PHARM 3235 Fluorometry

PHARM 3235
Md. Imran Nur Manik
Lecturer
Department of Pharmacy
Northern University Bangladesh

Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 1
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Fluorometry
Terminologies
Luminescence: 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 –
1. Fluorescence
2. 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. The extent of fluorescence can be
measured by fluorometry.
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
antibonding molecular orbital. If the excited electron in the antibonding orbital has
spin opposite to the electron present in the bonding orbital of ground sate, then the
excited state is called Excited singlet state.
Ground state Ground state
Excited state
Energy
What is excited singlet state?
At room temperature, in normal condition, molecules will be at ground state and bonding
electrons will spin in opposite directions. Energy level is lowest. Excitation requires energy
which must be supplied in the form of UV or visible light. Following light absorption, a
chromophore is excited to some higher vibrational energy level of S1 or S2. Then, due to
vibrational relaxation, the molecules will descend to the lowest vibrational energy level of the
excited state. This process is radiationless but energy is lost in other forms.
From the lowest vibrational level of excited state, molecules will return to the ground state by
emission of radiation. Since, little energy is lost during vibrational relaxation, the radiation
emitted has lower energy than the radiation absorbed. Hence, in fluorescence emitted radiation
has longer wavelength.
In fluorescent molecules, luminescence stops within 10-8
to 10-4
seconds. It is important to
remember that, the molecule will undergo vibrational relaxation to the lowest vibrational
energy level before returning to the ground state to give fluorescence. So
Md.
Imran
Nur
Manik

Fluorometry
Excited state
Ground state
Energy
Green arrows show
vibrational relaxation
Red arrows indicate
fluorescence
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.
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.
Excited singlet
state
Ground state
Energy
Green arrows show
Red arrows indicate
phosphorescence
Excited triplet
state
Singlet state: 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.
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.
Md.
Imran
Nur
Manik

Fluorometry
Triplet state: Triplet state is a state lying at an energy level intermediate between ground and excited
state and characterized by an impairing 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 seconds.
What is excited triplet state?
In the excited singlet state of a molecule, the electron in the excited state
and the electron the ground state spin in opposite direction i.e. they are
still paired. In some compounds, the molecule may convert from the lowest
vibrational level of excited state to a triplet state. In the triplet state, the
electron in excited state spins in the same direction as the electron in the
ground state.
Ground state Ground state
Excited state
Energy Triplet state
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.
Vibrational energy level:
Even at ground state a molecule is always vibrating. Therefore the energy at ground state is not a single
discreet value rather a set of discreet values. In another words there are different energy levels in the
molecule duo to its vibration.
Whether the molecule is in ground state or excited state, the molecule contains many energy levels
which are called vibrational energy levels.
Excited state
Ground state
Energy
There may be many Occupied Molecular Orbitals (bonding orbitals) and many Unoccupied
Molecular Orbitals (antibonding orbitals-electrons transit to these during excitation). Now,
when a molecule is in ground state, the electrons will not transit to the antibonding orbitals. But
they may transfer from one bonding orbital to another. This causes vibration of the molecules.
Again, when a molecule is in excited state, one electron from each pair of bonding electrons will
transit to the antibonding orbitals. When they are in these antibonding orbital, they may transfer
from one antibonding orbital to another.
Md.
Imran
Nur
Manik

Fluorometry
Vibrational relaxation: Vibrational relaxation is the transition of molecule from any of the vibrational
energy levels to the lowest vibrational energy level of the excitatory state.
Excited state
Ground state
Energy
Green arrows show
Energy lost in this process is thought to be via thermal process probably lost to solvent molecules.
Resonance fluorescence: Resonance fluorescence is the phenomenon where the molecule absorbs
and emits equal amount of energy. Practically resonance fluorescence doesn’t occur or occur rarely as
vibrational relaxation occurs.
Excited state
Ground state
Energy
Green arrows show
absorbed light
Red arrows show
emitted light
Internal conversion: The phenomenon of excited molecule to return to the ground state by losing
energy by means other than photo radiation is termed internal conversion.
Intersystem crossing: The transfer of a molecule present in the lowest vibrational energy level of the
excited singlet state to an excited triplet state is called intersystem crossing.
Differences between fluorescence and 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 seconds.
Afterglow Not present. Occurs and luminescence slowly fades.
Analytical
application
Yes. No.
Quantum efficiency: Quantum efficiency is defined as the ratio of number of light quanta emitted and
the number of light quanta absorbed.
absorbedlightofEnergy
emittedlightofEnergy
absorbedquantalightofNo.
emittedquantalightofNo.
orQ 
Its significance is that, it is an indicator of how fluorescent a molecule is. If Q is near 1, the molecule is
highly fluorescent molecule and if Q is near 0, the molecule is a very low fluorescent molecule.
Md.
Imran
Nur
Manik

Fluorometry
Fluorometry
The method of analysing a sample by measuring its fluorescence i.e. intensity and composition of light
emitted by it, is called fluorometry.
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 below –
Md.
Imran
Nur
Manik

Fluorometry
Excited singlet state
Excited triplet state
Ground state
Blue lines: Vibrational energy level within a state
Green lines: Vibrational relaxation
Sky blue line: Intersysten crossing
Purple lines: Internal conversion (radiationless)
Orange lines: Resonance fluorescence
Red line: Phosphorescence
Black line: Fluorescence
Theory of fluorometry
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.
Md.
Imran
Nur
Manik

Fluorometry
1. 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.
2. Radiationless vibrational relaxation to the lowest vibrational energy level of the excited singlet state:
Molecules initially undergo a more rapid process, a radiationless loss of vibrational energy and so
quickly falls to the lowest vibrational energy level of the excited state, known as vibrational relaxation.
3. Radiationless internal conversion (from excited singlet state to ground state followed by vibrational
relaxation): 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 (Followed by vibrational relaxation):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.
5. Intersystem crossing (From excited singlet state to excited triplet state): 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. Vibrational relaxation (to the lowest vibrational energy level of the excited triplet state): 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 seconds (Longer than corresponding singlet state).
Md.
Imran
Nur
Manik

Fluorometry
7. Radiationless internal conversion from excited triplet state to ground state followed by vibrational
relaxation: Here energy is released in the form of heat radiation.
8. Phosphorescence (Followed by vibrational relaxation). The emission of radiation emitted in the
transition of a molecule from a triplet excited state to a singlet ground state is called Phosphorescence.
It is characterized by afterglow because of the long life of the triplet state.
Relationship between fluorescence and chemical structure
Definite correlations between chemical structure and fluorescence can’t be made. But there is influence
of structural features on the fluorescence of organic compounds.
Degree of conjugation: Conjugation is necessary for fluorescence. This is because mobile π electrons 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.
Napthalene
(strongly fluorescent)
Benzene
(weakly
fluorescent)
Cyclohexane
(non-fluorescent)
Delocalization of electron: In mono-substituted benzene derivatives following rules can apply –
 Methyl and other alkyl groups have little effect on fluorescence intensity.
 Electron donating groups i.e. ortho-para directors increase fluorescence intensity as they
increase electron density (increase electron delocalization). E.g. fluoro, amino, hydroxy, methoxy
group etc.
 Exception: Halogen substitution specifically chlorine, bromine and iodine substitution
decreases/diminishes fluorescence by causing intersystem crossing. Thus they show
phosphorescence.
 Electron withdrawing groups i.e. meta directors decrease fluorescence intensity as they decrease
electron density (causes π electron localization). E.g. carboxyl, nitro, sulfonyl, aldehyde group etc.
Exception: Nitrile group even though meta directing, increases fluorescence intensity.
It was postulated that electrons of the CN group interacted with the π electrons of the benzene ring to result in a
distribution that favoured fluorescence.
In di-substituted benzene derivatives fluorescence is
unpredictable. For example, aniline is a fluorescent
compound. When a meta directing group e.g.
sulfamoyl group is added (then the compound is
sulfanilamide) fluorescent intensity increases 5 times.
Although it may be expected that substitution of the fluorescent compound, aniline, with a meta-directing group —
SO2NH2 would result in a compound which would fluoresce to a lesser degree than aniline.But Sulfanilamide, however,
was found to be five times as fluorescent as aniline.
Compound Fluorescence compare to benzene
Higher Lower
Benzaldehyde 
Chlorobenzene 
Aniline 
Nitrobenzene 
Benzoic acid 
Phenol
Md.
Imran
Nur
Manik

Fluorometry
 Molecular geometry:
Rigidity and planarity: 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.
(Fluorescein , is highly fluorescent, while phenolphthalein is nonfluorescent. The oxygen bridge in fluorescein imparts rigidity and
planarity that is not present in phenolphthalein. the vibrational energy is greater.)
OO O
COO
O
COO
O
Fluorescin
(strongly fluorescent)
Phenolphthalein
(non-fluorescent)
cis-trans isomerism: It also affects fluorescence intensity. Generally trans isomers have greater
fluorescence than corresponding cis isomers. This is due to non-planar character of cis isomers.
CH
HC
HC
HC
cis-stilbenetrans-stilbene
Heterocylic compounds:
N
H
O S
Decreased fluorescence
intensity
Pyrrole
N
2H-PyrroleFuran Thiophene
Increased fluorescence intensity
A double-bonded nitrogen (=N—) generally decrease the fluorescence intensity but  S,O,NH
generally increase the fluorescence intensity.
Ionization: Many compounds show fluorescence at ionized state. But this is dependent upon pH of the
solution.
Complexation: 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.
Tetracycline→ complexes
(non fluorescent) (fluorescent)
Md.
Imran
Nur
Manik

Fluorometry
Chemical conversion
Acid treatment-
Hydrocortisone is not fluorescent itself but they from strongly fluorescence compound in concentrated
in the prescence of ethanol.
Hydrocortisone → strongly fluorescent compound.
Oxidation
By oxidation and hydroxylation epinephrine forms strongly fluorescing compound.
Epinephrine→ Highly fluorescent compound.
Thiamine is not itself fluorescent ,but it`s oxidation product thiochrome is fluorescent.
Thiamine → Thiochorme
Instrumentation
In fluorometry the intensity of radiation emitted as fluorescence related to the concentration of the
fluorescing species is measured. The instrumentation is for measuring the intensity of fluorescence as a
function of the wavelength of the radiation.
The chief components are:
a) Light source
b) Filter (Primary Filter) /monochromater
c) Sample holder
d) A emission filter (Secondary Filter) / emission
monochromater
e) Detector
f) Recorder and Amplifier
Md.
Imran
Nur
Manik

Fluorometry
Radiation source:
To produce exciting light, radiation source is required. The radiation source must be intense and stable.
Mercury arc and Xenon arc lamp are commonly used.
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.
Md.
Imran
Nur
Manik

Fluorometry
Excitation filter:
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 holder:
Glass cells are used for most analysis. If measurement is to be under 320nm wavelength then quartz
cells are used.
Emission filter:
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.
Detector:
A photomultiplier and phototube is used to detect the fluorescent light and amplify it.
(The detector is placed at a right angle to the direction of travel of beam of exciting light.)
Recorder:
The output of the detector is connected to a meter, a digital display or a recorder. Recorder gives the
intensity of radiation in terms of electrical signal produced by the detector.
Factors influencing intensity of fluorescence
1. Concentration of fluorescing species
2. Presence of other solutes or impurities
3. pH of the sample solution
4. Stability of the sample compound
5. Solvent effects
6. Temperature
Mirror image rule
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.
Md.
Imran
Nur
Manik

Fluorometry
1. Concentration of fluorescing species:
Fluorescence intensity (F) can be described as follows –
1).........(I)-(IkF
I)-(IF
So,
I)-(IF
and,
F
0
0
0







constantalityProportionk
lightedtransmitttheofIntensityI
lightincidenttheofIntensityI
efficiencyQuantum
intensityceFluorescenF
Here,
0






The exponential form of the Beer’s law (Beer-Lambert law) is
).(2..........eII εbc
0


solutionincompoundsampleofionConcentratc
lengthpathb
lightincidentofhwavelengtat thecompoundoftyabsorptiviMolar
Here,



By putting the value of I from equation (2) in equation (1) we get –
Thus we can see that the relationship between fluorescence intensity and concentration is quite
complex. But from the above equation,
When c increases,
εbc
e
1
value decreases and thus F value increases. So we can say that fluorescence
will increase with increase of concentration of fluorescing species.
lower conc.
Medium conc.
High conc.
Conc.
F
Sharp change
Sharpness decreases
Practically constant
Concentration reversal:
Concentration reversal is the phenomenon where the fluorescence intensity decreases as a result of
increase in concentration.
For some chemical species, if the supplied energy is fixed but the concentration is increased gradually
then at one point the fluorescence will decrease. This is because; the supplied energy fails to excite all
the molecules present in the solution at a time. So when the excited molecules emit energy (this is the
fluorescence), the previously unexcited molecules will absorb that energy. The emitted energy measured
is less i.e. the fluorescence intensity is less.


 
-εbc
0 0
-εbc
0
εbc
F = k (I - I e )
F = k I (1- e )
1
F (1 - )
e
Md.
Imran
Nur
Manik

Fluorometry
Conc.
F
Figure: Conc. reversal
For example the supplied energy can excite 20 molecules. If 25 molecules are present in the
solution then, the energy will excite 20 molecules and 5 molecules will remain unexcited. When the
excited molecules emit energy the unexcited molecules will absorb that energy.
However, if the sample is concentrated, sufficient light absorption might occur so that the portion sensed by
the detector is only weakly irradiated. This results in the phenomenon of concentration reversal.
2. Presence of other solutes/impurities:
A. Fluorescent impurities:
The sample solution may contain components other than the sample which is fluorescent. These
interfere with accurate measurement of fluorescence of the sample compound. Thus precautions such
as use of pure solvent and chemical reagents, cleanliness in all operations should be taken.
B. Inner-filter effect:
It is the reduction in the fluorescence intensity due to presence of non-fluorescent solutes which retard
penetration of light to or from fluorescent molecules.
Non-fluorescent molecules either prevent incident light from reaching the fluorescent molecules
(absorption retardation) or prevent emitted light detection.
Remedy: the non-fluorescent absorber must be eliminated or be maintained constant from sample to sample and a standard curve must
be used which was determined at that concentration of absorber. Or, the wavelength of excitation or emission radiation to minimize
this effect.
C. Chemical quenching:
Chemical quenching is the decrease of fluorescence intensity due to presence of any chemical in the
sample solution. i.e.
It is a chemical process where a chemical species reduce fluorescence intensity.
The chemical responsible for quenching is called quencher.
There are two types of quenching –
a. Collisional quenching: When the quencher absorbs the energy emitted by the excited fluorescent
molecules, it is called collisional quenching. Halide ions e.g. iodide, chloride ions cause it.
A molecule of “quencher" interacts with an excited molecule of the potentially fluorescing substance. Interaction results in the
dissipation of excitation energy not by fluorescence but by transfer of energy to the quenching molecule.
b. Static quenching: When the quencher absorbs the incident light in place of fluorescent
molecules, it is called static quenching. Xanthines (caffeine) and purines cause it for vitamin B12.
Md.
Imran
Nur
Manik

Fluorometry
F
(ground state)
F*
(excited state)
F + Fluorescence
Quencher, Q
Q + F* Q* + F
Quencher, Q
Q* + F
Static quenching
Collisional quenching
Supplied energy
(incident light)
(Transmitted light)
+
3. pH of the sample solution:
Intensity of fluorescence is dependent upon the pH of the solution. This is due to two reasons –
A. Degree of ionization: In weak electrolytes pH affect the degree of ionization. Now, ionized and
unionized species may have different fluorescence intensity. Again, it is possible that ionized
species is fluorescent but the unionized species is not and vice versa. Thus pH may affect the
fluorescence intensity of a compound.
Exemplary, 2-naphthol ( 5.9apK ) shows fluorescence in both ionized and unionized forms. But
ionized form give fluorescence peak at 429µm whereas unionized form gives fluorescence peak at
359µm. So, if we measure fluorescence at 429µm (actually a filter is used to omit radiation below
415µm) then only the fluorescence of ionized species can be detected; this is detected at pH 8.5 and
above [Degree of ionization is detectable at pH equal to 1)(pKa  ].
B. Excited-state dissociation: Sometimes, it is possible that a compound has different acid strength
in ground state and in excited state. So, if the excited state acid strength is greater, then the
compound will dissociate more easily when in excited state. Then, difference in the fluorescence
intensities of ionized and unionized species will cause change in fluorescence.
Exemplary, when fluorescence is measured at 429µm (a filter is used to omit radiation of below 415µm)
fluorescence is detected in the pH range of 2-8.5. But ground state 2-naphthol undergoes detectable
ionization at pH 8.5 and above. Excited state 2-naphthol underwent ionization in the pH range 2-8.5,
which is why we get fluorescence in that range.
4. Stability of the sample compound (Degradation of Sample)
If the compound being analysed is unstable in the experimental condition then fluorescence intensity will
change. Causes of instability may be due to –
 Solvolytic degradation
 Auto oxidation
 Photo decomposition
 Chemical degradation
Photodecomposition can be reduced by decreasing the intensity of the incident light. Also all
measurements should be completed as quickly as possible to avoid above problems.
Md.
Imran
Nur
Manik

Fluorometry
5. Solvent effect:
 Presence of impurities
 Fluorescing impurities
 Quenching impurities e.g. oxygen
 Polarization effect
 Quenching effect
 Hydrogen bonding with analyte compound
6. Temperature:
Temperature reduces the fluorescence intensity. This is because of –
A. Increased internal conversion
Increased
temperature
Thermal motion of
molecules increased
Chances of intermolecular
collision is increased
Internal
conversion
Radiation
decreased
Fluorescence
decreases
B. Reduction in vibrational relaxation
In general, a 1⁰C rise in temperature results in a decrease of fluorescence intensity by 1%.
Comparison of Fluorometry with spectrophotometry
Sensitivity: Fluorometry is significantly more sensitive as an analytical tool than spectrophotometry.
The points included are.
In fuorometry the intensity of fluoresced light is measured directly by a fluorometer.
In spectrophotometry the intensity of light transmitted by a sample is measured and compared to that
transmitted by a blank.
The directly measured intensity can be amplified more readily and accurately in fuorometry than
the intensity difference measured in spectrophotometry.
In case of spectrophotometry the lower limit of detectability is determined by the smallest
concentration that will yield a detectable intensity difference between sample and blank. Here
small errors made in measuring the difference between the two intensities result in large errors
in calculated concentration.
The lowest limit of conc. that can be detected with accuracy is established by the molar
absorptivity in spectrophotometry (12mg/dl). The lower limit of conc. in fluorometry is
established by characteristics of the instrument and not usually by characteristics of the following
species.
Fluorescence measurements can offers sensitivity increases of 103-104 over absorbance
measurements.
Specificity:
Fluorometric assay can offer a degree of specificity that might not be attainable with a corresponding
spectrophotometric technique. The equations relating fluorescence intensity to concentration hold for
any region of the spectrum.
Md.
Imran
Nur
Manik

Fluorometry
Experimental Variables:
There are large no of experimental variable that must be controlled in fluorometric methods of analysis
than in corresponding spectrophotometric methods.
The temperature and the intensity of incident light must be maintained reasonably constant in a
fluorometric method, but not in a spectrophotometric procedure.
Extraneous solutes can markedly affect the intensity of fluorescence by quenching affects, but it
does not happen in spectrophotometry.
The influence of pH on fluorescence can be more complex than on absorbance and might
necessitate closer control of pH in fluorometric procedures than in spectrophotometric assays.
Difference between Absorption spectroscopy and Fluoroscence 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 and
selectivity
Less sensitive and less specific. More sensitive and highly specific.
Applications of fluorometry
Application in Chemistry:
Fluorometry is used in chemistry for –
1. Determination of metal ions: Complexes of metals ions may give strong fluorescence which is
utilized for this purpose.
2. Separation and identification: In many cases, after separation, chemicals are identified using
fluorometry. e.g. aminocrine.
Md.
Imran
Nur
Manik

Fluorometry
Application in Biopharmaceutics:
1. Measurement of drug in blood, urine and other body fluids.
2. Study of the rate and mechanism of drug absorption, metabolism and excretion.
3. Selection of toxic compounds.
Pharmaceutical applications:
Fluorometry is used for quantitative analysis of –
1. Hormones: Adrenaline, aldosterone, testosterone
2. Alkaloids:
a. Opioids: Morphine, codeine etc.
b. Rauwolfia alkaloids: Reserpine
c. Others: Atropine, emetine etc.
3. Vitamin: Riboflavin and thiamine are indicated for fluorometric assay by USP and BP.
4. Antibiotics: tetracycline, sulfonamide etc.
5. Cardiac glycosides: Such as digoxin, digitoxin, etc.
Fluorometry is also used for qualitative analysis of these drugs.
6. 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
1. Sensitivity: In case of Fluorescence, detectability to parts per billion or even parts per trillion is
common for most analytes. This extraordinary sensitivity allows the reliable detection of fluorescent
materials (chlorophyll, aromatic hydrocarbons, etc.) using small sample sizes. Also, field studies can be
performed in open waters without sample treatment. Fluorometers achieve 1,000 to 500,000 times
better limits of detection as compared to spectrophotometers.
2. Specificity: Spectrophotometers merely measure absorbed light and as many materials absorb light, it
becomes difficult to isolate the targeted analyte in a complex matrix. Fluorometers are highly specific
and less susceptible to interferences because fewer materials absorb and also emit light (fluoresce).
And, if non-target compounds do absorb and emit light, it is rare that they will emit the same wavelength of light as
target compounds.
3. Wide Concentration Range: Fluorescence output is linear to sample concentration over a very broad
range. Fluorometry can be used over three to six decades of concentration without sample dilution or
modification of the sample cell.
6. Simplicity and Speed: Fluorometry is a relatively simple analytical technique. Fluorometry's sensitivity
and specificity reduce or eliminate the sample preparation procedures often required to concentrate
analytes or remove interferences from samples prior to analysis. This reduction in or elimination of
sample preparation time not only simplifies, but also expedites the analysis.
7 Low Cost: Reagent and instrumentation costs are low when compared to many other analytical
techniques, such as gas chromatography and HPLC.
Reagent costs are low because, due to the high sensitivity of fluorometers, fewer reagents can be used. And,
small laboratory filter fluorometers can now be purchased for less than $3,000 USD.
Md.
Imran
Nur
Manik

Fluorometry
Limitations of Fluorometry
1. Molecules should be fluorescent to measure by the fluorescence spectroscopy.
2. All kind of materials on substances cannot be detected by it.
References
Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. 3 Id., p. 7.
Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular
Biology, School of Medicine.
G. K. Turner, "Measurement of Light From Chemical or Biochemical Reactions," in Bioluminescence
and Chemiluminescence: Instruments and Applications, Vol. I, K. Van Dyke, Ed. (CRC Press, Boca
Raton, FL, 1985), pp. 45-47.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 51-57.
Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, chap. 2.
Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, pp. 23-26.
Stotlar, S. C. 1997. The Photonics Design and Applications Handbook, 43rd Edition, Laurin Publishing
Co., Inc., Pittsfield, MA, p. 119.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 63.
Iain Johnson, Product Manager, and Ian Clements, Technical Assistant Specialist (May 1998
communication from Molecular Probes, Eugene, Oregon).
Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), pp. 14-15.
Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), p. 21.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York., p. 28.
Teitz Textbook of Clinical Chemistry and Molecular diagnosis (5th Edition)
Dr.B.K.Sharma, Instrumental methods of chemical analysis.
Gurdeep R Chatwal, Instrumental methods of chemical analysis
http://en.wikipedia.org/wiki/Fluorescence
http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence
http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm
Md.
Imran
Nur
Manik

PHARM 3235 Fluorometry

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to PHARM 3235 Fluorometry

Similar to PHARM 3235 Fluorometry (20)

More from Imran Nur Manik

More from Imran Nur Manik (20)

Recently uploaded

Recently uploaded (20)

PHARM 3235 Fluorometry