Fluorimetry.pptx by Saloni Kadam Nanded taluka

FLUORIMETRY
Presented By
Prof. Rajashree V. Lode
Assistant Professor
HTCOP, Chandrapur

INTRODUCTION
 A large numbers of substances are known which can absorb UV or Visible light
radiation. But these substances lose excess energy as heat through collisions
with neighboring atoms or molecules.
 However, a large numbers of important substances are also known which lose
only part of this excess energy as heat and emit the remaining energy as
electromagnetic radiation of a wavelength longer than that absorbed.
 This process of emitting radiation is collectively known as luminescence.
 Luminescence is the emission of light by a substance. It occurs when an electron
returns to the electronic ground state from an excited state and loses it's excess
energy as a photon.
 In luminescence, light is produced at low temperature; therefore the light
produced by this process is regarded as “light without heat” or “cold light”.
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 Luminescence spectroscopy is a collective name given to three related
spectroscopic techniques. They are:
• Molecular fluorescence spectroscopy
• Molecular phosphorescence spectroscopy
• Chemiluminescence spectroscopy
 Merits of Fluorimetry:
1. It is highly sensitive analytical technique which can accurately determine the
sample even in dilute solution.
2. As the wavelength of excitation and emission are characteristic of every
individuals compound, it helps in the determination of specific element.
3. Certain non-fluorescent substances can be made fluorescent and analyzed.
 Demerits of Fluorimetry:
1. All the elements do not exhibit the property of fluorescence. Therefore, such
elements can not be estimated by fluorimetry.
2. Fluorimetric measurements are accurate only in dilute solutions.
3. Fluorescence is pH dependant.
4. Presence of iodides completely destroy fluorescence.
5. Dissolved oxygen decreases fluorescence by direct oxidation or quenching.
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Electronic States of Molecule
 Concept of Singlet, Doublet & Triplet Electronic States:
a) Singlet State-
• A state in which all the electrons in a molecule are paired ( ). In the ground state
is always a singlet state.
• In a singlet state, the two electrons spinning, the electric & magnetic fields
produced in a opposite directions are exactly the same in magnitude but opposite
in directions.
• When a one electron from a pair of electrons in the ground state is excited to the
higher energy level without the changes in its spin.
• As a result, two electrons, one into the ground state and another in the excited
state, still spin in the anticlockwise and clockwise directions and cancel the
electric and the magnetic fields of each other.
• The difference between the ground singlet state and the excited singlet state is
that the latter is associated with the higher amount of energy as compared to that
of former.
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b) A Doublet State-
• A state in which an unpaired electron is present ( or ). The odd number of
electrons present in molecule.
• As in case of a ground state of a free radical, a single electron can spin either
clockwise or anticlockwise & can assume two orientations in the magnetic field.
One along the direction of an external magnetic field and another in the direction
opposite to the direction of an external magnetic field, this results in the
productions of the field in two directions.
• An unpaired electron assumes two molecular electronic states, the system as a
whole is associated with two different quanta energy. Such a system is termed as
a doublet state.
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c) A Triplet State-
• A state in which unpaired electrons of same spin are present ( ).
• When one of the electron from a pair of electrons in the ground state is excited &
if the excitation of an electron occurs with the change in the spin, both the
electrons, one in the ground state & another in the excited state spin in the same
direction.
• An electron in the ground state & excited state can spin either clockwise or
anticlockwise & can assume two orientations.
• The energy level of an electron associated with excited state is higher compared to
the ground state.
• The excited triplet state is always less energetic compared to the corresponding
excited singlet state.
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Theory of Fluorescence
• At the rest or in ground state, a molecule possesses three energies, rotational,
vibrational and electronic.
• Each electronic level of the ground state in turn contains several vibrational
energy level (V0, V1, V2, V3…….).
• Each molecule prefers to remain at the lowest vibrational energy level of a
ground state.
• When each molecule absorbs UV or Visible light, it gets raised to its excited
electronic state, each of which also contains several vibrational energy state (V'0,
V'1, V'2, V'3…….).
• Most of the molecules after absorbing energy jump to a higher energy level,
which in the absence of magnetic field, results in the formation of singlet,
doublet or triplet state. This states can be defined as number of unpaired
electrons in the absence of magnetic field.
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• A molecule when irradiated absorbs energy and moves from the ground state to
the first excited singlet electronic state. However, since the lifetime of a molecule
in its excited state is very short, it can lose its energy by any one of the following
three processes.
1. The molecule from the excited singlet state may fall to its original ground state
by collisional deactivation without emitting any radiation.
2. The molecule from the excited singlet state may lose its energy by emitting a UV
or visible photon and transit to singlet ground state. The intensity of emitted
radiation is lower than that of the energy absorbed and the emitted radiation is
of longer wavelength. This process is known as fluorescence.
3. In this, the electrons from the stable excited singlet state undergo transition to a
metastable triplet state i.e., intersystem crossing along with the emission of a UV
or visible light photon. The process is known as phosphorescence. The triplet
level is lower than the singlet state. Therefore phosphorescence occurs at a
longer wavelength when compared to the normal absorption & fluorescence.
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Fluorescence
 Fluorescence is the phenomenon of emission of radiation when electrons
undergo transition from singlet excited state to singlet ground state.
 Absorption of UV/Visible radiation by a molecule excites it from a vibrational
level in the electronic ground state to one of the many vibrational levels in the
electronic excited state.
 This excited state is usually the first excited singlet state and is not stable.
 A molecule in a high vibrational level of the excited state will quickly fall to the
lowest vibrational level of this state by losing energy to other molecules through
collision.
 Fluorescence occurs when the molecule returns to the electronic ground state,
from the excited singlet state, by emission of a photon or radiation of longer
wavelength than the incident or absorbed radiation.
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 This is because the energy of emitted radiation is less than that of incident or
absorbed radiation because a part of energy is lost due to vibrational or
collisional processes. Hence the emitted radiation has longer wavelength (less
energy) than the absorbed radiation.
 The wavelength of absorbed radiation is called excitation wavelength (λex) and
that of emission radiation is called as emission wavelength (λem).
 These two wavelengths are specific or characteristic for a given substance under
ideal conditions.
 If a molecule, which absorbs UV/Visible radiation, but does not fluoresce it
means that it must have lost its energy as some other way. These processes are
called radiation less transfer of energy.
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Phosphorescence
 Phosphorescence is the phenomenon of emission of radiation when electrons
undergo transition from triplet state to singlet ground state.
 The spin of an excited electron can be reversed, leaving the molecule in an
excited triplet state, this is called intersystem crossing.
 The triplet state is of a lower electronic energy than the excited singlet state.
 A molecule in the excited triplet state may not always use intersystem crossing to
return to the ground state. It could lose energy by emission of a photon.
 A triplet/singlet transition is much less probable than a singlet/singlet
transition.
 The lifetime of the excited triplet state can be up to 10 seconds, in comparison
with 10 average lifetime of an excited singlet state.
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Chemiluminescence:
 Chemiluminescence occurs when a chemical reaction produces an
electronically excited species, which emits a photon in order to reach the
ground state.
 The number of chemical reactions, which produce chemiluminescence, is
small.
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Factors Affecting Fluorescence
Various factors affecting fluorescence & Phosphorescence are as follows-
 Absorbancy:
• Absorbance of a molecule is directly proportional to the intensity of its
luminescence . Fluorescence or phosphorescence is exhibited by only those
molecules that have the tendency to absorb UV or visible radiations. Therefore,
conjugated (unsaturated) molecules with π electrons can absorb UV or visible
radiations & can exhibit fluorescence. (eg. Alkenes with double bonds)
 Functional Groups:
• Electron donating groups (OH, OCH3, CN, NH2, NHR, etc.) improve
fluorescence, electron withdrawing groups (COOH, CHO, COOR, SH, I, NO2
etc.) decreases or completely destroy fluorescence whereas groups like SO3, H,
NH4 show no effect on the intensity of fluorescence as well as phosphorescence.
 Molecular Weight:
• Elements with high atomic number exhibit decreased fluorescence. Therefore, it
can be said that with the decrease in molecular weight of compounds, the ability
of elements to exhibit fluorescence increases.
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 pH:
• pH has a marked effect on the ability of compounds to emit fluorescence.
Depending upon the acidity or alkalinity, a substance can be in ionized or
unionized form & hence can be fluorogenic or non-fluorogenic.
• Example-
a) Phenol in alkaline medium undergoes ionization and exhibits intense
fluorescence, whereas in acidic medium it is unionized and does not give
fluorescence.
 Temperature & Viscosity:
• A decrease in viscosity or an increase in temperature results in an increase in the
intermolecular collisions and may deactivate the excited molecules ultimately
destroying the fluorescence.
• Also certain non-fluorogenic compounds may exhibits fluorescence at
temperatures lower than room temp. or in a viscous solvent.
 Oxygen:
• The intensity of fluorescence decreases in presence of oxygen. This may be due
to-
a) Direct photochemical oxidation of fluorogenic material to non-fluorogenic
material.
b) Indirectly due to quenching (decrease in intensity & sensitivity of fluorescence.
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 Impurities:
• Compounds other than solute molecules are considered as impurities. Presence
of small amounts of impurities pose negligible effect on fluorescence.
• However, presence of high amount of impurities extinguishes fluorescence as
the impurities absorb major proportion of the incident radiations leaving very
few radiations to excite electrons for fluorescence.
 Concentration:
• There exists a linear relationship between the concentration of sample and
fluorescence. This condition holds good only for samples with low concentration.
• In dilute solutions, the radiations distribute uniformly throughout the solution &
get absorbed uniformly giving high intense florescence.
• But in highly concentrated solutions, the upper layers of the solution absorb
more radiations & therefore less amounts of radiations are transferred to the
lower layers. Thus, there is no uniformity in the absorption of radiations which
results in decreased fluorescence.
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• This is because in highly concentrated solutions, the intra-molecular
collisions cause a loss of vibrational energy & certain amount of emitted
fluorescence is reabsorbed resulting in decreased intensity of fluorescence.
This is called concentration or self quenching.
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Quenching
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 Quenching refers to decrease in the intensity of fluorescence due to decrease in
the sensitivity of constituents of the solution. Quenching can be due to pH,
temperature, viscosity, high concentration of solution, presence of specific
functional groups, chemical reaction etc.
 It is classified into the following types.
1. Chemical Quenching-
Chemical quenching may be due to,
a) pH- Aniline when exposed to excitation radiations of 290 nm, exhibits blue
fluorescence between pH 5- 13. It does not exhibit fluorescence at conditions of
pH below and above this range.
b) Oxygen- Many organic compounds in the presence of dissolved oxygen get excited
from excited singlet state to triplet state, due to the paramagnetic property of
oxygen, ultimately resulting in quenching.
c) Halides- Iodide ion is an extremely effective quencher. It causes a drastic decrease
in the intensity of fluorescence.

d) Heavy Metals- Heavy metals cause collisions & excitation of the molecules from
excited state to triplet ground state, resulting in loss of energy, thereby resulting
in quenching.
2. Collisional Quenching-
 High temperature of the sample and presence of heavy metals, very low viscosity
samples etc, causes increased collisions between the molecules resulting in
deactivation of excited molecules. This causes a decrease in fluorescence.
3. Self/ Concentration Quenching-
 There exists a linear relationship between fluorescence & concentration of sample.
Although linearity is observed in dilute solutions, it is not seen in high
concentrations. This is called as concentration dependant quenching.
3. Static Quenching-
 The substance to be examined may form a complex with the quencher molecule
inhibiting the excitation of the substance and hence decreases the fluorescence.
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Instrumentation
 Fluorimeters or fluorophotometers are the instruments used for measurement of
fluorescence.
 Depending upon the performance, characteristics sophistication and cost of
instruments, fluorimeters are mainly of two types, filter fluorimeter &
spectrofluorimeter.
1. Radiation Source
a) Mercury Vapour Lamp-
• It is widely used radiation source in filter fluorimeters. Both high pressure & low
pressure mercury vapour lamps are available.
• Low pressure mercury vapour lamps are equipped with a fused silica window.
When electric discharge occurs, radiations of various wavelengths are obtained.
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b) Xenon-arc Lamp-
• It is widely used as a radiation source in spectrofluorimeters. Application of low
voltage results in the production of an intense arc between the electrodes of the
lamp, leading to production of ultraviolet light.
• Xenon lamp emits intense and stable radiations in the wavelength range of 300-
1300 nm.
c) Laser Light-
• It is used when monochromatic light or high intensity radiations are desired.
Laser-excited fluorescence is used in the determination of ultra-trace inorganic
ions.
d) Tungsten Lamp-
• It is used when radiations above 450 nm are required. Tungsten lamp gives a
group of lines (broad band) unlike the sharp lines of mercury vapour lamp.
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2. Monochromators & Filters
 Filters or monochromators are used in fluorimeters to separate the exciting
radiations from the emitted radiations.
 Absorption and interference filters are used in fluorimetry.
 Absorption filters allow broad band whereas interference filters allow a narrow
band of wavelength to pass through.
 To isolate the wavelength of excitation, excitation monochromator is placed
between the radiation source & sample & it absorbs visible radiations &
transmits UV radiations.
3. Sample Holding System
 Cuvettes or cells which are cylindrical or rectangular in shape with an area of 1
cm and made of good quality glass or quartz are employed in fluorimeters.
 Cuvettes are provided with a lid to prevent vapourization of volatile materials.
 These are placed in a compartment having light absorbing surface so as to
reduce the scattered radiations from reaching the detector.
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4. Detector
Photomultiplier Tube-
 Construction-
• It consists of a light sensitive cathode and 10 anodes (dynodes) maintained at a
potential of 75 to 100 volts.
• Photomultiplier tube being sensitive can detect extremely weak signals also,
therefore it is used in intricate instruments.
 Working-
• It works on the principle of multiplication of the photoelectrons by secondary
emission of electrons.
• When a beam of light falls on the photocathode, photoelectrons are generated
which are accelerated toward the anodes.
• At each stage of their acceleration from one anode to another, the emission of
electrons is increased by a factor of 4 to 5 due to secondary emission of
electrons.
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Figure : Components of fluorimeters

Types of Fluorimeters
 Filter Fluorimeters-
1. Single Beam Filter Fluorimeters
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 Working-
• A beam of light from radiation source is allowed to pass through the condensing
lens to make them parallel. The radiations are then received by a primary filter,
which transmits UV radiation & absorbs visible radiations. UV radiations are
excitation radiations which fall on the sample cell.
• A secondary filter placed at right angles to the sample cell receives emitted UV
and fluorescent radiations. It absorbs UV radiations & transmits visible
radiations.
• The 90º geometry of filter separates wavelength of excitation & wavelength of
emission.
• The fluorescent radiations are then received by a suitable detector
(photomultiplier tube).
• The detector is connected to a suitable sensitive read out device like
galvanometer, which reads the fluorescence.

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2. Double Beam Filter Fluorimeter

• Backman’s radiofluorimeter which is double beam filter fluorimeter consists of
modified mercury vapour lamp with two anodes opposite to each other from the
centre.
• The lamp is illuminated by alternating current such that the two anodes provide
equal radiations to sample & reference at alternate half-cycles of exciting
voltage.
• Two beams of light from radiation source are passed through two primary filters
to reference and sample cells.
• Primary filter absorbs visible radiations and transmit UV radiations to the
respective cells.
• The radiations from reference and sample cells are simultaneously focused on a
common secondary filter, which absorbs UV radiations & transmit visible
radiations.
• The transmitted radiations are sent to the detector connected to a suitable read
out system.
• The fluorescent signal obtained from sample and reference cells are compared
and analyzed.
• In double beam system, variations in temperature and fluctuations in source &
detector can be neglected to a certain extent.
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 Spectrofluorimeters
In spectrofluorimeter, 2 grating monochromators replace the filters of fluorimeter.
1. Single Beam Spectrofluorimeter
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• Aminco-Bowman spectrofluorimeter is the most commonly used single beam
spectrofluorimeter.
• It employs high pressure xenon arc with stable direct current supply as a source of
radiation.
• Moreover, most of the spectrofluorimeters are equipped with one or two grating
monochromators. Grating monochromators used with 90º geometry.
• Gratings with 600 grooves/mm is used excitation radiation has a wavelength of 300
nm & emission radiation has a wavelength of 500 nm.
Working-
 Radiations from source enter into the excitation monochromator through slits and
mirrors. The excitation monochromator transmits the excitation wavelength of the
molecule to be analyzed into the sample cell through a slit.
 The sample absorbs the excitation radiations and emits fluorescent radiations which
are passed to emission monochromator through slits and mirrors.
 The radiations are finally passed to the detector through mirror & exit slits. Signals
from the detector are then amplified & sent to a suitable read out system.

2. Double Beam Spectrofluorimeter
Construction-
 Double beam spectrofluorimeter is an electro-optical system that splits radiations
into reference & sample beams, both travelling equivalent to the optical path.
 The arc of 150 W xenon lamp acts as a source of excitation radiation.
 Excitation monochromator & emission monochromator are also present.
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 An optical rotatory chopper & sector mirror are also present that splits the
excitation radiations & transmit light to sample & reference cells alternatively.
Working-
 Excitation radiations produced from xenon lamp are directed to the excitation
monochromator through a mirror. This radiations fall on the excitation
monochromator, through the entrance slit.
 A second mirror is present after the exit slit of excitation monochromator.
 Sector mirror and chopper are rotated by common shaft.
 The chopper creates alternate current signals by blocking the radiations to
reference cell & transmitting it to the sample cell & vice versa.
 During first rotation of chopper, sector mirror reflects the beam to reference cell.
 during second rotation of chopper, second mirror opens and radiations fall on
the sample cell.

 In each resolution of chopper, the opaque portion of the disc is measured as
zero.
 Lattice mirror reflects the sample beam (emission from the sample cell) &
transmits the reference beam (emission from the reference cell) to the
emission monochromator.
 Radiations emitted from the sample and reference cells are focused one after
the other on the entrance slit of emission monochromator via front surface
mirrors.
 Reference and sample beams are then focused on photomultiplier tubes
(detector) by passing through exit slits of emission monochromator.
 The signal from photomultiplier tube is measured by a photometer &
recorded using suitable read out system.
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APPLICATIONS
 Analysis of Organic & Biological Compounds
 Estimation of Thiamine (Vitamin B1)-
o Thiamine is a non-fluorescent compound. In alkaline potassium ferrocyanide
[K4Fe(CN)6] solution it undergoes oxidation to from thiochrome, which
exhibits fluorescence. This property is utilized in the estimation of thiamine in
food samples.
 Estimation of Riboflavin (Vitamin B2)-
o Riboflavin is estimated by fluorimetry. In this method, unknown amount of
sample is added to known amount of standard in the same solution &
fluorescence is measured.
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 Estimation of Diphenylhydantoin (Phenytoin)-
o Diphenylhydantoin is oxidized by alkaline KMnO4 to form benzophenone,
which exhibits fluorescence.
 Estimation of Methyldopa-
o Methyldopa is converted to fluorescent dihydroxy indole derivative which is
estimated by fluorimetry.
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 Estimation of Tetracycline in Serum:
o Tetracycline an antibiotic is converted to anhydrotetracycline which is further
complexed with aluminium. It is then determined by fluorimetry at a wavelength
of 475 nm.
o Calibration curve method is used for estimation.
o Fluorescence is due to both anhydrotetracycline and complex of
anhydrotetracycline with aluminium. However, aluminium complex exhibits 30
times greater fluorescence than anhydrotetracycline.
 Analysis of Inorganic Compounds
 Estimation of Uranium Salts-
o The given sample of uranium is evaporated with nitric acid in order to get an
oxidized product which is fused with sodium fluoride. The fusion gives melts of
sodium fluoride & uranium fluoride. On cooling, the melts solidifies, which is
examined in a specially designed fluorimeter.
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 Certain non-fluorescent inorganic ions can be made fluorescent by complexing
them with non-fluorescent organic reagents. Such elements can be analyzed by
fluorimetry.
 Miscellaneous Applications
 Measurement of Fluorescence by Fluorescent Indicators-
o The colour and intensity of fluorescence of many substances depends upon pH of
the solution. Therefore, such substances can be analyzed in fluorimeters by the use
of fluorescent indicators.
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 Determination of Chemical Structure-
o Hydrogen bonding, geometrical isomerism, polymerization, tautomerism &
reaction rates etc. can be studied using fluorimetry.
o For example, absorption of radiation varies with time which determines the rate
of reaction. Hence any change in the absorption causes a proportionate change
in the fluorescence. This helps in determining the reaction rates.
 Preparation of Fluorogenic Derivative from Non-fluorogenic Drug-
o Examples:
a) Complex of atropine with eosin is soluble in chloroform & exhibits fluorescence.
b) Other non-fluorogenic drugs which can be analyzed are morphine & codeine.
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