This document discusses fluorescence and fluorimetry. It begins with an introduction and overview of fluorescence as the emission of radiation when molecules are excited by radiation at certain wavelengths. It then discusses fluorimetry as the measurement of fluorescence intensity at a particular wavelength using a fluorimeter or spectrofluorimeter. Finally, it covers factors influencing fluorescence intensity, types of quenching, instrumentation components, and applications of fluorimetry in pharmaceutical analysis.

1. Dept of Pharmaceutical Analysis
Sree Dattha Institute Of Pharmacy
Sheriguda, Ibrahimpatnam.
Presented by
Salma
16U21R0001
Under the guidance of
Asst.Professor Mrs. Sadhana
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Flourimetry

2.  Introduction
 Principle
 Factors influencing flourescence intensity
 Quenching of flourescence and types
 Instrumentation
 Applications
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3.  FLOUESCENCE
It is a phenomenon of emission of radiation when the molecules are exited
by radiation at certain wavelength.
 FLOURIMETRY
It is measurement of fluorescence intensity at a particular wavelength with
the help of a filter fluorimeter or a spectrofluorimeter.
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4.  FLOURESCENCE
• Excited singlet state is highly unstable. Relaxation of electrons from
excited singlet to singlet ground state with emission of light .
 PHOSPHORESCENCE
• At favourable conditions like absence of oxygen and low temperature
there is transition from excited singlet state to triplet state which is called
as intersystem crossing. The emission of radiation when electrons
undergo transition from triplet state to singlet ground state is called as
phosphorescence.
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6. • Based upon the wavelength of emitted radiation when compared to
absorbed radiation
• A.Stoke`s flourescence: The wavelength of emitted radiation is
longer than the absorbed radiation
• B. Anti-stoke`s flourescence: The wavelength of emitted radiation
is shorter than the absorbed radiation.
• C.Resonance florescence: When the wavelength of the emitted
radiation is equal to the absorbed radiation.
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7.  Conjugation
A molecule must have unsaturation so that uv/vis radiation can be
absorbed. If there is no absorption of radiation, there will not be
flourescence
 Nature of substituent groups
Electron donating groups like amino, hydroxyl groups enhance
flourescence intensity.
electron withdrawing groups like nitro carboxylic group reduce flourescene
intensity.
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 Rigidity of structures
Rigid structures - more flourescence intensity
Flexible structures – less flourescence intensity
 Effect of temperature
Increase in tempeture leads to increase in collisions of molecules,
therefore deviation, which results in decrease in florescence
intensity on the other hand, decrease in temperature leads to
decrease in collisions of molecules, which results in increased
flourescence intensity.
 Viscosity
Increase in viscosity leads to decreased collisions of molecules, which
leads on enhancement of flourescence intensity. Decrease in viscosity
causes increased collisions of molecules, which results in decreased
flourescence intensity.

9.  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.
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QUENCHING OF FLOURESCENCE AND TYPES

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SOURCE OF LIGHT
MERCURY VAPOUR LAMP
Mercury vapour at high pressure give continuos background
above 350nm .Low pressure mercury vapour gives an additional
line at 254nm
XENON ARC LAMP
It gives more intense radiation than mercury vapour lamp.
TUNGSTEN LAMP
If excitation has to be done in visible region this can be used.

12.  FILTERS
• Filters works on the principle of absorption of unwanted light and
transmitting the required wavelength of light.
 PRIMARY FILTER
• Absorbs visible radiation and transmit uv radiation.
 SECONDARY FILTER
• Absorbs uv radiation and transmit visible radiation.
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13. • They convert polychromatic light into monochromatic light.
• They isolate a specific range of wavelength or a particular wavelenth of
radiation from a source.
 EXCITATION MONOCHROMATORS
• Provides suitable radiation for excitation of molecule.
 EMISSION MONOCHROMATORS
• Isolate only the radiation emitted by the flourescent molecules.
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14.  These are meant for holding liquid samples.These are made up of quartz
and can have various shapes like cylindrical or rectangular etc.
 DETECTORS
• The most commonly used detectors are
• 1.Barrier layer cell
• 2.Photo tubes
• 3.Photo multiplier tubes
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PHOTO MULTIPLIER TUBE
The principle employed in this detector is that multiplication of photo
electrons by secondary emission of electrons.
This is achieved by using a photo cathode and a series of anodes
[dyanodes].upto 10 dyanodes are used.

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INSTRUMENTS
 The most common types are
 Single beam fluorimeter
 Double beam fluorimeter
 Spectrofluorimeter

17.  Tungsten lamp as sorce of light.
 The primary filter absorbs visible radiation and transmits uv radiation.
 Emitted radiation measured at 90 by secondary filter.
 Secondary filter absorbs uv radiation and transmits visible radiation.
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18.  Similar to single beam instrument.
 Two incident beams from light source pass through primary filters
separately and fall on either sample or reference solution.
 The emitted radiation from sample or reference pass separately through
secondary filter.
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19.  In this primary filter in double beam flourimeter 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.
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APPLICATIONS
1.Determination Of Thiamine Hcl
2.Determination of phenytoin.
3.Determination of indoles,phenols and phenothiazines.
4.Determination Of Ruthenium Ions In Presence Of Other Platinum
Metals
5.Determination of napthols,proteins,plant pigments and steroids.
6.Respiratory tract infections.

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