Fluorometry

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The mechanisms by which molecules absorb electromagnetic radiation in the visible and ultra violet regions
of the spectrum and are, as a result, raised to excited electronic states are as same as of absorption
spectrophotometry. The reverse process, loss of energy and concomitant transition of molecules from excited
states to ground states of energy can occur with reemission of radiation. Such emission is known as
luminescence. The Intensity and composition of the emitted radiation can be measured and such measurements
form the basis of a sensitive method of analysis called fluorometry. Fluorometric methods of analysis have
found application in many situations of pharmaceutical interest such as in the analysis of riboflavin, thiamine
and reserpine in drug dosage forms.
Fluorometry is an analytical technique for identifying and characterizing minute amounts of a substance by
excitation of the substance with a beam of ultraviolet/visible light and detection and measurement of the
characteristic wavelength of fluorescent light emitted.
SOME RELATED TERMS:
☻Luminescence: 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. Luminescence is
the phenomenon of a chemical species to absorb radiation of UV or visible region and emit a radiation of
longer wavelength. Loss of energy and concomitant transition of molecules from excited states to ground
states with emission of radiation is called luminescence.
What happens here is, energy excites the molecules (more specifically electrons of the molecules). When the
molecules return to the normal state, they emit radiation-light.Luminescence can be divided into two types
depending on the lifespan of the excited state
 Fluorescence  Phosphorescence
☻Fluorescence: Fluorescence is defined as the emission of radiation by a chemical species during its
transition from an excited singlet state to the ground (singlet) state. Prompt fluorescence: S1→ S0 + h𝝂
The release of electromagnetic energy is immediate or from the singlet state.
Delayed fluorescence: S1 →T1 →S1 → S0 + h𝝂

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This results from two intersystem crossings, first from the singlet to the triplet, then from the triplet to the
singlet.
Fluorescence is a kind of a luminescence, which is the emission of photons from electronically excited states.
Fluorescence occurs when the electron is transferred from a lower energy state into an "excited" higher energy
state. The electron will remain in this state for 10⁻⁸ sec. then the electron returns to the lower energy state and
it releases the energy in form of fluorescence. In ultraviolet absorption spectroscopy when molecule absorbs
UV radiation at one wavelength and it’s immediately re-emission, usually in a longer wavelength. Some
molecules fluoresce naturally and others can be modified to make fluorescent compounds.
The phenomenon of radiation emission during
transition from the lowest vibrational energy level of
the excited singlet state to the ground state is called
fluorescence.
☻Fluorometry: It is measurement of
fluorescence intensity at a particular wavelength
with the help of a filter fluorimeter or a
spectrofluorimete. It’s measured by a fluorometer or
fluorimeter.
☻Phosphorescence: Phosphorescence is defined as the emission of radiation by a chemical species during
its transition from the excited triplet state to the singlet ground state. The triplet state of a molecule has a lower
energy than its associated singlet state so that transitions back to the ground state are accompanied with the
emission of light of lower energy than from the singlet state. Therefore, we would typically expect
phosphorescence to occur at longer wavelengths than fluorescence. Phosphorescence is often characterized
by an afterglow because of the long life of the triplet state, 10-4
-10 seconds.
An important feature of phosphorescence is
afterglow. Light is emitted from phosphorescent
molecules after radiation energy source is removed.
This is because the luminescence continues for 10-4
seconds to 10 seconds as the triplet state has greater
longevity. In phosphorescence, similar to the
fluorescence, vibrational relaxation must occur.
So, Phosphorescence may be defined as emission of
radiation resulting from transition of molecule from
excited triplet state to ground state.
☻Singlet state (SS): Singlet state
is the state in which all of the electrons
are paired and in each pair the two
electrons spin about their own axis in
opposite directions.
At the ground state, the molecular orbitals are occupied by two electrons. The spins of the two
electrons in the same orbital must be antiparallel.This implies that the total spin, S, of the molecule
in the ground state is zero [½ + (-½)]. This energy state is called “singlet state” and is labeled as S0.

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☻Excited singlet state: When two electrons of the singlet state are goes to the excited state it is called
excited singlet state. In excited singlet state electrons remain as in exciting position. In the ground state of a
molecule, the two electrons responsible for bonding lie in the bonding molecular orbital in opposite spins.
Now when energy is applied to excite the molecule, one of the electrons will transit to the excited state i.e. the
anti-bonding molecular orbital. If the excited electron in the anti-bonding orbital has spin opposite to the
electron present in the bonding orbital of ground sate, then the excited state is called excited singlet state.
☻Triplet state: Triplet state is a state lying at an energy level intermediate between ground and excited
state and characterized by an unpairing of two electrons. In contrast to the singlet state, there is a spin reversal
involving one electron of the pair and the pair of two electrons spins about their axis in the same direction.
The life time of the molecule in the triplet state in 10-4
to 10-2
seconds.
Basically the triplet state is the excited state between the ground state and the excited singlet state and electron
in this state spins in the same direction as that of ground state.
☻Chemiluminescence: Chemiluminescence is another phenomenon that falls in the category of
luminescence. This refers to the emission of radiation during a chemical reaction.
However, in such cases theexcited state is not a result of absorption of electromagnetic radiation. The oxidation
of luminol (3-aminophthalhydrazide) in an alkaline solution is an example of chemiluminescence.
☻Bioluminescence: Bioluminescence is the production and emission of light by a living organism.
Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi,
microorganisms including some bioluminescent bacteria, and terrestrial arthropods such as fireflies.
☻Fluorescein: Fluorescein is a fluorescence label that absorbs light at 490 nm and releases this energy at
520 nm.
Theoretical consideration /Basic concept / Principal:

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Fluorescence spectroscopy aka fluorometry or spectrofluorometry is an analytical technique for identifying
and characterizing minute amounts of a fluorescent substance by excitation of the substance with a beam of
ultraviolet light and detection & measurement of the characteristic wavelength of the fluorescent light emitted.
It is a spectrochemical method. These terms are explained with the illustration above (Theory of Fluorescence
and Phosphorescence)
When energy is applied to certain molecules in the form of UV or visible electromagnetic radiation, the
molecules temporally transit to an excited singlet state where the excited electron is in paired condition with
the ground electron. In the excited state, the molecules lose energy in radiationless manner to descend to the
lowest vibrational energy level of the excited state. The excited state lasts only 10-8
to 10-4
seconds and then
the excited molecule will return to ground state by losing energy through emitting radiation. This is termed
fluorescence and the emitted radiation is of longer wavelength. By measuring the emitted wavelength we can
determine the presence and amount of a compound in a sample.
1. Absorption of radiant energy: The absorption of UV-Vis radiation is necessary to excite molecules
from the ground state to one of the excited states. When a molecule absorbs radiant energy it is got promoted
from the ground state to the excited state and gets distributed in the various vibrational energy levels mostly
to the excited singlet state. Average time of molecule stay in excited state is 10-8
sec.
2. Radiationless vibrational relaxation (RVR): Absorption of radiation will excite molecules to
different vibrational levels of the excited state.This process is usually followed by successive vibrational
relaxations (VR) as well as internal conversion to lower excited states. Molecules initially undergo a more
rapid process, a radiationless loss of vibrational energy (loss energy by emitting photons) and so quickly falls
to the lowest vibrational energy level of the excited state, known as vibrational relaxation.
3. Radiationless internal conversion: From the lowest vibrational energy level of the excited singlet state,
a molecule can return to the ground state by photoemission or by radiationless process followed by vibrational
relaxation. When an excited molecule undergo a radiationless loss of vibrational energy, sufficient to drop to
the ground state then it is termed internal conversion
4. Fluorescence: After vibrational relaxation to first excited electronic level takes place, a molecule can
return to the ground state by emission of a photon, called fluorescence (FL).The radiation emitted in the
transition of a molecule from a singlet excited state to a singlet ground state is called fluorescence.The
radiation emitted as fluorescence is of lower energy and therefore of longer wavelength than that originally
absorbed.The fluorescence lifetime is much greater than the absorption time and occurs in the range from10-7
to 10-9
s.All absorbed energy will not emitted as fluorescence.
5. Intersystem crossing: Molecule in the lowest vibrational energy level of the excited singlet state converts
to a triplet state (the state lying at an energy level intermediate between ground state and excited).This process
is called intersystem crossing. Here molecules do not losses energy.
6. Radiationless vibrational relaxation: Once intersystem crossing has occurred, a molecule so quickly
falls to the lowest vibrational energy level of the excited triplet state by vibrational relaxation. The lifetime of
molecule in the triplet state is 10-4
to 10-2
seconds (Longer than corresponding singlet state)
7. Radiationless internal conversion: It is a process whereby excited molecules lose their energy due to
collisions with other molecules or by transfer of their energy to solvent or other unexcited molecules.After
RVR molecule goes to the ground state from triplet state.Energy is released here in the form of heat radiation.
8. Phosphorescence: Electrons crossing the singlet state to the triplet state can follow one of three choices
including:
 returning to the singlet state

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 relax to ground state by internal or/and external conversion
 lose their energy as a photon (phosphorescence) and relax to ground state
It requires a much longer time than fluorescence (10-4
s to up to few s).
Instrumentation for fluorescence spectroscopy:
Figure: A diagrammatic representation of an instrument
used to measure intensity of fluorescence.
In contrast to spectrophotometry, the intensity of light transmitted by a sample is not of direct concern in
fluorometry. Rather, it is the intensity of radiation that is emitted as fluorescence that is measured and related
to the concentration of fluorescing species. The components of instruments which are used in fluorometry are,
however, quite similar in design and function to those employed in spectrophotometers and colorimeters. A
diagrammatic representation of such a device is shown in figure above. The chief components are:
☻Light sources: The lamp or light source provides the energy that excites the compound of interest by
emitting light. The light from an excitation source passes through a filter or monochromator, and strikes the
sample. The light source must be intense and stable. Light sources include:
a. Gas discharge lamps:
 Xenon arc lamp (all wave length)
b. Incandescent lamps: Tungsten wire filament lamp
c. Laser: tunable dye laser

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 High pressure mercury vapor lamp d. X-ray source for X-ray fluorescence
The emission of a mercury lamp is concentrated in several very intense bands. Among those having a
wavelength of 254-365 nm are of a great value as excitation radiation is evenly distributed over a wide range
of wavelengths.
☻Excitation Filter (or monochromator): The excitation filter is used to screen out the wavelengths of
unabsorbed light by the compound being measured. It filters the source light and isolates the band of exciting
light that is to be passed to the sample holder. If the instrument uses coarse monochromator then the instrument
is called fluorometer.If grating or prism monochromator is used then the instrument is called
spectrofluorometer, spectrophotofluorometer or florescence spectrometers. Usually glass filters are used.
☻Sample compartment: The sample cell or cuvette holds the sample. The cuvette material must allow the
compound's absorption and emission light energy to pass through. Also, the size of the sample cell affects the
measurement. The greater the path length (or diameter) of the cell, the lower the concentration that can be
read. Glass cells (300nm) are used for most analysis. If measurement is to be under 320nm wavelength then
quartz cells (200-800nm) fused silica are used.
☻Emission filter/monochromator: It selects the band of fluorescence which is to be detected. It is usually
placed at right angle (90º) to the beam of exciting (transmitting) light but other arrangements are possible.
Stray light such as Rayleigh and Raman scatter is also emitted from the sample.
☻Detectors: The detector is placed at a right angle to the direction of travel of beam of exciting light. The
light detector is most often a photomultiplier tube or photoconductive target vidicon or return beam vidicon
or intensified target vidico though photodiodes are increasingly being used. The light passing through the
emission filter is detected by the photomultiplier or photodiode. The light intensity, which is directly
proportional (linear) to the compound's concentration, is registered as a digital readout.
☻Recorder & amplifier: The output of the detector is connected to a meter, a digital display or a recorder.
Recorder (indicates the detector response) gives the intensity of radiation in terms of electrical signal produced
by the detector.
Amplifier is used to amplify the detector response before recording.
Some most common types of fluorimeter are:
Single beam (filter) fluorimeter: It contains tungsten lamp as a source of light and has an optical system
consists of primary filter. The emitted radiations is measured at 90° by using a secondary filter and detector.
Primary filter absorbs visible radiation and transmit UV radiation which excites the molecule present in sample
cell. Single beam instruments are simple in construction cheaper and easy to operate.
Double beam (filter) fluorimeter: it is similar to single beam except that the two incident beams from
a single light source pass through primary filters separately and fall on the another reference solution. Then
the emitted radiations from the sample or reference sample pass separately through secondary filter and
produce response combinly on a detector.
Spectrofluorimeter (double beam): In this primary filter in double beam fluorimeter is replaced by
excitation monochromator and the secondary filter is replaced by emission monochromator. Incident beam is
split into sample and reference beam by using beam splitter.
Structural factors affecting fluorescence:
☻Fluorescence is expected in molecules that are aromatic or multiple conjugated double bonds with a high
degree of resonance stability. Conjugation is necessary for fluorescence.This is because mobile π electrons

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are responsible for UV-Vis absorption characteristics of compounds.Thus cyclohexane (saturated, no π
electron) is not fluorescent, benzene is weakly fluorescent and anthracene is highly fluorescent.
☻Fluorescence is also expected in polycyclic aromatic systems.
 Simple heterocyclic do not exhibit
fluorescence.
 Fusion of heterocyclic nucleus to benzene ring
increases fluorescence.
☻Substituents such as –NH3, –OH, –F, –OCH3, –NHCH3, & –N(CH3)2 groups, often enhance fluorescence.
☻On the other hand, these groups decrease or quench fluorescence completely: –Cl, –Br, –I, –NHCOCH3, –
NO2, –COOH.
☻Changes in the system pH, if it affects the charge status of chromophore, may influence fluorescence.
☻Many compounds show fluorescence at ionized state. But this is dependent upon pH of the solution.
☻The higher the rigidity the greater is the
fluorescence intensity. This is because, rigidity and
planarity will prevent vibration and free rotation of
aromatic rings hence less energy is dissipated in
radiationless manner.
☻Substances fluoresce more brightly in a glassy state or viscous solution.
☻Formation of chelates with metal ions also promotes fluorescence. Complexation increases rigidity and
minimizes internal vibration hence fluorescence intensity is increased.
e.g. Tetracycline has a weak native fluorescence but
complexes of the antibiotic with Ca2+
and a
barbiturate fluorescence quiet intensely.

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Environmental factors affecting fluorescence:
☻Temperature:
 A rise in temperature is almost always accompanied by a decrease in fluorescence.
 The change in temperature causes the viscosity of the medium to change which in turn changes the n
umber of collisions of the molecules of the fluorophore with solvent molecules.
 The increase in the number of collisions between molecules in turn increases the probability for deac
tivation by internal conversion and vibrational relaxation.
 Temperature of the reaction must be regulated to within +/- 0.1℃.
 In general, a 1⁰C rise in temperature results in a decrease of fluorescence intensity by 1%.
☻PH
:
 Relatively small changes in pH can sometimes cause substantial changes in the fluorescence intensity
and spectral characteristics of fluorescence.Example: Serotonin shows a shift in fluorescence emission
maximum from 330 nm at neutral pH to 550 nm in strong acid without any change in the absorption
spectrum.
 In the molecules containing acidic or basic functional groups, the changes in pH of the medium change
the degree of ionisation of the functional groups. This in turn may affect the extent of conjugation or
the aromaticity of the molecule which affects its fluorescence. Example: Aniline shows fluorescence
while in acid solution it does not show fluorescence due to the formation of anilinium ion.
 Therefore, pH control is essential while working with such molecules and suitable buffers should be
employed for the purpose.
☻Dissolved oxygen:
 Dissolved oxygen often decreases fluorescence dramatically and is an interference in many
fluorometric methods.
 The paramagnetic substances like dissolved oxygen and many transition metals with unpaired
electrons dramatically decrease fluorescence and cause interference in fluorimetric determinations.
 The paramagnetic nature of molecular oxygen promotes intersystem crossing from singlet to triplet
states in other molecules.
 The longer lifetimes of the triplet states increases the opportunity for radiationless deactivation to
occur.
 Presence of dissolved oxygen influences phosphorescence too and causes a large decrease in the
phosphorescence intensity.
 It is due to the fact that oxygen at the ground state gets the energy from an electron in the triplet state
and gets excited.
 This is actually the oxygen emission and not the phosphorescence. Therefore, it is advisable to make
phosphorescence measurement in the absence of dissolved oxygen.
 Other paramagnetic substances, including most transition metals, exhibit this same effect.
☻Solvents:
 Solvents affect fluorescence through their ability to stabilize ground and excited states differently,
thereby changing the probability and the energy of both absorption and emission.
 The changes in the “polarity” or hydrogen bonding ability of the solvent may also significantly affect
the fluorescent behaviour of the analyte.
 The difference in the effect of solvent on the fluorescence is attributed to the difference in their ability
to stabilize the ground and excited states of the fluorescent molecule.

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 Besides solvent polarity, solvent viscosity and solvents with heavy atoms also affect fluorescence and
phosphorescence.
 Increased viscosity increases fluorescence as the deactivation due to collisions is lowered.
 A higher fluorescence is observed when the solvents do not contain heavy atoms while
phosphorescence increases due to the presence of heavy atoms in the solvent.
 Some solvents e.g Ethanol, also cause appreciable fluorescence.
 Other sample matrix, e.g. proteins and bilirubin are more serious contributors to unwanted
fluorescence.
Quantum efficiency of fluorescence:
Quantum efficiency is defined as the ratio of number of light quanta emitted and the number of light quanta
absorbed. It is denoted by ∅.
∅ =
emitted light quanta
absorbed light quanta
Its significance is that, it is an indicator of how fluorescent a molecule is.
 if ∅ is near 1, the molecule is highly fluorescent molecule
 if ∅ is near 0, the molecule is a very low fluorescent molecule
Q-How you can chemically convert a non-fluorescence compound to fluorescence compound?
1. Complexation: Tetracycline is weak fluorescence but complexes with Ca2+
and barbiturate makes it
fluorescence.
2. Acid treatment: Hydrocortisone usually not fluorescence but they form strongly fluorescence compound
in sulphuric acid in presence of ethanol.
3. Oxidation: By oxidation and hydroxylation epinephrine forms strongly fluorescing compound.
Mirror image rule:

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Vibrational levels in the excited states and ground states are similar.
An absorption spectrum reflects the vibrational levels of the electronically excited state.
An emission spectrum reflects the vibrational levels of the electronic ground state.
Fluorescence emission spectrum is mirror image of absorption spectrum.
Q-Why absorbed energy and emitted energy is not identical?
 rearrangement of atoms in different energy levels is not similar
 triplet state
When an electron absorbs energy either from light (photons) or heat (phonons), it receives that incident
quantum of energy. But transitions are only allowed between discrete energy levels such as the two shown
above. This leads to emission lines and absorption lines.
Emitted energy: Photon is emitted and electron drops to lower quantum energy state. Emission spectra always
involve electrons going down in energy level.
Absorbed energy: Photon is absorbed and excites electron to higher quantum energy state. Absorption spectra
always involve atoms going up in energy level.
Common problems of fluorescence measurements:
Reference materials and sample:
 Reference materials is as fluorescent as the sample Contaminating substances, Raman scattering,
Rayleigh scattering.
Fluorescence reading is not stable:
 Fogging of the cuvette when the contents are much colder than the ambient temperature.
 Drops of liquid on the external faces of the cuvette.
 Light passing through the meniscus of the sample.
 Bubbles' forming in the solution as it warms.
Self-quenching:
 It results when luminescing molecule collide and lose their excitation energy by radiationless due to
presence of impurities like molecular oxygen.
Inadequate sensitivity:
 Fluorometry is significantly more sensitive as an analytical tool
Absorption of radiant energy:
 Absorption either of the exciting or of the luminescent radiation reduces the luminescent signal.

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Self-absorption:
 Attenuation of the exciting radiation as it passes through the cell can be caused by too concentrated an
analyte.
Excimer formation:
 Formation of a complex between the excited-state molecule and another molecule in the ground state,
called an excimer, causes a problem when it dissociates with the emission of luminescent radiation at
longer wavelengths than the normal luminescence.
Applications of fluorometry:
☻Application in Chemistry:
Fluorometry is used in chemistry for –
 Determination of metal ions: Complexes of metals ions may give strong fluorescence which is utilized
for this purpose.
 Separation and identification: In many cases, after separation, chemicals are identified using
fluorometry. e.g. aminocrine.
☻Application in Biopharmaceutics:
 Measurement of drug in blood, urine and other body fluids.
 Study of the rate and mechanism of drug absorption, metabolism and excretion.
 Selection of toxic compounds.

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☻Pharmaceutical applications:
Fluorometry is used for quantitative analysis of
 Hormones: Adrenaline, aldosterone, testosterone
 Alkaloids:
a.Opioids: Morphine, codeine etc.
b. Rauwolfia alkaloids: Reserpine
c.Others: Atropine, emetine etc.
 Vitamin: Riboflavin and thiamine are indicated for fluorometric assay by USP and BP.
 Antibiotics: tetracycline, sulfonamide etc.
 Cardiac glycosides: Such as digoxin, digitoxin, etc.
 Fluerometry is widely used in the analysis of drugs in systems (physiological systems) other than
dosage forms. The sensitivity of the method of analysis is applied for a large number of
pharmacological, biochemical, toxicological, pharmacokinetic (ADME) & biopharmaceutical studies
for the analysis of amount of drugs in biological fluids and tissues.
Advantages of fluorometry:
 Sensitivity: In case of Fluorescence, detectability to parts per billion or even parts per trillion is
common for most analytes.
 Specificity: Fluorometers are highly specific and less susceptible to interferences because fewer
materials absorb and also emit light (fluoresce).
 Wide Concentration Range: Fluorescence output is linear to sample concentration over a very broad
range.
 Simplicity: Fluorometry is a relatively simple analytical technique.
 Low Cost: Reagent and instrumentation costs are low when compared to many other analytical
techniques, such as gas chromatography and HPLC.
Limitations of Fluorometry:
 The extent of applicability of this technique is limited, because of the fact that all elements and
compounds are unable to exhibit fluorescence
 The method is not suited for determination of major constituents of a sample, because the accuracy is
very less for large amounts
 Careful buffering is necessary as fluorescence intensity may be strongly dependent
 The presence of dissolved oxygen may cause increased photochemical destruction
Selection criteria:
 Fluorometry only for fluorescence compound
 Conversion to fluorescence.
 Fluorometry low conc
 Electronic transition
 Chemistry
Fluorometry is more sensitive tool:
 Fluorometer directly measures intensity of light
 Measures at low con
 More sensitive
 More specific
 Amplifier is used

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☻Comparison of fluorometry with spectroscopy:
Fluorometry Spectrophotometry
More sensitive Less sensitive
Fluorometer directly measures intensity of light Cannot directly measures intensity of light
Intensity can be amplified Intensity cannot be amplified
More specific Less specific
Large numbers of variables can be controlled Small numbers of variables can be controlled
Temperature must be controlled Temperature don’t need to be controlled
Intensity of light maintain constant Intensity of light don’t maintain constant
Extraneous solutes affects Extraneous solutes don’t affects
Influence of pH is complex Influence of pH is not complex
☻Difference between absorption spectroscopy & fluorescent spectroscopy:
Features Absorption spectroscopy Fluoroscence spectroscopy
Theoretical consideration Measurement of amount of light
absorbed
Measurement of intensity of
fluorescence
Wavelength of light used Which gives maximum
absorption
Which gives maximum
fluorescence
Instruments Determines only the absorption
of light
Determines absorption of light as
well as emission of radiation
Light source Tungsten, H2-discharge lamp Mercury arc lamp, Xenon arc
lamp
Cell used Silica cell Glass and metal cells
Detector Phototube or photo multiplier is
used to detect the radiation
absorbed
Emission filter is used to separate
the emitted light from the
transmitted light
Concentration Concentration depends on the
molar absorptivity.
Concentration depends on the
characteristics of the instrument
Electrical transition Applicable for both π→π* &
n→π* transition.
Not applicable for the compound
containing n→π* transition
Experimental variables
(temperature & Extraneous solution)
Not so restricted Highly restricted
Sensitivity & selectivity Less sensitive & less specific. More sensitive & highly specific.
☻Differences between fluorescence & phosphorescence:
Property Fluorescence Phosphorescence
Transition Molecule transits from excited
singlet state to ground state.
Molecule transits from excited
triplet state to ground state
Lifespan Fluorescence is continued for only
10-8
to 10-4
seconds.
Phosphorescence continues for
10-4
seconds to 10-2
seconds.
Afterglow Not present. Occurs and luminescence slowly
fades.
Analytical application Yes No

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Fluorometry

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