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 Luminescence
 Luminescence is the emission of light by a substance.

 It occurs when an electron returns to the ground state from

an excited state & loses its excess energy as a photon.

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 Luminescence…
 A certain chemical systems are Luminescence; that is, they can be excited by

EMR or chemical rxn & , as a consequence, reemit radiation either of the same
or longer λ.
 In florescence & phosphorescence excitation takes place through the absorption

of photons.
 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.

 Absorbing a photon promotes one of the electrons to a singlet excited state.

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 Luminescence…
 Singlet, doublet and triplet are derived using the equation for

multiplicity, 2S+1, where

 S = total spin angular momentum (sum of all the electron spins)

 Individual spins are denoted as spin up (s = +1/2) or spin down (s = -1/2).

 Two electrons in an atom cannot have the same four quantum
 The Principal Quantum Number (n), Azimuthal Quantum Number (ℓ),
magnetic moment (mℓ), and spin (ms).
 Only 2 electrons can occupy each orbital where they must have
opposite spin states.
 These opposite spin states are called spin pairing

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 Luminescence…
 Individual spins are denoted as spin up (s = +1/2) or spin

down (s = -1/2).
 If we were to calculate the S for the excited singlet state,

the equation would be 2(+1/2 + -1/2)+1 =2(0)+1 = 1

 If the spin multiplicity for the excited triplet state was

calculated, we obtain 2(+1/2 + +1/2)+1 = 2(1)+1 =3,

which gives a triplet

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 Cont…

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 Cont…

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 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,
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phosphorescence may continue for some time after removing the excitation source.
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 Radiationless Transitions: Internal Conversion
The electron can hop over from one state to the other, without

releasing a photon.
 This event is called a "radiationless transition", because it occurs

without release of a photon.

 The electron simply slides over from a low vibrational state of the

excited electronic state to a high vibrational state of the electronic

ground state.
 If the electron simply keeps dropping a vibrational level at a time

back to the ground state, the process is called "internal conversion".

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 Florescence vs phosphorescence
 Florescence:
 Emission not involving spin change (S S)
 Efficient
 Very probable
 Fast rate
 Short lifetime
 Phosphorescence:
 Emission involving spin change (S T)
 Improbable (forbidden transition)
 Slower rate
 Longer lifetime ( approximately 104 s fold difference)

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 Fluorescence spectroscopy

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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.

 The fluorescence spectrum of a molecule is, ideally, a mirror image of the

longest wavelength band in the absorption spectrum of the molecule but often
the spectrum is distorted due to partial overlap b/n the absorption & the
emission spectra.
 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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 FLUOROMETRY

Figure 1: Idealized absorption and emission spectra

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 Electronic transitions to and from the lowest vibrational states are often
referred to as 0–0 (zero zero) transitions and have the same energy in both
absorption and fluorescence. Whereas all other absorption transitions require
more energy than any transition in the fluorescence emission. We can
therefore expect the emission spectrum to overlap the absorption spectrum at
the wavelength corresponding to the 0 – 0 transition and the rest of the
emission spectrum to be of lower energy, or longer wavelength
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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 with Monochromators.

 Instruments using filters are generally called fluorometers,

 While instruments equipped with Monochromators are called

spectrofluorometers since they provides both excitation & 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 & so/n, &
avoid any interference from the transmitted light from the light source.
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 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

18 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= fluorescence quantum efficiency = # molecules emitting/total # molecules excited

 ε(L/mol-cm) & b (cm) have their usual meanings

 Po in incident radiant power density

 These three determine the sensitivity of fluorometry for the analyte.

 The measurement situation in fluorometry distinguishing b/n a 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 fluoresce
 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 - decreases 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 - decreases fluorescence by deactivation of the excited

molecular collision

 Higher To - increases thermal motion - deactivation through

heat

 A rise in 1oC results in decrease in intensity of fluorescence

24 by 1%
 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 & the potentially
luminescent molecule react subsequent to photoexcitation of the latter
& 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 & 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 & 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 & uncharged species is generally
different. (i.e. change in pH alter the ratio of charged & uncharged species
 Both the λ and the emission intensity are likely to be d/t for the ionized forms of
the cpd.
 The presence of dissolved oxygen often reduced the intensity of fluorescence in
a so/n.
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 Factors…
 It is not entirely possible to predict how strongly fluorescent a

molecule will be.

 E.g. adrenaline & noradrenaline differ in their structure by only a

single methyl group but noradrenaline exhibit fluorescence nearly 20x

intensely than adrenaline

Adrenaline Noradrenaline

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 Factors…
 Generally, fluorescence is associated with an extended chromophores /

auxochrome system & rigid structure

 Quinine is an example of a strongly fluorescent molecule as might be

expected from its extended chromospheres & rigid structure

 The chromophores in ethinylestradiol is just an aromatic ring but the

presence of a phenolic hydroxyl group in combination with rigid ring

structure in the rest of the molecules render it fluorescent.

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 Application of fluorescence spectrophotometry
 Direct analysis methods
 The natural fluorescence of the fluorescent analytes is measured.
 Such cpds show reasonable intrinsic fluorescence to be determined by the
direct method.
 Indirect analysis methods
 Two strategies:
 The analyte to be determined is suitably derivatised to a fluorescent
species.
 Then the fluorescence of the complex is measured.
 A fluorescent species whose fluorescence is quenched by the analyte.
 The fluorescence intensity of the target molecule is measured as a
function of the concentration of the analyte.

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 Application of fluorescence spectrophotometry in
pharmaceutical analysis

 Determination of fluorescent drugs in low dosage

formulation
 Limited if impurity ( Fluorescent or converted fluorescent

cpd)
 Determination of small amounts of drugs in biological

fluids
 Used in the study of the chemical equilibrium & kinetics

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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

measuring the emission at 320nm

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 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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 Phosphorescence spectroscopy

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 Phosphorescence spectroscopy

 This involves an inter-system crossing or transition from

the singlet to the triplet state after the excitation.

 A triplet results when the spin of one electron changes,

 So that the spins are the same or unpaired.

 The phosphorescence is emitted at wavelengths longer

than those of flouroscence.

 Since the energy level of the triplet state lies below the

excited state levels.

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 Cont…
 Phosphorescence emission is characterized by its frequency, lifetime,

quantum yield & vibrational pattern.

 These properties form the basis for qualitative analysis.

 Whereas the correlation of intensity with concentration gives the bases for

quantitative measurements.

 Phosphorescence is measured in a similar manner as that of flouroscence

 Except for employing a mechanical method for distinguishing between

them by their time delay

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 Con…
 Phosphorescence is not observed at room Temp., as

the energy of the triplet state is readily lost by

collisional deactivation process involving the solvent.
 Therefore, phosphorescence is normally observed at

reduced Temp. in solidified samples.

 Several commercial fluorimeters are provided with

phosphorescence attachments.
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 Application of phosphorimetry
 Although low-temperature phosphorimetry is inherently a very
sensitive analytical method, it too has yet to attract a substantial
group of users.
 However, there are a substantial number of molecules of
pharmaceutical interest that:
 Phosphoresce but do not fluoresce (e.g., metronidazole) or
 Fluoresce weakly & phosphoresce intensely (e.g., warfarin), &
 It is probably worthwhile to pursue phosphorimetry as a valuable
aspect of molecular luminescence spectroscopy.
 Applied to the determination of:
 Varities of alkaloid, chinese traditional medicine, tetracyclines, quinolone,
riboflavin, anticancer drugs, naphazoline, naproxen, naftonyl dipyridamole

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