This document provides an overview of fluorescence spectroscopy. It begins with a brief introduction to fluorescence as a type of luminescence involving emission of light from electronically excited states. It then discusses the Jablonski diagram, which provides the scientific foundation for fluorescence. Several key characteristics of fluorescence emission are described, including Stokes shift and Kasha's rule. The document outlines some common applications of fluorescence spectroscopy and describes the basic components and operation of fluorescence spectrometers, including light sources, monochromators, and photomultiplier tubes. It concludes by noting that fluorescence intensity can decrease at extremely high sample concentrations due to factors like self-quenching.

1. FLUORESCENCE
SPECTROSCOPY
JAYAKRISHNA J
MPHIL STUDENT
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF KERALA
KARIAVATTOM CAMPUS
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J Cell Sci. 124 (2): 157–160

2. PHENOMENON OF FLUORESCENCE
INTRODUCTION TO FLUORESCENCE
1. Luminescence is the emission of light from any substance and occurs from electronically
excited states.
1. FLUORESCENCE
2. PHOSPHORESCENCE
Spin in the ground and excited states
Fluorescence –ground state to singlet state and back.
Phosphorescence - ground state to triplet state and back.
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JABLONSKI DIAGRAM
Scientific foundation for fluorescence was given by Alexander Jablonski
Professor Jablonski is regarded as the father of Fluorescence Spectroscopy
J. R. Lakowicz, Introduction to Fluorescence, Principles
of Fluorescence Spectroscopy, 3rd Ed. Springer US
 Regarded as the starting point for
discussing light absorption and emission .
 Also defined the term ‘anisotropy’

4. Fluorescenc
eEmission of Photons
 S1 → S0 radiative transition
 Timescale of 10-10 to 10-7 s
 Rapid Vibrational Relaxation
And Internal Conversion
D. C. Harris, Quantitative Chemical Analysis, 7th Ed, W.
H. Freeman and Company, New York (2006).
Fluorescence occurs at higher wavelength
(lower energy) compared to Absorption
A Stokes shift is the difference between the
absorption and emission peaks of a molecule.
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A radiative transition between two
electronic states of the same spin
multiplicity.

5. Blue glass Filter
Church Window!
<400nm
Quinine
Solution
Yellow glass of wine
Em filter, transmits > 400 nm
Adapted from HORIBA JobinYvon Inc., Leading the 21 st Century in Time-
Resolved Fluorescence Instrumentation Dr. Adam M. Gilmore
Applications Scientist Fluorescence slide share
FIRST OBSERVED FROM QUININE BY SIR J.W.HERSHEL IN 1845
3.The first observation of fluorescence was reported by Sir John Frederick William
Herschel in 1845
4.He states that superficial colour presented by a homogenous liquid internally
colourless

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.
Fig Adapted Article by: Hobart M. King, Ph.D., RPG
The term fluorescence comes from the MINERAL FLUORSPAR (CALCIUM FLUORIDE)
coined by SIR GEORGE G. STOKES.
Fluorescent fluorite: Tumble-polished
specimens of fluorite in normal light (top)
and under short-wave ultraviolet light
(bottom).
J. R. Lakowicz, Introduction to
Fluorescence, Principles of Fluorescence

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CHARACTERISTICS OF FLUORESCENCE EMISSION
1. Stokes Shift
2. Kasha Rule
Fluorescence Typically occurs at lower energies or longer
wavelengths
J. R. Lakowicz, Introduction to Fluorescence, Principles of
Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp
Stokes Shift:
Difference in energy/wavelength
between absorption max and emission
max.
Kasha’s Rule:
Emission predominantly occurs from the
lowest excited state (S0 OR T1)

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Fluorescence is now a dominant methodology used in
1. Environmental Monitoring,
2. Medical Diagnostics,
3. DNA Sequencing,
4. Forensics,
5. Genetic Analysis,
6. Sensing Applications.
1. High sensitivity
2.Fluorescence intensity, Fluorescence maximum, spectral shape,
fluorescence anisotropy, fluorescence lifetime, time-dependence of
anisotropy, fluorescence correlation spectroscopy
3. Relatively simple experimentation
4. Lower cost of equipment compared to similar other techniques

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FLUORESCENCE SPECTROSCOPY
Steady-State Experiments Time Resolved Experiments
Excitation Spectra Emission Spectra
(Fluorescence Spectra)
D. C. Harris, Quantitative Chemical Analysis, 7th Ed, W. H.
Freeman and Company, New York (2006).

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Fluorometer
The Filter Fluorometer The Spectrofluorometer
A-1 filter fluorometer Perkin-Elmer 204
A filter fluorometer will use filters while a spectrofluorometer will
use grating monochromator.
https://www.edinst.com/blog/fluorescence-
measurements-introduction/

11. A-1 filter fluorometer
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https://www.edinst.com/blog/fluorescence-
measurements-introduction/

12. Perkin-Elmer 204
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https://www.edinst.com/blog/fluorescence-
measurements-introduction/

13. Example of a COMPACT SPECTROFLUOROMETER (FS5) and a more
ADVANCED PHOTOLUMINESCENCE SPECTROMETER (FLS1000).
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https://www.edinst.com/blog/fluorescence-
measurements-introduction/

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J. R. Lakowicz, Instrumenation for Fluorescence
Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer
US (2006) pp 27-61

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 The primary use of a Spectrofluorometer is to record excitation and emission
spectra.
 Excitation and emission spectra are x-y plots of fluorescence intensity
vs. wavelength.
 To obtain accurate spectra, the components must have the following
characteristics
An ideal Spectrofluorometer
J. R. Lakowicz, Instrumentation for Fluorescence
Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer
US (2006) pp 27-61
Properties of the ideal components of spectrofluorometer

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J. R. Lakowicz, Instrumentation for Fluorescence
Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer
US (2006) pp 27-61
 Light Sources, Monochromators, And Photomultiplier Tubes With Such Ideal
Characteristics Are Not Available.
 There is need to correct for the non ideal response of the instrument .
 Fluorescence Intensity Measurements Are Absolute, Not Relative.
• In absorption spectrum, the intensity of light transmitted by the sample is
measured relative to that of a reference (or blank).

17. https://www.edinst.com/blog/fluorescence-
measurements-introduction/
Introduction to Instrumentation
The figure above shows the layout of
an FLS1000 Photoluminescence
Spectrometer
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Light Sources
1. Arc and Incandescent Xenon Lamps
2. Pulsed Xenon Lamps
3. High Pressure Mercury Lamps
4. LED Light Sources
5. Laser Diodes
J. R. Lakowicz, Instrumentation for Fluorescence
Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer
US (2006) pp 27-61
 At present the most versatile light source for a steady-state spectrofluorometer
is a high-pressure xenon (Xe) arc lamp.
 light output from 250 to 700 nm
 number of sharp line 450 nm and above 800 nm.

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J. R. Lakowicz, Instrumentation for Fluorescence
Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer
US (2006) pp 27-61
Spectral output of a continuous Xenon arc lamp
• The wavelength-dependent output of Xe lamps is a major reason for distortion
of the excitation spectra of compounds that absorb in the visible and
ultraviolet.

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J. R. Lakowicz, Instrumentation for Fluorescence
Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer
US (2006) pp 27-61
 Xenon lamps are usually contained within specially designed housings.
 A xenon lamp that is on should never be observed directly.
 The extreme brightness will damage the retina, and the ultraviolet light
can damage the cornea.

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MONOCHROMATORS
J. R. Lakowicz, Introduction to Fluorescence, Principles of Fluorescence
Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61
 Monochromators are used to disperse polychromatic or white light into the
various colors or wavelengths.
 This dispersion can be accomplished using prisms or diffraction gratings.
 The monochromators in most spectrofluorometers use diffraction gratings
rather than prisms
https://www.shimadzu.com/an/uv/support/fundamentals/singl
e_double.html

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J. R. Lakowicz, Introduction to Fluorescence, Principles of Fluorescence
Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61
Grating may be PLANAR or CONCAVE. Planar
gratings are produced MECHANICALLY and may
contain IMPERFECTIONS.
Concave gratings are produced by
HOLOGRAPHIC or PHOTORESIST methods and
have less imperfections.
 Few Reflecting Surfaces
 Lower stray lightThe distance between adjacent grooves and the
angle the groove influence both the dispersion
and efficiency of a grating.

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Efficiencies of two MCs blazed at
different angles
J. R. Lakowicz, Introduction to Fluorescence, Principles of Fluorescence
Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61
The transmission efficiency of a grating and monochromator is dependent on
wavelength and on the design of the grating.
For a mechanically produced plane grating the efficiency at any given wavelength can
be maximized by choice of blaze angle, which is determined by the shape and angle of
the tool used to produce the grating.
By choice of this angle one may obtain maximum diffraction efficiency for a given
wavelength region

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Second order diffraction of 300 nm light
occurs at 600 nm. Hence a peak will
appear at 600.
500 600550 700
Wavelength
The 600 nm peak can be avoided by
placing a cut off filter (eg. <400 cut off)
in the emission channel.
500 600550 700
Wavelength
Corrected spectrum
Assume that you are recording the emission spectrum of a sample. Excitation
WL is 300 nm. Sample emits in the 500-700 nm region

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PHOTOMULTIPIER TUBES
J. R. Lakowicz, Instrumenation for Fluorescence Spectroscopy, Principles of
Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61
Almost all fluorescence spectrometers use PMTs as detectors and it is
essential to understand their capabilities and limitations.
PMT is a device that converts light intensity into electrical current,
the current generated being proportional to the photon intensity.

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PMT
Xenon Lamp
Excitation
Monochromator
Emission
Monochromator
Sample
ExGrating
Em Grating
Procedure
1) White light source on
2) Shift excitation grating to desired
wavelength (excitation wavelength)
3) Light enters sample chamber
4) Light Hits the Sample
5) Emission from the sample enters
emission monochromator
6) Set emission grating
7) Detect emitted light at PMT
8) Raster emission grating
Measuring Emission Spectra

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EFFECT OF CONCENTRATION
J. R. Lakowicz, Introduction to Fluorescence, Principles of Fluorescence
Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61
At extremely high sample concentrations, the
Fluorescence Intensity can actually decrease
because most of the light is absorbed in the
front layers.
Reabsorption of the shorter wavelength.

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COMMON ERRORS IN SAMPLE PREPERATION
J. R. Lakowicz, Instrumenation for Fluorescence
Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer
US (2006) pp 27-61
With the Instrument
• Stray light
• Slit Widths
• Signal/Noise

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Fluorescence & Structure:
 usually aromatic compounds
 low energy transition
 quantum yield increases with number of rings and degree of
condensation.
 fluorescence especially favored for rigid structures
 fluorescence increase for chelating agent bound to metal.
N H
N
H2
C
N
O
Zn
2
quinoline indole fluorene 8-hydroxyquinoline

30. The fluorescence spectrum and intensity of
a molecule often depend strongly on the
molecule’s environment.
FACTORS AFFECTING
FLUORESCENCE AND
PHOSPHORESCENCE
Temperature
pH
Dissolved oxygen
Solvent
30

31. Temperature:
 As temperature increases fluorescence decreases.
 Due to change in temperature viscosity of the medium changes.(less
Viscosity)
 Change in viscosity increases the number of collisions of the molecules of
the fluorophore with solvent molecules.
 No. of collisions increases the probability for deactivation by internal
conversion and vibrational relaxation.
 To overcome this , it is recommended to use thermo stated cell holders.
31

32. pH
 Relatively small changes in pH can cause considerable changes in
the fluorescence intensity and spectral characteristics of fluorescence.
 The molecules containing acidic or basic functional groups undergoes
ionization due to the changes in pH of the medium.
 It may affect the extent of conjugation or the aromaticity of the
molecule which affects its fluorescence.
 For 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
32

33. Dissolved Oxygen
 Oxygen and many transition metals with unpaired electrons are
paramagnetic which 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.(phosphorescence)
 Presence of dissolved oxygen influences phosphorescence too and causes a
large decrease in the phosphorescence intensity. This is actually the oxygen
emission and not the phosphorescence.
 Therefore, it is advisable to make phosphorescence measurement in the
absence of dissolved oxygen.
33

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Solvent:
The changes in the “polarity” or hydrogen bonding ability of the solvent affect the
fluorescent behaviour of the analyte.
Solvent viscosity and solvents with heavy atoms also affect fluorescence and
phosphorescence.
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.
J. Chem. Educ. 2011, 88, 731–738

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APPLICATIONS
Naked eye and smartphone applicable detection of toxic mercury ions using
fluorescent carbon nanodots
doi:10.3906/kim-1701-46
1.Band Gap Determination
2. Impurity level and defect detection
3.Surface Structure and Excited States.

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https://www.nobelprize.org/prizes/chemistry/
2014/press-release/

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