This document discusses the principles and applications of fluorimetry. It defines fluorescence as the emission of light from excited electrons returning to ground state. Fluorimetry involves using ultraviolet light to excite sample molecules, which then emit light of lower energy. Factors that influence fluorescence intensity are discussed. Common instrumentation includes light sources, monochromators, cuvettes, detectors, and data analyzers. Applications include determining inorganic substances, proteins, and steroids. Two case studies on analyzing glucose in milk and silver in food/water samples using fluorimetry are presented.

2. Presentation on “ Fluorimetry and
its Application

3.  Definition
 Principle
 Classification
 Instrumentation
 Parameters
 Applications
 Pros and Cons

4. Fluorescence:
 When a beam of light passes through a
substances, the particles would absorbs some form
of energy and emits the radiation.
 It is phenomenon in which the excited singlet
electrons(the electrons are paired) emits emission
radiation when brought down to
ground level at the certain wavelength.
 Fluorescence occurs at (10-9 – 10-6 sec).

5.  It is a type of electromagnetic spectroscopy that
analyses the fluorescence from the sample.
 It involves the use of beam of light, usually ultra
violet light that excites the electrons in the molecules
of certain compounds (fluorescence substances) and
that produces a light of lower energy

6.  The principle involved is Vibrational relaxation.
 Molecules have bonding (σ) electrons and
(π)electrons and n non bonding electrons.
 When the excited electrons absorbs the radiant
energy, the bonding electrons gets down to anti
bonding orbital.
 Has high energy but less stable.
 it is a instantenous process

7. The factors effecting fluorescence intensity are
 Concentration
 Quantum yield of fluorescence
 Intensity of incident light
 Adsorption
 Oxygen
 pH
 Temperature & Viscosity
 Photodecomposition
 Quenchers
 Scatter

8. It is the process of reduction of fluorescence
intensity by the presence of substances in the
sample other than fluorescent analyte.
Forms of quenching
Self quenching
Collision quenching
Static quenching
Inner fluorescent effect

9. Contd.,
Self quenching:
Fluorescence intensity with
concentration.
Static quenching:
Due to complex formation between fluorescent
analyte and substances.
Collision quenching:
Collision between fluorescent substances and
halide ions.
Inner fluorescent effect:
Absorption of incident light by filters
decreases the fluorescent intensity.

10.  Test solutions prepared for fluorescence
spectrophotometer are usually 10 times to 100 times
less concentrated than those used in absorption
spectrophotometer.
 Concentrations of 10-5-10-7 mol/l are frequently
used.
 Blank should be not less than 0.40 and not more than
2.50.

12. The basic components of fluorimeter includes
An excitation light source
An excitation monochromator
A cuvette
An emission monochromator
A detector
A data analyser

14.  Mercury lamps are the most commonly employed
light sources.
 They have the property that their spectral output
depends upon the pressure of the filler gas.
 Mercury arc lamp-radiation @254 nm
 Xenon arc lamp- spectrum over the range between
250nm-600nm
 Tungsten lamp- intensity of lamp is low
 Turn able dye laser- radiation about 360nm-650nm

15.  Monochromator produces monochromatic light by
removing unwanted wavelengths from the source light
beam.
 The function of the monochromator is to isolate a single
atomic resonance line from the spectrum of lines emitted by
the hollow cathode lamp.
 Excitation monochromators:
Isolates only the radiation absorbed by the molecule
 Emission monochromators:
Isolates only the radiation emitted by the molecule

16. The proper selection of filters requires
familiarity with the emission spectrum and
the excitation spectrum.
Primary filter:
It absorbs the visible light and transmits UV light.
Secondary filter:
It absorbs the UV light and transmits visible light.

17.  Cuvettte are cylindrical or rectangular cells fabricated
of silica or glass.
 They are meant for holding the diluted samples.
 Path length is about 10mm or 1cm
 Cells and other glassware used for fluorimetric
analysis should be carefully cleaned, preferably by
boiling in 50% nitric acid followed by thorough
rinsing in distilled water.

18. Detectors are device or instrument designed to
detect the presence of a particular object or
substance.
Three forms of detectors are
 Photovoltaic cell
 Phototube
 Photomultiplier tube.

19.  A photomultiplier tube is a photo emissive device in which
the absorption of a photon results in the emission of an
electron.
 These detectors work by amplifying the electrons generated
by a photocathode exposed to a photon flux.
 Cathode voltage is 75-100 V.
 Amplification of 10^6 is obtained.
 Electron_Multiplier_Continuous_Dynode.mp4

20. SINGLE BEAM
FLUORIMETER
DOUBLE BEAM
FLUORIMETER
 In single beam
fluorimeter, all the
light waves pass through the
sample.
 Primary filter– absorbs
visible light.
 Secondary filter– absorbs
UV light.
 Reference and sample cant
be analyzed simultaneously
 In double beam fluorimeter the
light beam splits into two
parts and only one part passes
through the sample.
 Primary filter– absorbs the
light and passes to the sample.
 Secondary filter – absorbs the
light from the sample and sent
to detector.
 Reference and sample can be
analyzed simultaneously

21. Single beam fluorimeter Double beam fluorimeter

22. The fluorescence intensity is proportional to the initial
radiation times abc.
log(Fo/Fo-F)= abc
Fo-100 (standard)
F- fluorescence measured
b- cell length
c- concentration
a- proportionality constant
By Simplification,
abc=2-log(100-F)
InShot_20191128_123357970.mp4

23.  Determination of inorganic substances;
 Determination of thiamine
 Determination of indoles, phenols, phenothiazines.
 Napthols ,proteins ,plant pigment and steroids can be
determined.
 Detection of impurities at nanogram quantities.
 Detection of respiratory tract infection.
 Determines phenyltoin.

24. Case study 1

25. Objective:
Analyses of free glucose and glucose 6
phosphates in cow’s milk using fluorimetry.
Materials and methods:
Reagents-tris buffer
 The samples were excitated with 544 nm
monochromatic light and read at 590 nm light.

26.  The present enzymatic-fluorometric methods for
determinationof total glucose and glucose 6-P in milk
are reliable analytical methods
 This method matches the indigenous methods for the
glucose analysis.
 The free glucose level in milk obtained in the present
study, mean 331 lmol/l, inter percentile 143–529
lmol/l.

28. Objective:
 Determination of Ag(I) in Some Food and Water
Samples using fluorimeteric detection.
Methods and materials:
 Ag(I) stock solutions (1000 mg/L)
 DKMS complex– fluorimetric reagent
Rhodamine B hydrazide (RH) +ferrocenecarboxaldehyde
(1 mmol, 0.21 g) in absolute ethanol (50 mL) in the
presence of catalysis acetic acid at refluxing
temperature for 24 h.

31.  The procedure is highly sensitive, as well as selective,
because it does not require the preparation of samples.
 Rapid response and high specificity
 The new sensitive system is inexpensive
 Has a low LOD (micrograms per liter).
 Easy in construction.
 Allows wide sample variation because it is
interference-free.
 This system minimizes analysis time and the manpower
needed

32. Fifield&kealey., principlkes and practice of analytical chemistry
(2000)
Syed&haddad ., textbook of analytical chemistry,(2011)
https://www.slideshare.net/sreevidyavemuri/fluorimetry-45064390
http://www.authorstream.com/Presentation/nalinisahoo-
2331005-fluorimetry/
https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Fl
uorescence%20Spectroscopy.pdf
http://srmbiotech25.yolasite.com/resources/Applications_of_Fl
uorimetry_and_Phosphorimetry.pdf
https://www.nature.com/subjects/fluorescence-spectrometry
https://link.springer.com/journal/10895
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662353/