Fluorometry/Florescence
Spectrophotometry
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Introduction
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 its excess
energy as a photon.
It is of 3 types:
Fluorescence spectroscopy
Phosphorescence spectroscopy
Chemiluminescence spectroscopy
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Introduction…
A certain chemical systems are Luminescence; that is, they can be excited by
EMR or chemical rxn and, as a consequence, reemit radiation either of the
same or longer λ.
In florescence & phosphorescence excitation takes place through the
absorption of photons.
In chemoluminscence an excited species is formed in a chemical rxn.
Absorption of an ultraviolet or visible photon promotes a valence electron
from its ground state to an excited state with conservation of the electron’s
spin.
For example, a pair of electrons occupying the same electronic ground state
have opposite spins and are said to be in a singlet spin state.
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Absorbing a photon promotes one of the electrons to a singlet excited state.
Introduction…
Emission of a photon from a singlet excited state to a singlet ground state, or
between any two energy levels with the same spin, is called fluorescence.
The probability of a fluorescent transition is very high, and the average lifetime
of the electron in the excited state is only 10-5–10-8 s.
Fluorescence, therefore, decays rapidly after the excitation source is removed.
In some cases an electron in a singlet excited state is transformed to a triplet
excited state in which its spin is no longer paired with that of the ground state.
Emission between a triplet excited state and a singlet ground state, or between any
two energy levels that differ in their respective spin states, is called
phosphorescence.
Because the average lifetime for phosphorescence ranges from 10-4–104 s,
phosphorescence may continue for some time after removing the excitation source.
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FLUOROMETRY
Fluorometry is an analytical method , w/c utilizes the ability of
some sub to exhibit fluorescence.
Light of a λ d/t from the irradiating light is released or emitted ff
the absorption process.
Most often the irradiating light is UV light and the emitted light is
visible light.
In a molecule containing a number of UV absorption bands, the
longest λmax is the one associated most strongly with the
production of fluorescence.
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Instrumentation
Instruments for measuring fluorescence contain the same basic
components as UV/Vis spectrophotometers.
The only d/c is presence of additional wavelength selector or dispersion
device after sample cell.
The dispersion can be achieved with filters or monochromators.
Instruments using filters are generally called fluorometers, while
instruments equipped with Monochromators are called
spectrofluorometers since they provides both excitation and emission
spectra.
It is most convenient to measure the fluorescence at a right angle to the
excitation beam to decrease the scatter from the cell walls and so/n &
7 avoid any interference from the transmitted light from the light source.
FLUOROM....
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Instrum…
Source
The signal produced by an analyte is proportional to the number of excited
analyte molecules formed per unit time.
An intense continuum source used in most commercial fluorometers is the
xenon arc lamp.
Wavelength selectors
Two monochromators; one to select the wavelength to be used for
excitation of the sample, the other to scan the wavelength range of the light
emitted by the sample.
Detector
A key requirement for a detector is its ability to detect weak optical signals.
A photomultiplier tube is used as the detector in most fluorescence
9 spectrophotometer.
Analytical Information
The main analytical application of molecular spectrofluorometry is detection
& quantification of species present at concentration so low that most other
techniques are not useful.
Φ= quantum efficiency = # molecules emitting/total # molecules excited
ε(L/mol-cm) and b (cm) have their usual meanings
Po in incident radiant power density (watts/cm2).
These three determine the sensitivity of fluorometry for the analyte.
The measurement situation in fluorometry distinguishing b/n a small signal and
zero signal is more favorable than that encountered in absorption spectroscopy
( measuring a small d/c b/n two large number).
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Advantages
A. Sensitivity
Substances that are reasonably fluorescence may be determined at
concentration up to 5000 times lower than those required for absorption of
spectrophotometer
In Spectrofluorimetric measurement the detector measures single light
intensity w/c may be amplified electronically many times without
introducing significant noise
B. Selectivity
Not all substances that absorb in the uv-vis fluorescence
Wavelength of excitation /emission can be easily varied to selectively
measure the fluorescence
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Factors that affecting the degree of fluorescence
Structural rigidity
It is found empirically that fluorescence is particularly
favored in molecules that possess rigid structures.
One part of a non-rigid molecule can undergo low-
frequency vibrations with respect to its other parts; such
motion undoubtedly account for some energy loss.
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Factors….
Solvent effect
Variation in viscosity will cause variation in the frequency of
collision b/n molecules
Decrease in viscosity ==== decrease fluorescence by deactivation of the
excited molecular collision
If the excited state of a polar molecule has a higher dipole moment
than its ground state (most molecules are in this class), the excited
state will be more stabilized by interaction with a polar solvent
than will the ground state.
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Factors….
As a result, upon going from a less polar to a more polar solvent,
the fluorescence spectrum will shift to longer wavelengths.
In a few cases, the ground state of a solute is more polar than the
excited state.
In this case, going to a more polar solvent stabilizes the ground
state more than the excited state, causing a shift to shorter
wavelengths with increasing solvent polarity.
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Factors….
Temperature effect
Variation in temperature will cause variation in the frequency of
collision b/n molecules
Increase To ==== decrease fluorescence by deactivation of the excited
molecular collision
Higher To === increase thermal motion ==== deactivation through
heat
A rise in 1oC results in decrease in intensity of fluorescence by 1%
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Factors….
Quenchers
Quenching is the process whereby emission from excited
molecule is decreased by energy transfer to another molecule
(quencher)
The emission of light by photoexcited luminescent molecules
may be decreased or even eliminated by interactions with other
chemical species.
E.g. presence of dissolved oxygen
F + hv--------F*
F*------F + hv
F* + Q ----------- F + Q
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Factors….
Two kinds of quenching:
Static quenching - complexation b/n the potentially luminescent
molecule & the quencher takes place in the ground state.
The complex, when excited, fails to luminesce.
The efficiency of quenching is governed by the formation constant of
the complex as well as by the conc of the quencher.
Eg. the quenching of the fluorescence of doxorubicin by Fe(III).
Dynamic quenching - the quenching species and the potentially
luminescent molecule react subsequent to photoexcitation of the latter
and during the lifetime of its excited state.
Its efficiency depends on the viscosity of the solution, the lifetime of
the excited state of the luminescent species, and the conc of the
quencher.
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Factors….
The fluorescence of a molecule is decreased by solvents
containing heavy atoms or other solutes with such atoms in their
structure e.g. carbon tetrabromide and ethyl iodide.
Heavy atoms in solution quench fluorscence by colliding with
excited molecules so that their energy is dissipated, i.e. chloride
or bromide ions in so/n cause collision quenching
Oxygen
The presence of oxygen may interfere in two ways
By direct oxidation of fluorescence substances to non-fluorescence
products
By Quenching of fluorescence
De aerated solution
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Factors….
Concentration Effect
If the conc of a so/n prepared for florescence measurement is too high, some of the light
emitted by the sample as fluorescence will be reabsorbed by other unexcited molecules in so/n.
For this reasons, fluorescence measurement are best so/n as a sample 10-100 weaker than those
w/c would be used for measurement by UV spectrophotometry
Effect of pH
Flurophores that contain ionizable groups are affected by the pH
The fluorescence of an aromatic cpd with acidic or basic ring substituents is usually pH
dependent.
Fluorescence intensity from excited states of charged and uncharged species is generally different. (i.e.
change in pH alter the ratio of charged and uncharged species)
Both the λ and the emission intensity are likely to be d/t for the ionized forms of the cpd.
19 The presence of dissolved oxygen often reduced the intensity of fluorescence in a so/n.
Factors…
It is not entirely possible to predict how strongly fluorescent a molecule
will be.
For example adrenaline and noradrenaline differ in their structure by
only a single methyl group but noradrenaline exhibit fluorescence
nearly 20x intensely than adrenaline
Generally, fluorescence is associated with an extended
Chromophores/Auxochrome system and rigid structure
Quinine is an example of a strongly fluorescent molecule as might be
expected from its extended chromospheres and rigid structure
The Chromophores in Ethinylestradiol is just an aromatic ring but the
presence of a phenolic hydroxyl group in combination with rigid ring
20 structure in the rest of the molecules render it fluorescent
Application of fluorescence
spectrophotometry in pharmaceutical
analysis
Determination of fluorescent drugs in low dosage formulation
Limit if impurity ( Fluorescent or converted fluorescent cpd)
Determination of small amounts of drugs in biological fluids
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Application of fluorescence
spectrophotometry in pharmaceutical
analysis…
Determination of dissolution rate of digoxin tablet
Determination of stability of peptide drugs in so/n
Determination of aluminum in water for injection as a fluorescent
complex
Determination of Ethinylestradiol tablets
The BP utilizes a fluorescence assay to determine Ethinylestradiol
in tablets
The tablet contain low dosage of the drug so that interference by
excipients is likely to cause a problem in UV/Visible
spectrophotometric measurement
The sample is measured using an excitation λ of 280nm and
22 measuring the emission at 320nm
Application…
After the fluorescence of the sample extract in methanol has been
determined, 1M NaOH so/n is added to the sample so/n and the fluorescence
is determine again
The addition of NaOH removes the fluorescence by ionizing the phenol
groups of the Ethinylestradiol and thus any residual fluorescence w/c is due
to excipients can be subtracted from the reading
In the BP assay the Ethinylestradiol content of the tablet extract is
determined by comparison with the fluorescence of a so/n containing a
known amount of Ethinylestradiol standard analysed using the same
condition
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