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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5
 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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