spectrofluorimetry, M. Pharm. (1st sem.)

GURU GOBIND SINGH COLLEGE OF PHARMACY
SPECTROFLUORIMETRY
SUBMITTED TO: SUBMITTED BY:
Dr. Ashwani K. Dhingra Prabhjot Kaur
HOD, Professor M-507

Content
 Introduction
 History of spectrofluorimetry
 Theory/Principle of FLUORESCENCE
 Factors affecting FLUORESCENCE
 Quenching & it’s types
 Instrumentation
 Applications

Introduction
Absorption of UV-Visible radiation causes transition of
electrons from ground state to excited state.
As excited state is not stable so, excess energy is released by
Collisional deactivation and photoluminescence.
This measurement or study of emitted radiation is the
principle of Flourimetry.
 Phosphorescence is also related phenomena, which is the
study of emitted radiation when electron undergoes
transition from triplet state to ground state.

Fluorescence and Fluorimetry
FLUORESCENCE
When a beam of light is incident on certain substances, they emit visible
light or radiations. This is known as fluorescence.
Fluorescence starts immediately after the absorption of light and stops as
soon as the incident light is cut off.
 The substances showing this phenomenon are known as fluorescent
substances.
FLUORIMETRY
 Fluorimetry is an analytical method for the measurement of
fluorescence, based upon the emission of absorbed radiation by the
molecules.
 The instrument used is called as spectroflourimeter.

History :
 The first observation of fluorescence from a quinine solution in sunlight was
reported by Sir John Frederick William Herschel in 1845.
Fig: Quinine
 The quinine in tonic water is excited by the ultraviolet light from the sun.
Upon return to the ground state the quinine emits blue light with a
wavelength near 450 nm.

Fluorescence excitation & emission spectra
for a solution of quinine

Theory of FLUORESCENCE
THEORY OF FLUORESCENCE
A molecule either at ground state or rest is said to
possess 3 different types of energy levels. They are:
1.Rotational energy
2.Vibrational energy
3.Electronic energy

When an electromagnetic radiation falls on the molecule it
eventually brings about the changes in the energy levels during
the process of absorption. The theory of fluorescence is
explained best by Jablonski diagram.
►SINGLET GROUND STATE: A molecular electronic state
in which all the electrons are paired.
 ► DOUBLET STATE: When a molecule from the G.S
absorbs uv/visible radiation, one or more of the paired electron
raised to an excited state. Here the electrons are unpaired.
They can be of singlet excited state and triplet excited state.

 The main theory/principle involved in the fluorescence is when an incident
light absorbed by the sample it undergoes the transition from the singlet
ground state to singlet excited state.
 Where the singlet excited state is not stable one and the molecules present
in excited state immediately return to the ground state by emitting the
energy.
 A part of energy is lost due to Vibrational transitions and the remaining
energy is emitted as UV/visible radiation of longer wavelength than the
incident light.
 This is because the energy of emitted radiation is lesser than that of incident
or absorbed radiation because a part of energy is lost due to Vibrational
collision.
 The wavelength of absorbed radiation is called as excitation wavelength
and emitted radiation is called as emission wavelength. It is spontaneous
process.

Partial energy diagram for a
photoluminescent system

Factors affecting FLUORESCENCE
1. Nature of moleculesature of molecule
2. Effect of substitution
3. Effect of concentration
4. Absorption
5. Photo decomposition
6. Oxygen
7. Ph
8. Conjugation
9. Temperature
10. Viscosity
11. Quantum yield of Fluorescence
12. Intensity of incident light
13. Path length

1. Nature of molecules
Only the molecules absorbs uv/visible radiation can show the
fluorescence. Greater the absorbency of molecule more intense its
fluorescence. Unsaturated molecules with ir bond and good
resonance stability can exhibit fluorescence.
Eg: Alkenes with conjugate double bond exhibit fluorescence
whereas saturated molecules with sigma bond do not exhibit
fluorescence.
Aliphatic unsaturated cyclic organic compounds exhibit
fluorescence.

2.Nature of Substituent
Electron donating groups like amino, hydroxyl groups enhance fluorescence
intensity.
While electron withdrawing groups like halogens, nitro, carboxylic groups will
reduce fluorescence intensity.
3.Effect of concentration
Beers law state that in a solution of an absorbing substances the absorbance is
directly proportional to the concentration.
Fluorescent intensity is directly proportional to concentration of the substance
but this is true in low concentrations, in high concentration it doesn’t obey
linearity and hence fluorescence intensity decreases.

4.Oxygen
Oxygen decrease the fluorescence intensity in two ways: It oxidizes fluorescent
substance to non fluorescent substance. It quenches fluorescence because of the
paramagnetic properties of molecular energy.
5.PH
In the molecules containing acidic and basic functional group the changes in the
pH of the medium change the degree of ionization of the functional group.
6.Conjugation
A molecule must have unsaturation i.e. 7 electrons so that UV/visible radiation can
be absorbed. If there is no absorption of radiation, there will not be fluorescence

7.Effect of temperature
 Increase in temperature leads to increase in collisions of molecules
therefore deviation, which results in decrease in fluorescence intensity. On
the other hand decrease in temperature leads to decrease in collisions of
molecules, which results in increased fluorescence intensity.
8.Viscosity
 Increase in viscosity leads to decreased collision of molecules, which leads
to enhancement of fluorescence intensity. Decrease in viscosity causes
increased collision of molecules which results in decreased fluorescence.

Quenching
 Quenching refers to any process that reduces the fluorescence
intensity of a given substance.
 This may occur due to various factors like:
1. pH
2. Concentration
3. Temperature
4. Viscosity
5. Presence of oxygen
6. Heavy metals
7. Specific chemical substances etc.

Types of quenching
 Self Quenching or Concentration Quenching
 Chemical Quenching
 Static Quenching
 Collisional Quenching

Self Quenching or concentration quenching
 Concentration quenching is a kind of self quenching.
 It occurs when the concentration of the fluorescing
molecule increases in a sample solution. The fluorescence
intensity is reduced in highly concentrated solution (>50
µg/ml).

Chemical quenching
Chemical quenching is due to various factors like change in pH, presence
of oxygen, halides and electron withdrawing groups, heavy metals etc
 Change in pH: Aniline at pH (5-13) gives fluorescence when excited at
290 nm. But pH <5 or, pH 13 it does not show any fluorescence.
 Oxygen: Oxygen leads to the oxidation of fluorescent substance to non
fluorescent substance and thus, causes quenching.
 Halides and electron withdrawing groups: Halides like chloride ions,
iodide ions and electron withdrawing groups like -NO2, - COOH, -CHO
groups lead to quenching.
 Heavy metals: presence of heavy metals also lead to quenching
because of collision and complex formation.

Static and Collisional Quenching
 Static quenching: In this process, the quenching agent
forms a non-fluorescent complex with the quenching
agent.
 Collisional quenching: Quenching take place when the
number of collisions is increased due to several factors
like the presence of halides, heave metals, increased
temperature, and decrease in viscosity.

Instrumentation of FLUORESCENCE
Spectrophotometer
1. Source of light
2. Filters & Monochromators
3. Sample cells
4. Detectors

Schematic diagram of FLUORESCENCE
Spectrometer

Diagram of Fluorescence spectrometer

Continue....
1. Source of light
The source of light should emit a radiation over a continuous region and be of
adequate intensity and stability.
A. Mercury vapour lamp – Mercury vapour at high pressure(8 atmospheres)
gives intense lines on a continuous background above 350nm. Lines are seen at
365,398,436,546,579,690, and 734nm. Low pressure mercury vapour gives an
additional line at 250nm.
B. Xenon arc lamp - It gives a more intense radiation when compared to
mercury vapour lamp. It is used as the source of light in Fluorescence
spectrophotometers.
C. Tungsten lamp- If excitation has to be doe in visible region, this can be used.
It does not offer uv - radiation, more over the intensity of this lamp is too low.

Continue....
2. Filters and Monochromators
- In Flourimetry two things are important i.e. Excitation wavelength and emission
wavelength. As these two wavelengths are different in most cases, a filter or
monochromator is used for the purpose.
- In an expensive fluorimeter primary and secondary filter are present. Primary
absorbs visible radiation and transmits uv radiation. Secondary absorbs uv and
transmits visible radiation.
- In spectrofluorometer, excitation monochromators and emission monochromator
re present which have gratings. Excitation monochromator- provides a suitable
radiation for excitation of molecule which is absorbed by molecule.
- Emission monochromator- It provides only the radiation emitted by the
fluorescent molecule.

Continue....
3. Sample cells
Sample cells are used to hold sample solutions. They are cylindrical or polyhedral.
The cells are made up of color corrected fused glass and pathlength is normally 10mm
or 1cm. It need not be made up of quartz, science we are measuring only the emitted
radiation and not the absorbed radiation. Hence even if there is absorption of radiation
by glass, it will not affect the results.
4. Detectors
The emitted radiation is mostly visible radiation and sometimes uv – radiation to
measure the intensity of such radiation, Photovoltaic cell, Phototubes, or
Photomultiplier tubes can be used. But science, we use low concentration of substances
and the intensity of emitted radiation is weak, only Photomultiplier tubes are best and
accurate. In inexpensive instruments like Photoflourimeters, Photo tubes can be used.

Applications
1. Determination of inorganic substances - Examples (Aluminum ion,
Zinc ion, tin ion).
2. Determination of organic substances - Aromatic polycyclic
hydrocarbons, indoles, naphthols, proteins, plants pigments steroids etc.
can be determined at low concentrations by Fluorimetry.
3. Pharmaceutical applications -
Compounds which are fluorescent e.g. p-amino Salicylate,
Desipramine, Aminacrine, Morphine Quinine etc.
Compounds converted to fluorescent products by chemical reaction e.g.
Chlordiazepoxide, Chloroquine, Methyldopa etc.

Applications
Separate the mixture of medicinal substances , various options
available are :
-A wavelength at which only one drug is excited is chosen.
-Using chemical reaction
-Using separation methods and analyzing the compounds.
4. Miscellaneous applications :
Fluorescent indicators: The intensity and colour of many
fluorescent substances depends on pH range and hence are used in
acid-base titrations
Eg: Eosin, Fluorescein, Quinine sulphate.

Applications
5. Other applications :
1. Determination of fluorescent drugs in low-dose formulations
in the presence of non-fluorescent excipients.
2. In carrying out the limit tests where the impurity is
fluorescent.
3. Useful for studying the binding of drugs to component in
complex formulations.
4. Widely used in bioanalysis for measuring small amounts of
drug and for studying drug-protein binding.

spectrofluorimetry, M. Pharm. (1st sem.)

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spectrofluorimetry, M. Pharm. (1st sem.)