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2. Spectrofluorimetry
Both fluorescence and phosphorescence are types of
photoluminescence (luminescence)
Luminescence: It is the process of reemission of previously absorbed light
When the molecules in the ground state absorb UV light, they are
transferred to the excited state, then, reemission of the previously absorbed
light takes place and the molecules return to the ground state where
fluorescence or phosphorescence takes place
Molecular Emission:
After the absorption of UV Visible light, the excited molecular
species are extremely short-lived and deactivation occurs due to:
a- Internal collision (internal conversion)
b- Cleavage of chemical bonds initiating photochemical reactions
c- Re-emission of light (luminescence)
d- Heat
e- Interaction between the solute and the solvent
Molecules on excitation normally posses’ higher vibrational energy
than they had in the ground state. This extra vibrational energy is lost (Fig.l )
by collision after which the molecules return to the ground electronic state with
the emission of light as fluorescence. Deactivation as fluorescence is a rapid
process occurring within 10-6 - 10-9 seconds of the excitation. Figure 1 shows
the energy transfer during the absorption, fluorescence and phosphorescence
of UV-Visible radiation. The lowest set of energy levels represents the ground
electronic level and its associated vibrational levels. The upper set of
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 91
energy levels represents the first excited electronic state and its associated
vibrational sub-levels.
Photoluminescence should involve both photo-excitation and emission
processes
1- Photoexcitation process:
It may occur by absorption of one of the following forms of radiant
energy:
a- sunlight
b- visible radiation
c- UV
d- X-rays
2- The emission process:
It is the emission of radiant energy from an excited electronic state.
Photoluminescence is called fluorescence when the spin of the
excited electron does not change as the photoexcited species undergoes a
transition from the excited state to the ground state
Singlet and triplet state
Most of the organic compounds that fluoresce or phosphoresce are
aromatic. Some highly unsaturated aliphatic compounds with JI electronic
systems also yield luminescence. Each occupied orbital of a ground-state
molecule has a pair of electrons. The Pauli exclusion principle states that
two electrons in an orbital must have opposing spins and therefore, the net
spin for most ground state molecule is zero.
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 92
In the excited molecules, which exhibit fluorescence, the spin of π electron
and that of π* electron, which together constitute a pi bond in the
chromophore system, are in opposite directions, i.e., they are anti-parallel,
and the molecules are in the singlet states. Some excited molecules
particularly at low temperature, may undergo a slow intersystem crossing
(the triplet excited state) in which the spin of π and π* electrons are
unpaired (parallel). Return from the triplet excited state to the singlet
ground state results in the emission of phosphorescence.
Intersystem crossing process is a slow process than fluorescence, and
consequently phosphorescence occurs after 10-8 seconds and may observed
even several minutes or hours after the source of excitation is removed. The
difference in the energy level (E) between the excited and unexcited
states during excitation (absorption), fluorescence and phosphorescence
are in the order: E (absorption)> E (fluorescence)> E (phosphorescence)
As the wavelengths corresponding to the E values are inversely
proportional to the wavelengths, the order of the max are:,(absorption) <
,(fluorescence) < (phosphorescence)
For example, the wavelengths of maximum excitation, fluorescence and
phosphorescence of anthracene are 255nm, 425nm, 680nm respectively. The
techniques of spectrofluorimetry and phosphorimetry measure the intensity
of the light emitted from a system that has absorbed radiant energy.
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 93
Theory of fluorescence and phosphorescence:
The theory of luminescence is described by using a molecular-energy
interpretation. Fluorescence of organic molecules means emission of
radiant energy during a transition from the lowest excited singlet state S1
to the singlet ground state S0.
Phosphorescence of organic molecules means emission of radiant energy
during a transition from the lowest excited triplet state T1 to the singlet
ground state S0.
Figure 1: Partial energy diagram for a photoluminescent system
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 94
Theory of phosphorescence:
In phosphorescence, an intersystem crossing can take place readily
from S1 to one of the vibrational levels of T1 state that has very nearly the
same energy level (process III). This is followed by non radiative decay
(process IV) to the T1 level.
Intersystem crossing process involves a change in the spin of the
excited electron and thus a change in spin multiplicity.
The triplet state T1 is metastable, and molecules populating it have excess
energy. This energy can be lost by
a- Phosphorescence (process V)
b- Oxygen quenching: energy is transferred to molecular oxygen which
can easily undergo a transition since it has a triplet ground state. For
this reason oxygen must be excluded from the cuvet of
phosphorimeter.
c- By collision: collisions can be diminished by any process that makes a
sample rigid, so that the molecules are immobilized. This has been
done by increasing the viscosity of a suitable solvent by cooling it to a
point where a rigid glass is obtained. The solvent often used is "EPA" a
mixture of ethyl ether, isopropanol and ethanol in the ratio of 5: 5: 2
used at liquid nitrogen temperature (77K). This mixture does not,
crystallize.
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 95
Differences between fluorescence and phosphorescence:
1- Phosphorescence may sometimes persist for many seconds after the
excitation source is removed.
2- Fluorescence emission is always at shorter wavelength than that of
phosphorescence.
3- Fluorescence is usually observed at room temperature in liquid
solution, while phosphorescence is observed in rigid medium at very
low temperature.
4- Fluorescence life time is usually in the range 10-7-10-9 sec, while
phosphorescence lifetime is usually in the range 10-4 -10 sec.
Half life time: It is the time required for half of the molecules to emit
photons and thus return to the ground states.
Effect of molecular structure on luminescence properties:
Fluorescence may be expected generally in:
1- Aromatic molecules that contain conjugated double bonds
2- Polycyclic aromatic compounds (with great number of π electrons)
3- Substituents strongly affect on the fluorescence; substituents such
as NH2, NHCH3, N(CH3)2, OH and OCH3 groups enhance the
fluorescence, while electron with drawing group such as NO2 Cl-,
Br-, I- and COOH groups decrease the fluorescence
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 96
4- Rigid molecules are strongly fluorescent such as fluorescein and
eosin, while non rigid molecule such as phenolphthalein is not
fluorescent.
5- Formation of metal chelates promotes the fluorescence.
Phosphorescence may be expected generally in:
1- Aromatic hydrocarbons
2- Introduction of substituents such as NH2, SH, OH to aromatic
hydrocarbon enhance the phosphorescence and also aromatic nitro
compounds
3- Majority of aromatic aldehydes and ketons show phosphorescence.
Fluorescence Spectra:
Instruments that measure the intensity of fluorescence are called
fluorimeter. Those that measure the fluorescence intensity at variable
wavelengths of excitation and emission, and are able to produce fluorescence
spectra are called spectrofluorimeters
In the recording the fluorescence spectra, the limitations of light sources
and measuring devices assume real significance. These limitations are:
1- Variation of the intensity of available energy with .
2- Variation in the response of the detector to light of different
wavelengths.
In absorption spectrophotometry, both these factors are not
immediately evident, because comparison of blank and test solution is
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 97
carried out under identical conditions, and the absorption spectrum
recorded is true (within the limitation of the instrument). The excitation and
fluorescence spectra, may, however, be grossly distorted version of the true
spectrum if the instrument is not specially adopted.
a) Excitation Spectra:
Before a compound can fluoresce, energy must be observed, and with
an ideal light source, of constant intensity at different wavelengths, the
most intense fluorescence is produced by radiation corresponding in
wavelength to that of the absorption peak of the substance. Therefore, if
the intensity of the fluorescence is plotted as a function of the wavelength
of the radiation used to excite the fluorescence, an activation or excitation
spectrum will, result. This will
be identical to the absorption
spectrum when corrected for
instrumental effect,
because the fluorescence
efficiency is greatly
independent of .
As the intensity of the
fluorescence is measured at a
particular wavelength, the
disadvantage of variation in sensitivity of the detector with the
Figure Fluorescence excitation and emission spectra for a solution of quinine
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 98
wavelength does not appear. However, in practice, the light source is not
ideal and the output from the monochromator used to supply exciting
radiation will vary according to wavelength. The detector will therefore
respond to variation in the intensity of the fluorescence caused by more or
less absorption of energy by the sample, and also by more or less
excitation energy available from the light source. A curve of intensity of
exciting light as a function of wavelength can be prepared for the light
source and may be used to correct the apparent excitation curve
obtained.
b) Emission Spectra (Fluorescence)
When a monochromator source of constant light intensity is used to
irradiate a sample, the fluorescence may be analysed in a monochromator
at constant slit width to give apparent emission spectrum. The true
spectrum is obtained by applying a correction for change in detector
sensitivity with wavelength and for changes due to fluorescence
monochromator i.e., half band width of emergent light and light losses.
Fluorescence emission spectra arise because of transition from the
first excited state and their shapes are therefore independent of the light
used to excite fluorescence. If the substance has an absorption band, the
emission spectrum often bears a mirror-image relationship to it when
plotted on a frequency scale, but if several bands occur, this relationship
may be highly distorted because of overlapping of absorption and
fluorescence bands.
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 99
Instrumentation:
When both the excitation and emission spectra are to be recorded,
two monochromator are essential, one for the light source (excitation
monochromator) and one for the fluorescence (emission
monochromator). The light source must provided a high level of UV and
Visible radiation and a compact high pressure Zenon arc lamp is used.
The production of ozone by the photochemical conversion of
atmospheric oxygen in the lamp compartment presents a toxic hazard
unless the ozone is thermally decomposed or removed by adsorption onto
charcoal. As many experiments will almost certainly entail the
measurement of very weak fluorescence. The detector must be a highly
sensitive photomultiplier tube of low dark current.
If the main interest lies in the fluorescence emission spectra, one
monochromator may be dispensed with a suitable light source and filter
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 100
used instead. The rather poor luminosity associated with the
monochromator even with a xenon arc lamp is replaced by the much more
intense light from a source such as a mercury vapour lamp, from which a
suitable activation beam is isolated by means of the filter. This
arrangement partially overcome, one of the difficulties inherent in
spectrofluorimetry, i.e., that so much of the available light is lost.
Advantages of spectrofluorimetry:
1-High sensitivity:
Substances that are reasonably fluorescent may be determined at
concentration up to 1000 times lower than those required for absorption
spectrophotometry. In spectrofluorimetric measurement, the
photomultiplier tube measures a single light intensity (relative to a zenon
light intensity) which may be amplified electronically many times without
introducing significant noise. In UV-Vis absorption spectroscopy, the
photomultiplier tube measure two intensities I0 and IT. at very low
absorbance, the small difference between I0 and IT approach the noise of
the signal and cannot be measured with satisfactory precision.
The high sensitivity offered by spectrofluorimetry may be of no
advantage if the sample contains a sufficient quantity of the analyte for
assay by absorption spectrophotometry, the latter being generally the
more precise technique. For example, the highly fluorescent substance
quinine sulphate may be assayed with good accuracy and precision in
Quinine sulphate tablets (300 mg) by measuring the absorbance at 348
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 101
nm of the filtered extract of the tablet powder in 0.1 M HCl. However, low
dosage drug formulations containing less than 1 mg per dose unit and
biological samples (blood, urine, etc....) containing low concentration of
the drugs, may require the high sensitivity of spectrofluorimetry, thus the
spectrofluorimetric method is of choice for the determination of many
hormones, alkaloids and vitamins in formulations and biological fluids.
2-Selectivity:
Two factors confirm on spectrofluorimetry a greater selectivity than
that given by UV-Visb. Absorption spectrophotometry. First, not all the
substances that absorb in the UV-Vis region fluoresce. In non fluorescent
molecules, absorbed energy is lost by alternative radiationless pathways,
principally by internal conversion. Molecules require in addition to a
chromophore, a degree of rigidity in their structure to reduce the
dissipation of the absorbed energy by internal conversion.
Substances that are fluorescent are characterized by their
wavelengths of maximum excitation and emission. Different fluorescent
species may show different wavelengths of maximum excitation and /or
emission. The facility to vary independently the wavelength of excitation
and the wavelength of fluorescence allows the analyst to select the
optimum combination of wavelength for the analyte and to reduce
interference from other fluorescing species in the sample,
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 102
Quantitative Aspects:
Many of the quantitative aspects of spectrofluorimetry may be
understood by reference to the fundamental equation for the intensity of
the fluorescence emitted. This equation may be derived from that of the
Beer-Lambert law:
A= LogI0/IT = abC
Or I0/IT =10abc
IT=I0 x l0-abc
but fluorescence (F) = (I0- IT)
where is the quantum yield of the fluorescence
At very low absorbance (< 0.02), the equation will be
F= 2.3 I0 abC
For a fixed set of instrumental (I0 and b) and sample (a and) parameters,
the fluorescence is proportional to the concentration.
F= K C where K = 2.3 I0 a b
Factors Affecting Fluorescence Intensity:
1- Concentration:
The previous equations show that the fluorescence intensity of a
substance is proportional to concentration only when the absorbance in a 1
cm cell is less than 0.02. With increasing the absorbance, introduce an
increasingly significant error (The inner filter effect) and cause negative
curvature in calibration graphs. If the concentration of the fluorescent
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 103
substance is so great that all incident radiation is absorbed, the equation
will be:
F = I0
That is the fluorescence is independent of concentration, and
proportional to the intensity of incident radiation only, a property that may
be utilized to determine the approximate emission characteristics of a light
source.
A further problem ensures if the emission and excitation spectra
overlap, which results in the reabsorption of fluorescence and a negative
dependence of fluorescence on the concentration. The variation over a
wide concentration range is shown in Fig. 3.
Fig. 3 Diagrammatic representation of the variation of fluorescence
intensity with concentration.
Region (a): Proportional relationship Region (b): Negative deviation from linearity. Region (c): Fluorescence independent of concentration Region (d): Reabsorption of fluorescence
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 104
Quantum yield of fluorescence ()
This is the ratio:
Since some absorbed energy is lost by radiationless pathways, the
quantum efficiency is less than 1.
Highly fluorescent substances take value near 1, which shows that most
of the absorbed energy is re-emitted as fluorescence. For example,
fluorescein in 0.1 M NaOH and quinine in 0.05 M H2SO4 have, values of
0.85 and 0.54 respectively at 23°C. Non-fluorescent substances have = 0.
2- Intensity of incident light (I0):
An increase in the intensity of light incident on the sample produces a
proportional increase in the fluorescence intensity. The intensity of
incident light depends on the intensity of light emitted from the lamp. The
excitation monochromator transmission properties (which for a particular
instrument are constant) and the excitation slit width. The intensity of
incident light and sensitivity of a fluorescence measured are increased by
increasing the width of excitation slit. However, wide slit settings
introduce problems due to photochemical decomposition or to spectral
overlap, with consequent reduction of selectivity. The choice of the
excitation slit-width is therefore a compromise between sensitivity,
selectivity and stability.
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 105
3- Pathlength (b):
The symbol for pathlength (b) in the previous equation does not
refer to the dimension of the sample cuvette but to the internal volume of
sample solution, when the fluorescence is both generated and detected.
The effective pathlength viewed by the detector depends on both the
excitation and emission slitwidths. Therefore, the use of microcuvettes
does not necessarily reduce the fluorescence. In fact, if inner filter
quenching or self-absorption is significant, the use of microcells may
reduce these interferences and actually increase the measured
fluorescence.
4- Adsorption:
The extreme sensitivity of the method requires very dilute solution,
10-100 times, weaker than those employed in absorption
spectrophotometry. Adsorption of the fluorescent substance on the
container walls may therefore presents serious problems and strong stock
solutions must be kept and diluted as required. Quinine is a typical
example of a substance which is adsorbed onto cell walls.
4-Oxygen:
The presence of oxygen may interfere in two ways:
a) By direct oxidation of the fluorescent substance to non-fluorescent
products.
b) By quenching of fluorescence. It is a useful precaution, therefore,
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 106
to - check a de-aerated solution and compare the results obtained
with that from the oxygen-containing solution.
Anthracene is well known to be susceptible to the presence of oxygen.
5-pH:
It is to be expected that alteration of the pH of a solution will have a
significant effect on fluorescence if the absorption spectrum of the solute
is changed. Many phenols, for example, are fluorescent in both dissociated
and undissociated forms. Consequently, the fluorescence from a solution
of the phenol will show two peaks, one being due to the ionic form, acidic
solutions may be necessary to suppress the peak due to the ionic form.
6- Photodecomposition:
In absorption spectrophotometry, the intensity of the radiation
passing through solution is weak by photochemical standards, although
adequate for measurements; decomposition of the solute is therefore, not
very likely. Spectrofluorimetry, on the other hand, requires high intensity
illumination for irradiation, and the risk of photochemical change is
thereby increased. An error up to 20% could quite easily arise. It may be
possible in unfavourable cases to select radiation of a wavelength which is
not strongly absorbed so that the extent of photochemical change is
reduced, at the same time adequate sensitivity is retained.
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 107
7- Temperature and viscosity:
Variation in temperature and viscosity will cause variations in the
frequency of collision between molecules. Thus, an increase in the
temperature or the decrease in the viscosity is likely to decrease the
fluorescence by deactivation of the excited molecules by collision.
Similarly, many substances are not normally fluorescent at room temperature
are capable of emitting light when excited at a low temperature or when in a
viscous solvent or glassy matrix. The temperature coefficient of fluorescence
are typically-1%°C increase in the temperature.
8- Quenchers:
Quenching is the reduction of the fluorescence intensity by the
presence of substances in the sample other than the fluorescent
analyte(s). Absorption of the incident or emitted radiation quenches
the fluorescence by inner filter effect. Collisional quenchers reduce
the fluorescence by dissipating the absorbed energy or heat due to
collision with the quenching species. For example, quinine is highly
fluorescent in 0.05 M H2SO4 but not fluorescent in 0.1 M HC1 due to
collisional quenching by halide ions
Static Quenchers:
Form a chemical complex with the fluorescent substance and alter its
fluorescence characteristics. Certain xanthine derivatives e.g. caffeine,
reduce the fluorescence of riboflavine by static quenching. The application
of spectrofluorimetry for the study of binding properties (number of
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 108
binding sites, binding constant) of drugs and macromolecules e.g.
proteins, is based on the alternation of the fluorescence intensity of the
proteins or of the drug with the binding.
9- Scatter:
When the excitation and emission monochromators are at the same
wavelengths, scattered light of the same wavelength as the incident light
will be detected by the photomultiplier arising from colloidal particles in
the sample (Tyndall scatter) and from the molecules (Rayleigh scatter).
Even when the excitation and emission monochromators at set 20 nm or
vmore apart, a little Rayleigh- Tyndall scatter may be detected. Although it
is compensated by using a blank solution* it limits the sensitivity of the
measurements. Reduction of excitation and emission slit-widths to reduce
spectral overlap of excitation and emission spectra will reduce Rayleigh-
Tyndall scatter, at the expense of the sensitivity of the measurement.
Raman Scatter:
Arises from the conversion of some of the incident radiation into
vibrational and rotational energy by the solvent molecules. The resultant
scattered light is of lower energy and, consequently of longer wavelength.
Applications of spectrofluorimetry:
1- Compounds which are fluorescent are readily determined with simple
instruments as the solution for examination is normally obtained by
dissolution of the sample in a suitable solvent (Table 1)
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 109
Compound
PH
ax (nm)
Excitation Emission
Minimum
concentration
required (µg mL-1)
Adrenaline
1
295 335
0.1
Allyl morphine
1
285 355
0.1
Amylobarbitone
14
265 410
0.1
Chloroquin 11
335 400
0.05
Chlorpromazine
11
350 480
0.1
Cinchonidine
1
315 445
0.01
Cinchonine
1
320 420
0.01
Cyanocobalamine
7
275 305
0.003
Ergometrine
1
325 465
0.002
Folic acid
7
365 450
0.01
Menadione
280 320
0.07
Noradrenaline
1
285 325
0.006
Oxytetracycline
11
390 520
0.05
Reserpine
1
300 375
0.008
2- Single substances which are in themselves, non-fluorescent may be
determined as a result of chemical change (Table 2). This method is
useful for both inorganic and organic compounds, and many inorganic
compounds, form highly fluorescent complexes by combination with
organic reagents. The determination of selenium illustrates the increase in
the sensitivity which can be obtained with fluorimetry as as compared with
that for absorption. Thus 0.3 µgml-1 of selenium may be determined by
measurement of the absorbance of its complex with 3,3- diaminobenzidine,
but by using the fluorescence of the complex ,0.04 µg of selenium can be
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 110
measured. The sensitivity is further increased to 0.002 µg of selenium with
2,3 diaminonaphthalene as reagent.
e.g.:
1) Determination of primary amines, amino acids, peptides..etc. through:
Reaction with fluorescamine reagent
2) Determination of primary and secondary aliphatic amines through:
a- reaction with 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole ( NBD-CI ) give
yellow fluorescence
b- reaction with l-dimethylaminonaphthalene-5-sulphonyl chloride (Dansyl,
chloride)
Spectrofluorimetry
Instrumental analysis Dr. Hisham E Abdellatef
Page | 111
Thiamine HCI in pharmaceutical preparations such as tablets and elixirs
and in food stuffs such as flour is relatively easily determined by oxidation to
highly fluorescent thiochrome. The product is soluble in 2-methyl-propan-1-ol
and hence is easily extracted from the reaction mixture for measurements
For mixture of two components, it may be possible to select the exciting radiation
of appropriate wavelengths, such that only one compound fluoresces at any
time. Even if there is not possible, measurements of the fluorescence at two
wavelengths may be sufficient to determine the composition of the mixture.