Fluorimetry

it consists of material for fluorimetry principle, instrumentation, applications

1. FLUORIMETRY
 It is defined as the measurement of the emitted fluorescence light.
 The device which is used to measure the fluorescence is called
fluorimeter.
 Substance which can absorb ultraviolet or visible light energy lose one
part of energy as heat and another part of energy as electromagnetic
radiation of a wavelength longer than that of absorbed.
 This process of emitting radiation is collectively known as
luminescence.
 Luminescence is of two types:
1. Fluorescence
2. Phosphorescence
FLUORESCENCE:
 When a beam of light is incident on certain substance, they emit
visible light or radiations
 This phenomenon is known as fluorescence.
 The substances showing this phenomenon are known as fluorescent
substances.
 This is instantaneous and fluorescence starts immediately after the
absorption of light and stops as soon as the incident light is cut off.
PRINCIPLE:
 Fluorescence is the phenomenon of emission of radiation when there
is transition from singlet excited state to singlet ground state.
 Wavelength of absorbed  Excitation wavelength
 Wavelength of emitted  Emission wavelength
 These two are specific for a given substance under ideal condition.
VARIOUS ELECTRONIC STATES IN FLUORIMETRY:
 If there are (n) numbers of unpaired electrons, it means that (n+1) fold
degeneracy (equal energy states) will be associated with the electron
spin.
 Singlet ground state:
It is a state in which all the electrons in a molecule are paired.
i.e., no of unpaired electrons n=0
 Doublet state :
It is a state in which unpaired electrons are present.
 Triplet state :
It is a state in which unpaired electrons of same spins present.

2.  Singlet excited state:
It is a state in which electrons are unpaired out of opposite spin.


To achieve transition from excited state to ground state there are the
possibilities:
 Collisional deactivation:
It takes place from excited singlet state is relatively unstable
In such a situation, the excited molecules will return to the
ground state by Collisional deactivation without emitting any
radiation.
 Fluorescence:
 A part of energy is lost due to vibrational transition and the
remaining energy is emitted as UV/visible radiation of longer
wavelength from excited singlet state is called fluorescence.
 Phosphorescence:
 The molecule with relatively stable excited singlet state may
undergo transition to a metastable triplet state and sometimes
return to ground state.
 The emission of radiation when electrons undergo transition
from triplet state to ground state is known as
phosphorescence.
 Intersystem crossing:
 The process of crossing from a singlet state (no unpaired
electron) to a triplet state (two unpaired electrons) is termed
as intersystem crossing.
 The decay from the triplet to the ground state singlet is
forbidden by spin symmetry and is therefore slow.
 Thus, the life-time of phosphorescence is much longer than
fluorescence.
 The above mechanism of phosphorescence involving
singlet-triplet decay scheme has been confirmed by the
magnetic susceptibility and ESR measurements.
 According to Hund’s rule, the triplet level always lies
lower than the corresponding singlet level.
 Thus phosphorescence spectrum is not the mirror image of
the absorption spectrum and it always occurs at longer

3. wavelengths compared with the absorption and
fluorescence spectrum.


FACTORS AFFECTING FLUORESCENCE :
 Nature of molecules
 Nature of substituent
 Concentration
 Adsorption
 Intensity of incident Light
 Methods of illumination
 Oxygen
 pH
 photodecomposition
 temperature
 viscosity
APPLICATIONS:
 Determination of uranium in salts by fluorimetry which is used
extensively in the field of nuclear research.
 In general, inorganic ions do not exhibit fluorescence. However, some of
these inorganic ions form fluorescent chelates with non-fluorescent
organic molecules.
 Eg: 1) Determination of ruthenium ion in the presence of other
platinum metals where ruthenium forms the complex ion which
fluoresces strongly at pH 6.
2) Estimation of traces of boron in steel by means of the complex
formed with benzoin.

4. 3) Calcium can be estimated by fluorimetry with calcein solution.
Fluorescent Indicators:
 The intensity and colour of the fluorescence of many substances
depend upon the pH of the solution i.e., their colours depend upon the
pH range.
 These are termed as fluorescent indicators.
 These are mainly used in acid base titrations mainly in coloured
solutions in which the changes in colour of indicators get masked.
 Eg: Eosin, Fluorescein, Acridine, Quinine sulphate.
Fluorometric reagent:
 Fluorometric regents for cation analysis have aromatic structures with
two or more donor functional groups that permit chelate formation
with the metal ion.
 Eg: Benzoin, Flavanol,8-hydroxy quinoline
Determination of vitamin B:
 Vitamin B1 (Thiamine) is non-fluorescent whereas its oxidation-
product, thiochrome, fluoresces with blue colour.
 The property is used for the determination of vitamin B1 in the food
samples like meat, cereal etc.
Organic Analysis:
 It is used to carry out qualitative as well as quantitative analysis for a
great many aromatic compounds present in cigarette smoke, air-
pollutant, concentrates and automobile exhausts.
 Eg: determination of benzopyrene in the nanogram range

INSTRUMENTATION
1. Source of light:
The source of light should emit radiation over continuous region
and be adequate intensity and stability.
Mercury lamp:
 Mercury vapours at high pressure give intense lines on
continuous background above 390nm.
 Lines are seen at 365, 398,486, 579,690 and 734 nm.
 Low pressure mercury vapours gives an additional line at
254nm.
 It is used as source in filter type of flourimeters.

5. Xenon arc lamp:
 It gives more intense radiation when compared to mercury
vapour lamp.
 It is used as source of light is spectrofluorimeters.
Tungsten lamp:
 If excitation is to be done in visible region, this can be used.
 It does not offer UV radiation and more over the intensity of this
lamp is too low.
Filter and monochromators:
In fluorimetry, two things are important,
 Excitation wavelength
 Emission wavelength
 As these wavelengths are different in most cases, a filter or
monochromator is used for purpose.
 Inexpressive instrument like filter fluorimeter, primary filter and
secondary filter are present.
 Primary filter  absorbs visible radiation and transmits UV radiation.
 Secondary filter  absorbs UV radiation and transmits
Visible radiation.
 In spectrofluormeter, excitation monochormators and emission
monochromator are present which have gratings.
 Excitation monochromator  provides a suitable radiation for
Excitation of molecule
 Emission monochromator  Isolates the radiation emitted by the
Fluorescent molecule.
Sample cells:
 The sample cells are cylindrical or polyhedral like used in
colorimetry.
 The cells are made up of colour corrected fused glass and path length
is 10mm or 1cm
 It is need to be made up of quartz, since we are measuring only
emitted radiation and not absorbed radiation.
 Even if there is absorption of glass, it will not affect the results.
 All the surfaces are polished in fluorimetry, because emission
measurements are made at 90
0
angle.
Detectors:
 The emitted radiation is mostly visible radiation and sometimes UV
radiation.

6.  The measure the intensity of such radiation, photovoltaic cell, photo
tubers or photomultiplier tubes can be used.
 Since we use low concentration of substances and the intensity of
emitted radiation is weak, only photomultiplier tubes are best and
accurate.
Instruments:
The main common types are,
a) Single beam (filter) fluorimeter
b) Double beam (filter) fluorimeter
c) Spectrofluormeter (double beam)

Single beam fluorimeter:



 This instrument contains tungsten lamp as source of light and
Optical system consists of primary filter.
 The primary filter absorbs visible radiation and transmits UV radiation
which excites molecules in the sample cell.
 The emitted radiation(fluorescent radiation) s measured at 90
0
by using
secondary filter and a detectors
 The secondary filter absorbs UV radiation and transmits visible radiation,
emitted by the compound.
 Instead of 90
0
we use 180
0
symmetry as in colorimetry the secondary filter
has to be highly efficient, otherwise both unabsorbed UV radiation and
fluorescent radiation will produces detector response and give false result.
 To avoid this we use 90
0
symmetry between incident beam and emitted
beam of radiation.

7. Advantages:
 Simple is construction
 Cheaper
 Easy to operate
 Range of application can be widened by using different combinations
of primary and secondary filters
Disadvantages:
 It is not possible to use sample and reference solution at a time.
 Rapid scanning to get excitation or emission spectrum of the
compound is not possible.




Double beam fluorimeter:



 It is similar to single beam, except the two incident beams from single
light source pass through primary filter separately used fall in either
sample or reference solution.
 The emitted radiation from sample or reference solution pass separately
through secondary filters and produces response on the detectors.
Advantages:
 The sample and reference solution can be analysed simultaneously.
Disadvantages:
 Rapid scanning is not possible due to use of filters.

8. Spectrofluorimeter (double beam):


 The primary filter is double beam fluorimeter is replaced by excitation
monochromator
 The secondary filter is replaced by emission monochromator.
 The incident beam is split into sample and reference beam by using beam
splitter.
 The detector is photomultiplier tube.
Advantages:
 Rapid scanning to get excitation and emission spectrum
 More sensitivity and accuracy.