This document discusses spectrofluorimetry, which involves using fluorescence spectroscopy to study the emission of radiation from molecules. It begins by explaining fluorescence, where molecules absorb UV or visible light and emit light of a longer wavelength as they relax to the ground state. The document then defines key terms like singlet and triplet states. It describes the instrumentation used, factors that influence fluorescence intensity, and applications of spectrofluorimetry like determining organic and inorganic substances. Examples are given of using it to analyze minerals, plant pigments, and food and pharmaceutical samples.

1. Spectrofluorimetry
Dr. Chris
Phayao University, March 2016

2. Introduction
• Absorption of uv/visible radiation causes
transition of electrons from ground state (low
energy) to excited state (high energy).
• As excited state is not stable, excess energy is
lost by
– Collision deactivation
– Emission of radiation (Photo Luminescence)
• Emission Spectroscopy : emission of radiation
is studied.

4. Understanding the terms……..
• Singlet ground state : state in which electrons in a
molecule are paired. [ ]
• Singlet excited state: state in which electrons are
unpaired but of opposite spins. [ ]
• Triplet state: state in which unpaired electrons of
same spin are present. [ ]
• Excitation process: absorption of energy or light
followed by conversion from ground state to
excite state.
• Relaxation process: process by which atom or
molecule losses energy & returns to ground state.

5. Photo Luminescence
• Light without heat or cold light
• Basically of 2 types
– Fluorescence: part of energy is lost due to
vibrational transitions and remaining energy is
emitted as uv/visible radiation of longer
wavelength than incident light.
– Phosphorescence: under favorable conditions,
excited singlet state undergo transition to triplet
state. Emission of radiation when e- undergo
transition from triplet state to ground state.

6. Flourescence – emission at higher wavelength (lower
energy) than excitation
https://www.youtube.com/watch?v=SGFlr1jFNBM

7. http://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Fluorescence%20Excitation
%20and%20Emission%20Fundamentals.pdf

8. Instrumentation
Fixed λ
=> EXCITATION
Fixed λ
=> EMISSION

9. Excitation and Emission
For the Excitation spectra:
Detection wavelength is fixed, the excitation
wavelength is varied
For the Emission spectra:
The excitation wavelength is fixed, the detection
wavelength is varied

10. Classification
• Based on the wavelength of emitted radiation
when compared to absorbed radiation
– Stokes fluorescence: wavelength of emitted
radiation is longer than absorbed radiation
– Anti-stokes’s fluorescence: wavelength of emitted
radiation is shorter than absorbed radiation.
– Resonance fluorescence: wavelength of emitted
radiation is equal to that of absorbed radiation.

11. Actors affecting fluorescence intensity
• Conjugation: molecule must have conjugation ( π electron) so
that uv/vis radiation can be absorbed
• Nature of substituent groups:
– e- donating groups like NH2, OH groups enhance
fluorescence.
– e- withdrawing groups like NO2, COOH reduce fluorescence.
• Fluorescent intensity is directly proportional to concentration.
• Increase in viscosity leads to decreased collisions of molecules
there by increasing fluorescent intensity.
• More rigid the structure of molecule, more the intensity of
fluorescence.
• Increase in temp leads to increased collisions b/w molecules
decreasing fluorescent intensity.
• Presence of O2 decreases the fluorescence and so de-aerated
solutions must be used.

12. • Source of light
– Mercury vapour lamp : Hg vapour in high pressure (8
atm) gives intense lines on continuous background
above 350nm.
– Xenon arc lamp: gives more intense radiation.
– Tungsten lamp: used if excitation has to be done in vis
region.
• Filters and monochromators
– In fluorimeter 10 filter ( absorb vis radiation and
transmit uv radiation) and 20 filter (absorb uv
radiation and transmit vis radiation) are present.
– In spectrofluorimeters, excitation monochromators
and emission monochromator are present.

13. • Sample cells
– Sample cells are cylindrical or polyhedral made up
of colour corrected fused glass & path length
normally 10mm to 1cm.
• Detectors
– Photo voltaic cell, photo tubes or photo multiplier
tubes can be used.

14. Advantages
• More sensitive when compared to other
absorption techniques. Concentrations as low
as μg/ml or ng/ml can be determined.
• Precision upto 1% can be achieved easily
• As both excitation & emission wave lengths
are characteristic it is more specific than
absorption methods.

15. Example: Naphtalene in water
Uv/vis spectrum
https://www2.chemistry.msu.edu/facult
y/reusch/VirtTxtJml/Spectrpy/UV-
Vis/spectrum.htm
Flourescence spectrum
https://www.osapublishing.org/oe/ful
ltext.cfm?uri=oe-21-20-
24219&id=268663

16. Minerals:Minerals:
Cr 3+ emissions
from Al2O3
sapphires
Absorbs blue
and green light
Red color
Emits red color
http://www.eso.org/~rfosbury/
Article%20from%20Journal201
3.pdf

17. Applications of Spectrofluorimetry
• Determination of Organic substances
– Plant pigments, steroids, proteins, naphthols etc can be
determined at low concentrations.
– Generally used to carry out qualitative as well as
quantitative analysis for a great aromatic compounds
present in cigarette smoking, air pollutant concentrates &
automobile exhausts.
• Determination of inorganic substances
• Extensively used in the field of nuclear research for the
determination of uranium salts.
• Determination of vitamin B1 (thiamine) in food samples
like meat cereals etc.
• Determination of Vitamin B2 (riboflavin). This method
is generally used to measure the amount of impurities
present in the sample.

18. • Most important applications are found in the
analyses of food products, pharmaceuticals,
clinical samples and natural products.
• Fluorescent indicators:
– Intensity and colour of the fluorescence of many
substances depend upon the pH of solutions.
These are called as fluorescent indicators and are
generally used in acid base titrations.
– Eg: Eosin – pH 3.0-4.0 – colourless to green
– Fluorescein – pH 4.0-6.0 – colourless to green