of
solution. (e.g. Decomposition)
 e.g. Aniline gives fluorescence at pH between 5-13.
3. Static quenching
 It is due to formation of complex and decreases fluorescence intensity.
4. Collision quenching
 Increase in collision between molecules decreases fluorescence intensity.
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14. 1. Light source
2. FILTERS / Monochromators
3. Sample cells
4. Detector
5. Read out system
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15.  High intensity Radiant energy should be supplied from the light source, as
Fluorescence is directly proportional to intensity.
 Commonly used light sources:
 Laser lamp
 Deuterium lamp
 Mercury vapour lamp
 Xenon arc lamp, etc.
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16.  Filters basically absorb the incident light of undesired wavelength and passes the
light of desired wavelength.
 Act like slits.
Types of filters
1. Primary filter:
 Passes incident light to the sample.
 They take Visible light and passes UV light.
2. Secondary filter:
 Get light from the sample & passes to detector.
 They take UV light and passes Visible light. (ease of measurement)
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17.  Same as UV, Photomultiplier tube detector is commonly used.
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 Determination of inorganic substances i.e. Al, Li, etc.
 Determination of thiamine HCl.
 Determination of Phenytoin.
 Determination of Indole, phenols, phenothiazines.
 Determination of proteins, plant pigments.

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