Spectrum and Spectroscopy:

Spectrum: Different colors observed when the white

light dispersed through the prism. The changing of light
intensity as a function of frequency.

Spectroscopy: Spectroscopy is the study of the

absorption and emission of light and other radiation by
matter. It involves the splitting of light (or more
precisely, electromagnetic radiation) into its constituent
wavelengths (a spectrum), which is done in much the
same way as a prism splits light into a rainbow of colors.
Spectroscopy:
Types of spectroscopy:
1. Emission spectroscopy
2. Absorption spectroscopy
Absorption spectroscopy:
The absorption spectroscopy means that we observe the spectrum of the light right after it go
through the substance, (look at the pic.) black line means the photons which have those specific
kind of energy were absorbed by the substance; emission spectrum means the range of
electromagnetic spectra in which a substance radiates (emits). The substance first must absorb
energy. This energy can be from a variety of sources.
 Emission spectroscopy:
When the electrons in the element are excited, they jump to higher energy levels, however, in both
the atomic and molecular cases, the excited states do not persist.
After some random amount of time, the atoms and molecules revert back to their original, lower
energy state. In atoms, the excited electron returns to a lower orbital, emitting a photon.
In molecules, the vibrational or rotational mode decays, also emitting a photon.
As the electrons fall back down, and leave the excited state, energy is re-emitted, the wavelength of
which refers to the discrete lines of the emission spectrum
 Electrons ground level
Energy emission

Absorb energy

High energy level

Fig: Excitation and emission of electrons

 Jablonski Diagram:
Aleksander Jablonski was a Polish academic. He developed a
written representation that generally shows a portion of the
possible consequences of applying photons from the visible
spectrum of light to a particular molecule. These schematics are
referred to as Jablonski diagrams.
Every column usually represents a specific spin multiplicity for a
particular species.
Horizontal lines represent eigenstates for that particular
molecule.
Bold horizontal lines are representations of the limits of
electronic energy states.
Absorbance: The first transition in most Jablonski diagrams is the absorbance of a photon of a
particular energy by the molecule of interest. This is indicated by a straight arrow pointing up.
Absorbance is the method by which an electron is excited from a lower energy level to a higher
energy level. The energy of the photon is transferred to the particular electron. That electron then
transitions to a different eigenstate corresponding to the amount of energy transferred. Only certain
wavelengths of light are possible for absorbance, that is, wavelengths that have energies that
correspond to the energy difference between two different eigenstates of the particular molecule

Internal conversion: If vibrational energy levels strongly overlap electronic energy levels, a
possibility exists that the excited electron can transition from a vibration level in one electronic state
to another vibration level in a lower electronic state. This process is called internal conversion and
mechanistically is identical to vibrational relaxation. InternalConversion occurs because of the
overlap of vibrational and electronic energy states. As energies increase, the manifold of vibrational
and electronic eigenstates becomes ever closer distributed.

Fluorescence:Another pathway for molecules to deal with energy received from photons is to emit a
photon. This is termed Fluorescence. Fluorescence is a slow process on the order of 10-­9 to 10-­7
seconds;; therefore, it is not a very likely path for an electron to dissipate energy especially at
electronic energy states higher than the first excited state. While this transition is slow, it is an
allowed transition with the electron staying in the same multiplicity manifold. Fluorescence is most
often observed between the first excited electron state and the ground state for any particular
molecule because at higher energies it is more likely that energy will be dissipated through internal
conversion and vibrational relaxation.
Transition Time Scale Radiative Process?

Absorption s yes

Internal Conversion s-­s no

Vibrational Relaxation s-­s no

Fluorescence s-­s yes

Intersystem Crossing s-­s no

Figure given is a classic representation of the electronic levels of a molecule in solution or in gas phase (in solid
phase the energy levels collapse into “bands” although the basic concepts are still valid). The energy levels occupied
by an electron are named “singlet states” and the letters 0 S , 1 S , 2 S , ..., indicate the ground state, the first excited
state, etc.; upon absorption of a photon, an electron moves from the ground state 0 S to the excited states. Associated
with each electronic level, there are several vibrational and rotational levels, which differ in energy by a smaller
amount than the corresponding electronic levels.
Moreover, there are energy transitions that are not directly allowed (forbidden transitions). They are identified as
“triplet states” and indicated by 1 T , 2 T , …, etc.; they also feature associated vibrational and rotational levels. The
absorption probability of a photon in each electronic level is described within the framework of quantum mechanics
(energy separation between the levels, momentum and spin of the various levels).
The molecules interact when in presence of photons of the appropriate photon energy E , where,

In the relation, is the Planck constant (6.626 J s), is the speed of light (2.9979 m/s) while and are the frequency and
wavelength of the electromagnetic wave describing the photon.
For absorption to occur, E has to be of the order of magnitude of the separation between the excited level and the
ground state; that is,
The absorption process takes an amount of time of the order of the femtosecond or shorter. Once in the
excited electronic level, the molecules relax fairly rapidly (about s) to the lowest level of the first excited
state 1 S ; hence, they decay with rate R k to emit fluorescence. The characteristic time of the fluorescence is
of the order of one nanosecond.
The Perrin-Jablonski diagram (Figure 1) is instrumental to determine the law describing the decay time of
fluorescence. If is the population of the excited level , upon absorption of photons the population of the
level changes are described by the relation:

where 1 f is a function that describes the process of the excitation photons (pulsed source, continuous
wave source, etc.). By solving the equation (and disregarding 1 f ), we find the

where , the decay time of the excited state is defined as:

 The fluorescence quantum yield is the fraction of excited molecules that return to the ground state with
the emission of fluorescence. From direct examination of the Perrin-Jablonsky diagram, one simply
divides the rate of radiative emission by the total rates of deactivation, which includes both the radiative
and non-radiative contributions:

By using the definition of decay times, the quantum yield can also be expressed in terms of lifetimes:

One can say that the quantum yield is the ratio of the number of emitted photons over the total number of absorbed photons.
 Luminescence:
Luminescence is emission of light by a substance not resulting from heat is thus a form of cold body
radiation. It can be caused by chemical reactions, electrical energy, subatomic motions, or stress on a
crystal. There are two pre-requisites for luminescence:

 The luminescent material must have a semiconductor structure with a nonzero band gap.
[Metals do not provide luminescence if they have no band gap].
 The energy must be imparted to this material before luminescence can take place.
 Types of luminescence
a) By Mechanism:
Fluorescence Fluorescence and phosphorescence are
photon emission processes that occur during
Phosphorescence molecular relaxation from electronic excited
b) By Excitation Source: states. These photonic processes involve
transitions between electronic and
Chemiluminescence Lyoluminescence vibrational states of polyatomic fluorescent
Candoluminescence molecules (fluorophores). Fluorophores play
Cathodoluminescence
the central role in fluorescence spectroscopy.
Crystalloluminescence Fluorophores are the components in
Electroluminescence
Piezoluminescence molecules that cause them to fluorescence
Photoluminescence
Sonoluminescence
Bioluminescence
Radioluminescence
Electrochemiluminescence
Thermoluminescence
Fluorescence Spectroscopy
Fluorescence spectroscopy is a sensitive optical emission
technique in which sample molecules are excited with a
photon source. Those molecules that relax by radiant
emission can be subsequently detected by measuring the
intensity of that emission.
PRINCIPLE:
Absorption of UV or visible radiation causes transition of electrons
from singlet ground state to the singlet excited state. As this state is
not stable, it emits energy in the form of UV or visible radiation and
returns to singlet ground state. Fluorescence emission occurs as the
fluorophore decay from the singlet electronic excited states to an
allowable vibrational level in the electronic ground state.
 Instrumentation:
A. Source of light
 Mercury vapour lamp
 Xenon arc lamp
 Tungsten film
B. Filters and monochromators
 Primary filters and secondary filters
 Excitation monochromators and Emission
monochromators
C. Sample cells, Detectors
Light Source:
The typical light source utilized in a spectrofluorometer is a high-pressure xenon arc lamp. The
bulb of this lamp includes xenon at high pressure that is excited to higher level by the electrical
arc established by the current running through the electrodes. The emitted light is a continuous
spectrum from (depending upon the models and geometries) about 250 nm up to 1100 nm.
In the past several years lasers have replaced the xenon arc lamp, specifically for time-resolved
applications. Although they emit radiation only at specific wavelengths, their brightness is order
of magnitude higher than that of the lamp. A recent advancement is the supercontinuum laser (or
white laser) that delivers any wavelength in the range from 390 nm up to 2000 nm.
Light emitting diodes (LEDs) are also utilized as light sources especially in the region from 240
nm to 350 nm, where lasers are not available (with exceptions at 266 nm, 315 nm, 325 nm). They
are compact, relatively inexpensive and the source of choice when building an instrument
dedicated to a specific application.
 Monochromator:
Monochromators are utilized to select the wavelength used for irradiating the sample when using a
xenon arc lamp; in the collection channel of a spectrofluorometer they are utilized to record the range
of wavelengths emitted by a fluorophore (emission spectrum, see below). The simplest
monochromator includes a diffraction grating and slits at the entrance and at the output.
It is important to realize that the transmission of the light traversing a monochromator is affected by
two parameters:
1. the wavelength; the grating has a specific transmission curve and some wavelengths are
transmitted with a higher efficiency than other wavelengths, a feature to remember when
collecting excitation and emission spectra.
2. the polarization status of the radiation; the grating of the monochromator transmits differently
radiation with different planes of polarization.
 Light Detectors:
In all the instruments the fluorescence signal is converted into current by a photomultiplier tube
(PMT), or photodiode (instruments for lifetime measurements may utilize other types of
detectors too, such as hybrid PMTs, microchannel plate detectors or streak cameras).
Photomultiplier tubes are sensitive within a set wavelength range that is determined by the
material used in the photocathode.
A spectrofluorometer includes other optical elements such as lenses and
mirrors; moreover polarizers are utilized for anisotropy measurements. The
operational region of the instrument is given by the superposition of the
wavelength transmission of the various elements of the instruments. Even
within this region, the variation in transmission has to be taken into account
when measuring the fluorescence parameters. The procedures will be outlined
in the measurements sections below. Figure 4 displays the technical diagram
of the K2 Multifrequency Phase Fluorometer made by ISS, an instrument
capable of measuring all of the relevant fluorescence parameters. The standard
light source is a 300 W xenon arc lamp. Continuous wave (cw) lasers, pulsed
lasers (including the multi-photon laser) and light emitting diodes (LEDs) can
be coupled to the K2 as well; typically these sources are utilized for the
measurement of the decay times of fluorescence.

The light emitted by the source travels through the excitation channel that
comprises the monochromator, a filter holder and the polarizer holder; the
monochromator selects the wavelength of the light that excites the sample.
The fluorescence emitted by the sample is collected through the left or the
right channels; the right channel includes the emission monochromator. The
instrument includes polarizers’ holders, filters holders, shutters for blocking Figure: PC1 Photon Counting Spectrofluorometer
the light from reaching the sample and the detectors. All of these components (courtesy of ISS)
are required for automated measurement acquisition.
 Measurements:
Excitation Spectrum:
•The excitation spectrum displays the emission intensity distribution at one wavelength while scanning the
excitation wavelength over a range.
•Practically, for the acquisition of the excitation spectrum, the emission monochromator of the spectrofluorometer is
set at a fixed wavelength (in the sample emission range) and the excitation monochromator is scanned over a range
of wavelengths (the range that corresponds to the sample absorption range).
•Referring to the Jablonsky-Perrin diagram of Figure, when acquiring the excitation spectrum one detects photons
emitted by the molecules at a set wavelength (represented by one of the green lines), while scanning the
wavelength of the radiation (energy of photons) sent to the sample from high energy to low energy (blue lines).
•If there are no changes occur to the molecule in the excited state, then the excitation spectrum closely resembles
the absorption spectrum acquired with a spectrophotometer, yet, in most instances, it does not: in order for the two
to match, a suitable correction of the instrumental factors has to be applied. The main culprit of the differences is
due to the lamp; it features a peculiar emission spectrum of its own, that is, the intensity of the emitted radiation is
not constant at all the wavelengths.
•In order to correct for this effect, a small fraction of the excitation light is diverted in the
Reference channel of the spectrofluorometer where it passes through the quantum counter and it
is collected by the reference detector.
•The quantum counter, usually a stable fluorophore at a high concentration in solution, delivers a
number of photons proportional to the absorbed signal; therefore, at each wavelength, we have a
signal proportional to the signal emitted by the lamp; this signal is utilized to correct the
fluorescence signal collected in the emission channel.
•Although this correction addresses most of the concerns, it does not completely correct the
excitation spectrum as the beam splitter utilized to divert part of the excitation light into the
reference channel reflects differently the two planes of polarization.
•For a full correction to be implemented, one should place a cuvette with a scattering solution in
the sample compartment and acquire an emission spectrum over the wavelength range of interest;
then acquire the emission spectrum of the fluorophore and divide it by the spectrum of the
scatterer.
•In this way, the excitation spectrum is fully corrected. Practically, the correction introduced by
using the quantum counter and the reference channel is sufficient; one should nonetheless specify
the experimental conditions when publishing the spectrum
Figure: Excitation spectrum of
Rose Bengal in a water solution,
acquired using the PC1 Photon
Counting Spectrofluorometer
(courtesy of ISS). The spectrum
was acquired by scanning the
excitation monochromator from
400 nm to 600 nm in steps of 1
nm; at each position data were
acquired for 1 second. The
fluorescence was observed at
610 nm.
Emission spectrum:
The emission spectrum of a fluorophore is most likely the most popular experimental measurement carried out in
fluorescence. The spectrum is acquired by setting the excitation wavelength at a fixed value (one of the blue lines of Figure)
and then by scanning the emission monochromator over a range of emission wavelengths (the green lines of Figure ).
There are a few general rules that apply to emission spectra:
a) The emission of fluorescence occurs at wavelengths longer than the excitation wavelength (Stokes shift) .
b) The shape of the emission spectrum does not change by changing the excitation wavelength.
c) The emission spectrum is a mirror image of the excitation spectrum of lower energy.

Figure: Emission spectrum of Rose Bengal water

solution, acquired using the PC1 Photon Counting
Spectrofluorometer (courtesy of ISS). The excitation
monochromator was set at 490 nm. The emission
spectrum was acquired by scanning the emission
monochromator from 500 nm to 700 nm in steps of 1
nm; at each position data were acquired for 1
second.
• As for the excitation spectrum, the emission spectrum is affected by experimental artifacts, namely, the transmission of
the emission monochromator and the sensitivity of the light detector: The transmission of the monochromator varies
with the wavelengths and, moreover, it features different transmission for the two planes of polarization of the light (see
below for the definition of light polarization); the sensitivity of the light detector varies with the wavelength.
• All these variation have to be accounted for in order to acquire a “true’ emission spectrum. To this respect, one
distinguishes between technical spectrum (the spectrum acquired by an instrument) and the corrected spectrum (the
technical spectrum that has been corrected for the experimental artifacts).
• Manufacturers typically provide correction files for an instrument; these factors are embedded in the software and
corrected spectra can be acquired on line; or spectra can be corrected afterwards.
• Practically, one does not need to correct a spectrum unless it is meant for publications; even in that event, it is
completely acceptable to specify that the spectrum is a technical spectrum rather than a corrected one.
• There are some instances when corrected spectra are required; when calculating the quantum yield of a fluorophore one
has to calculate the area under the spectrum; the spectrum has to be corrected for providing the proper value.
Calculation of excitation and emission
wavelength:
The fluorescence emitted can be mathematically represented as follows;
  For a dilute solution with only 1 fluorophore (defined as a molecule/compound that exhibits fluorescence) the
fluorescence intensity is a function of both the excitation and the emission wavelength and can be written as;
                          

The fluorescence quantum yield Q is a measure of the proportion of molecules emitting fluorescence as compared to the total number of molecules excited.
Fluorescence intensity may be measured as a function of the excitation or emission wavelength, the two variables in the above equation.
• The excitation spectrum depicts the fluorescence intensity as a function of the excitation wavelength
for a fixed emission wavelength.

Since Q(lem) is a constant, the excitation spectrum looks like the absorption spectrum for that fluorophore.

• The emission spectrum depicts the fluorescence intensity as a function of the emission wavelength for a fixed
excitation wavelength.

                                 
Since the light absorption term is a constant, the emission spectrum represents the quantum yield for that fluorophore.
• Excitation emission matrices (EEMs) are assembled from a series of fluorescence emission spectra collected at
sequential excitation wavelengths.

This data results in a matrix where the first row consists of the excitation wavelengths, the first column consists of the
emission wavelength and the rest of the matrix contains the fluorescence intensities as shown below

          
EEMs form surface plots that are usually presented as contour plots where each contour line connects points of equal
fluorescence intensity (similar to isobar and isothermal charts).
Emission spectra may vary depending on the chemistry of the fluorophore and the environment. The position of the fluorescence maxima
is dependent on the environment and the dynamics of the fluorophore. A turbid sample such as tissue contains not only multiple
fluorophores which may or may not interact with each other but also contain scatterers and absorbers. Thus an EEM of such a material,
shows the fluorescence excitation emission maximum of each fluorophore present altered from its pure state by its environment. Any
scatterers present typically manifest themselves in a broad band attenuation of the collected spectrum. The presence of an absorber is
indicated by valleys at both excitation and emission wavelengths at which it absorbs. Thus, fluorescence gives a measure of the
composition as well as environment of the sample of interest.
Decay times of fluorescence:
Fluorescence lifetime (FLT) or Fluorescence Decay time (FDT) is the time a fluorophore spends in the excited state before
emitting a photon and returning to the ground state. FDT can vary from picoseconds to hundreds of
nanoseconds depending on the fluorophore.

The decay time is affected by many parameters of the microenvironment (temperature, ions, polarity,
viscosity, electric fields) and this is the reason it is widely utilized for studying molecular interactions.
the lifetimes can be used an analytical tool as well for the characterization of the presence of specific dyes or
simply for the quantitation of complex fluorescent mixtures (the type of crude oil provided by a well, the dye
in a hair spray or a soap, the production process of paper, counterfeiting of banknotes and of drugs, etc.)

Fig: Decay
fluorescence
curve
Quantum yield:
The fluorescence quantum yield is the ratio of photons absorbed to photons emitted through fluorescence. In other words
the quantum yield gives the probability of the excited state being deactivated by fluorescence rather than by another,
non-radiative mechanism.
Anisotropy and Polarization:
Anisotropy (or polarization) is a popular application of fluorescence spectroscopy as it allows for the measurement of the
rotation of molecules as well as of their shape and size and the rigidity of molecular structures.
Polarized light can be utilized for interesting experiments and applications. When polarized light with the proper energy
illuminates an ensemble of molecules, only molecules with the excited state dipole moment (or transition moment)
oriented in the same direction of the electrical field (polarization) can absorb the photons.

 If the direction of polarization of the excited beam and the direction of the dipole
moment of the molecule are perpendicular to each other, no absorption takes
place. In intermediate cases, the probability of the absorption is proportional to 2
cos  , where  is the angle between the vector E of the exciting light and the
vector M of the transition moment dipole

 Because of the preferential absorption rules of the molecules, a polarized light

introduces a photoselection of the molecules. As the distribution of the excited
fluorophores is anisotropic, the fluorescence is anisotropic too. Any change in the
direction of the transition moment during the time the molecule spend in the
excited level will result in a decrease of the anisotropy, that is the overall
polarization of the fluorophores solution will decrease.
Fig: Molecules with the electric dipole
featuring a component parallel to the
direction of the electric filed of the
excitation light have a probability for
absorption of a photon
CIE Graph:
The CIE color model is a color space model created by the
International Commission on Illumination known as the Commission
Internationale de l’Elcairage (CIE).

The CIE system characterizes colors by a luminance parameter Y and

two color coordinates x and y which specify the point on
the chromaticity diagram. This system offers more precision in color
measurement than do the Munsell and Ostwald systems because the
parameters are based on the spectral power distribution (SPD) of the
light emitted from a colored object and are factored by sensitivity
curves which have been measured for the human eye.

On the CIE chromaticity diagram at left, some annotation is made

about the significance of different parts of the diagram. The boundary
represents maximum saturation for the spectral colors, and the diagram
forms the boundary of all perceivable hues.
Calculation of CIE Chromaticity Coordinates

The calculation of the CIE chromaticity

coordinates for a given colored object
requires the multiplication of its spectral
power at each wavelength times the
weighting factor from each of the
three color matching functions. Summing
these contributions gives three values called
the tristimulus values, from which the
chromaticity coordinates are derived.
 How to draw CIE graph:
INSTALLATION
Download ChromaticityDiagram.opx file, and then drag-and-drop onto the Origin workspace. An icon will appear in the Apps Gallery window.

OPERATION
With a worksheet or a graph active, click the app icon. A toolbar with three buttons will appear.

Open Chromaticity Diagram Template

Click the first button, and it will open a workbook template. In the third sheet, two graph templates (CIE 1931 and CIE 1976) are inserted. Open the graph window by
double clicking on the worksheet's cell. If you want to show color temperature in CIE 1931, click Show/Hide Color Temperature button on the bottom right of the graph.
You can also add your own data to the graph template.
•Chromaticity Diagram with PL
• Click the second button, and it will open the PL2CIE dialog.
• Choose Photoluminescence Spectra (PL spectra) from the worksheet or the active graph. You can choose multiple PL spectra, and each PL spectrum corresponds to a point in the
chromaticity diagram.
• In Settings branch, select Spectral Type, two types are available: Radiance and Reflectance or Transmittance (0-1). The latter requires input to be between 0 and 1. For the latter,
select Spectral Power Distribution of Illuminant, four options are available, A, D50, D65 and Custom, for the custom type, user should replace PL sheet's column M with his
actual SPD data in the app folder's Chromaticity.ogwu file. Select Standard Observer: CIE 1931 2° or CIE 1964 10°, which defines the color matching function.
• In Graph branch, select CIE type for the diagram. Two options are available: CIE 1931 and CIE 1976. Choose plot type (Scatter or Line+Symbol) and determine whether to show
spectrum labels in the graph.
• Click OK button. A chromaticity diagram with points for PL data will be created. If you want to show color temperature in CIE 1931, click Show/Hide Color Temperature button on the
bottom right of the graph. Workbook data for the graph will be hidden. You can activate the workbook in Project Explorer.
• L*a*b* Plot
Click the third L*a*b* Plot button, a dialog will be opened and ask the user to choose a goal
2D Graphs

3D Graphs
Factors affecting fluorescence:
1. Conjugation
2. Rigidity of structures
3. Nature of substituent group
4. Effect of temperature
5. Viscosity
6. Oxygen
7. Effect of pH
1. Conjugation: Molecule must have unsaturation i.e. it must have π electrons so that UV/vis radiation can be
absorbed. If there is no absorption of radiation, there will be no fluorescence.
2. Rigidity of structures: Rigid structures will produce more fluorescence, while flexible structure will produce less
fluorescence.
3. Nature of substituent group: Electron donating groups like amino, hydroxyl groups enhance fluorescence activity.
Electron withdrawing groups like Nitro, carboxylic group reduce fluorescence. Groups like SO3H or on NH4+ have
no effect on fluorescence intensity.
4. Effect of temperature: Increase in temperature leads to increase in collisions of molecules and decrease in
fluorescence intensity while decrease in temperature leads to decrease in collisions of molecules and increased
fluorescence intensity.
5. Viscosity: Increase in viscosity leads to decreased collisions of molecules which will enhance fluorescence intensity
while decrease in viscosity causes increased collisions of molecules which cause decreased fluorescence intensity.
6. Oxygen: Oxygen decreases the Fluorescence intensity in two ways: Oxidizes fluorescence substances to non
fluorescence substances. It quenches fluorescence because of paramagnetic properties.
7. Effect of pH:
a) Aniline: Neutral or alkaline medium shows visible fluorescence while acidic conditions give fluorescence in UV
region only.
b) Phenols: Acidic conditions do not give fluorescence while alkaline conditions gives good fluorescence
Effect of concentration on fluorescence
intensity:
Thus, fluorescence intensity is directly proportional to the concentration of solution
Bandgap calculation using wavelength:
We use, for converting wavelength into energy manually
Where, = 1240
So, =
Suppose taking a data having excitation at 500nm wavelength
Calculating,
eV
Thus, calculating manually we are getting 2.48 eV as bandgap
Using origin:
Plotting graph using origin
Here wavelength (nm)
Is in X-axis and intensity
In Y-axis.
Here excitation wavelength
for which bandgap is to be
calculated is 500nm.
Marking the wavelength
For calculation of energy directly making a C column where C(Y) = 1240/Col(A)
All values are displayed for energy in column C(Y)
Graph has been plot for intensity and energy
Actual Band gap energy is 2.47 eV
There is 0.01 eV error for calculated value
Advantages:
It is one of the newer methods and its potentialities are still largely unexplored.
It also affects precision. Up to 1% can be achieved easily in Flourimetric.
The method is very sensitive and also possesses specificity because there is a choice of
wavelength not only for the radiation emitted, but also for the light which excites it.
Limitations:
Careful buffering is necessary as fluorescence intensity may be strongly dependent
 Ultraviolet light used for excitation may cause photochemical changes or destruction of the
fluorescent molecule .
The presence of dissolved oxygen may cause increased photochemical destruction.
 Traces of iodide and nitrogen oxides are efficient quenchers and therefore interfere.
 The method is not suited for determination of major constituents of a sample, because the
accuracy is very less for large amounts.
 The extent of applicability of this technique is limited, because of the fact that all elements and
compounds are unable to exhibit fluorescence.
 Precautions:
Fluorescence analysis is especially applicable to trace substances, care must be taken to eliminate
contaminations of sample.
Rubber and cork stoppers contain fluorescent materials and these are extracted if the solvent touches
them.
 Filter paper also contains fluorescent material which is extracted by solvents.
 Grease from stop cocks and other sources is a fluorescent contaminant.
 All glasses contain Al, Ca and SiO2 which may be extracted.
 The most important consideration is the concentration of the reagent. Concentration must be expressed
in micro molecules so that the ratio of the reagent to metal may be estimated easily.
 Large temperature change between unknown and standard should be avoided.
 It is also not desirable to expose the solution to ultra violet radiation for longerperiods.
 Conclusion:
Fluorescence spectroscopy is a sensitive optical emission technique in which sample molecules are excited
with a photon source. Those molecules that relax by radiant emission can be subsequently detected by
measuring the intensity of that emission.
The fluorescence decay time of typical fluorophores falls in a window (1 -20 ns) suitable for the
observation of several molecular processes of biological relevance.
The spectral properties of fluorophores are changed by several processes including collisions with other
molecules, rotational diffusion, and formation of complexes; moreover, the fluorescence properties are
sensitive to changes of the environment such as pH, electrical fields, concentration, temperature, polarity.
At first glance it seems easy to perform fluorescence experiments. However, there are numerous factors
that can compromise the data and invalidate the results. One needs to be constantly aware of the possibility
of sample contamination, or contamination of the signal from scattered or stray light. Collection of emission
spectra, and examination of blank samples, is essential for all experiments.
References:
1. Spencer R.D., Weber G., 1970. Measurements of sub nanosecond fluorescence lifetimes with cross
correlation phase fluorometer. Annals New York Acad. Sci. 158, 361-376.
2. Strickler J.S. and Berg R.A., 1962. Relationship between absorption intensity and fluorescence lifetime of
molecules. J. Chem. Phys. 37, 814-822.
3. David M. Jameson, 2014. Introduction to Fluorescence; CRC Press – Taylor & Francis Group, Boca
Raton.
4. Joseph R. Lakowicz, 2006. Principles of Fluorescence Spectroscopy; 3rd Edition, Springer–Verlag, New
York.
5. Bernard Valeur, 2005. Molecular Fluorescence; Wiley-VCH Verlag Gmbh, Weindheim.
6. Wolfgang Becker, 2005. Advanced Time-Correlated Single Photon Counting Techniques; Springer-
Verlag, Berlin/Heidelberg 2005.