Spectroflurometer

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.

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Spectroflurometer
Mr. Sagar Kishor Savale
(M. pharm First year Student)
(Department of Pharmaceutics, North Maharashtra University, college of
R.C.Patel Institute of Pharmaceutical Education and Research, Shirpur, 425405,
Dist.Dhule, Maharashtra.)
2015-016
[email protected]

1. Introduction
1. Fluorescence is the phenomenon whereby a molecule, after absorption radiation,
emits radiation of a longer wavelength.
2. A compound absorbs radiation in the UV-rgion and emits visible light.
3. Absorption of uv/visible radiation causes transition of electrons from ground state
(low energy) to excited state (high energy).
4. This increase in wavelength is known as the Stokes shift.
2. Important Terminology
1. Singlet ground state : state in which electrons in a molecule are paired.
2. Singlet excited state: state in which electrons are unpaid but of opposite spins.
3. Triplet state: state in which unpaired electrons of same spin are present.
4. Excitation process: absorption of energy or light followed by conversion from
ground state to excite state.
5. Relaxation process: process by which atom or molecule losses energy & returns to
ground state.

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3. 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. 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.
4. Function
1. It has been used for the direct or indirect quantitative and qualitative analysis by
measuring the fluorescent intensity F.
2. It is relatively inexpensive and sensitive (the sensitivity of fluorescence is
approximately 1,000 times greater than absorption spectrophotometric methods).
5. Instrumentation
1. Light source
2. Monochromator
3. Cuvettes
4. Detector

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5. Amplifier
6. Read out device

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1. Light source: Xenon arc lamp or mercury lamp
2. monochromators are used: primary monochromator and secondary
monochromator
3. Cuvettes: Usually quartz cuvettes are used.
4. Detector: A phototube or photomultiplier tube is used.
5. Read out device: A galvanometer or a potentiometer is used as read out device.
6. Advantages
1. Fluorimetric techniques have a high degree of specificity.
2. Precision upto 1% can be achieved easily
3. The tests based on fluorimetry are highly sensitive. It is possible to determine
concentrations of fluorescing species down to nanogram and epigram range.
7. Disadvantages
The susceptibility to environmental conditions and the virtual impossibility of predicting
whether a compound will fluoresce.
8. Applications
1. For the chemical modification such as oxidation , reduction, hydrolysis,
couping and self condensation
2. The determination and comparison of both excitation and fiuorescence spectra
of a compound may help to identify it.
3. The assay of vitamin in foodstuffs, NADH in mitochondria, microorganism,
hormones like cortisol, oestradiol, drugs, cholesterol and prophyrins.
4. Enzyme assays and kinetic analysis

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5. Study of protein structure
6. 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.