Fluorescence is the phenomenon whereby a molecule, after absorption radiation, emits radiation of a longer wavelength. A compound absorbs radiation in the UV-rgion and emits visible light. Absorption of uv/visible radiation causes transition of electrons from ground state (low energy) to excited state (high energy). This increase in wavelength is known as the Stokes shift.

1. TapeshwarYadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry
Spectroflurometer

2. History
The term fluorescence comes from the mineral fluorspar (calcium
fluoride) when Sir George G. Stokes observed in 1852 that
fluorspar would give off visible light (fluoresce) when exposed to
electromagnetic radiation in the ultraviolet wavelength.

3. Introduction
Fluorescence is the phenomenon whereby a molecule, after
absorption radiation, emits radiation of a longer wavelength.
A compound absorbs radiation in the UV-rgion and emits
visible light.
Absorption of uv/visible radiation causes transition of
electrons from ground state (low energy) to excited state
(high energy).
This increase in wavelength is known as the Stokes shift.

4. Understanding the terms……..
Singlet ground state : state in which electrons in a molecule are
paired.
Singlet excited state: state in which electrons are unpaid 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. Principle
It is an analytical device depends on the fluorescence
phenomenon which is a short-lived type of
photoluminescence created by electromagnetic excitation.
That is, fluorescence is generated when a molecule transmits
from its ground state So to one of several vibrational energy
levels in the first excited electronic state, S1, or the second
electronic excited state, S2, both of which are singlet states.
Relaxation to the ground state from these excited states
occurs by emission of energy through heat and/or photons.

6. Contd…
The difference between the excitation and emission
wavelengths is called the Stokes shift.
Stokes’ studies of fluorescent substances led to the
formulation of Stokes’ Law, which states that the wavelength
of fluorescent light is always greater than that of the exciting
radiation. Thus, for any fluorescent molecule, the
wavelength of emission is always longer than the wavelength
of absorption.

8. What is The fluorescence quantum yield (Φf)?
It is the quantitative expression of the fluorescence
efficiency, which is the fraction of excited molecules
returning to the ground state by fluorescence.
Quantum yields range from 1, when every molecule in an
excited state undergoes fluorescence, to 0 when fluorescence
does not occur.
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9. A molecule’s fluorescence quantum yield is influenced
by external Variables such as:
• temperature
• viscosity of solvent
• pH
Increasing temperature generally decreases Φf because more
frequent collisions between the molecule and the solvent
increases external conversion.
Decreasing the solvent’s viscosity decreases Φf for similar
reasons.
For an analyte with acidic or basic functional groups, a change
in pH may change the analyte’s structure and, therefore, its
fluorescent properties.
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12. What can Spectroflurometer do?
It has been used for the direct or indirect quantitative and
qualitative analysis by measuring the fluorescent intensity F.
 It is relatively inexpensive and sensitive (the sensitivity of
fluorescence is approximately 1,000 times greater than
absorption spectrophotometric methods).
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13. fluorescent intensity F is dependent on both intrinsic properties
of the compound (fluorescence quantum yield Φf), and on readily
controlled experimental parameters including:
• intensity of the absorbed light I0
• molar absorption coefficient Ɛ
• path length of the cell b
• concentration of the fluorophor in solution c
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14. At low concentrations of fluorophore, the fluorescence
intensity of a sample is essentially linearly proportional to
concentration.
However, as the concentration increases, a point is reached at
which the intensity increase is progressively less linear, and the
intensity eventually decreases as concentration increases
further.
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15. concentrationsconcentrations
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16. Components of Spectroflurometer
i. Light source
ii. Monochromator
iii. Cuvettes
iv. Detector
v. Amplifier
vi. Read out device

20. Light source: Xenon arc lamp or mercury lamp
A high pressure xenon arc lamp is used for
spectrophotometer, which provides intense, stable and
continuous beam in UV and visible region.
Filter fluorimeters generally use low-pressure mercury
vapour lamp which emits distinct line spectra in the UV and
visible region.

21. Monochromator
Two sets of monochromators are used: primary
monochromator and secondary monochromator.
Primary monochromator: It includes slits and dispersive
device to isolate the wavelength for excitation of sample.
Secondary monochromator: Isolates the wavelength of
emitted fluorescence.

22. Cuvettes: Usually quartz cuvettes are used.
Detector: A phototube or photomultiplier tube is used.
Read out device: A galvanometer or a potentiometer is used
as read out device.

24. Applications of Spectroflurometer
i. For the chemical modification such as oxidation ,
reduction, hydrolysis, couping and self condensation
ii. The determination and comparison of both excitation and
fiuorescence spectra of a compound may help to identify
it.
iii. The assay of vitamin in foodstuffs, NADH in
mitochondria, microorganism, hormones like cortisol,
oestradiol, drugs, cholesterol and prophyrins.
iv. Enzyme assays and kinetic analysis
v. Study of protein structure

25. 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.

26. 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
 Fluorescien – pH 4.0-6.0 – colourless to green

27. Advantages
Fluorimetric techniques have a high degree of specificity.
Precision upto 1% can be achieved easily
The tests based on fluorimetry are highly sensitive. It is
possible to determine concentrations of fluorescing species
down to nanogram and epigram range.
As both excitation & emission wave lengths are characteristic
it is more specific than absorption methods.

28. Disadvantages
The susceptibility to environmental conditions and the
virtual impossibility of predicting whether a compound will
fluoresce.
The other major problem is quenching, whereby the energy,
transferred to the other molecules.
Contamination can quench the fluorescence and hence give
false/no results