La fluorescence est un phénomène physique dans lequel un matériau absorbe de la lumière à une longueur d'onde spécifique et réémet de la lumière à une longueur d'onde différente. Cela se produit lorsque les atomes, les molécules ou les particules du matériau absorbent de l'énergie sous forme de photons (généralement de la lumière ultraviolette ou visible) et passent à un état électronique excité temporaire. Lorsque ces électrons retournent à leur état fondamental, ils émettent de la lumière à une longueur d'onde plus longue que la lumière absorbée, ce qui crée un phénomène de fluorescence.
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La fluorescence est un phénomène optique et lumineux où un matériau absorbe la lumière à une certaine longueur d'onde (lumière d'excitation) et réémet de la lumière à une longueur d'onde différente (lumière de fluorescence). C'est un processus qui se produit lorsque les atomes ou les molécules d'un matériau absorbent de l'énergie sous forme de photons de lumière et deviennent temporairement excités. Lorsqu'ils reviennent à leur état fondamental, ils émettent des photons de lumière à une longueur d'onde plus longue que celle de la lumière d'excitation.
Voici quelques points clés sur la fluorescence :
Absorption de la lumière d'excitation : Les atomes ou les molécules d'un matériau absorbent des photons de lumière d'excitation, ce qui les amène à un état énergétique supérieur.
Émission de lumière de fluorescence : Lorsque ces atomes ou molécules reviennent à leur état énergétique de base, ils émettent des photons de lumière à une longueur d'onde plus longue que la lumière d'excitation.
Longueur d'onde de fluorescence : La longueur d'onde de la lumière de fluorescence est caractéristique du matériau et peut être utilisée pour identifier des composés spécifiques. Cette propriété est largement exploitée dans la spectroscopie de fluorescence.
Applications de la fluorescence : La fluorescence est utilisée dans de nombreuses applications, y compris la microscopie à fluorescence (pour visualiser des cellules et des structures biologiques), l'analyse chimique (spectroscopie de fluorescence), la détection de certaines maladies (par exemple, la fluorescence induite par un marqueur), et même dans l'éclairage fluorescent.
Fluorophores : Les matériaux qui exhibent des propriétés de fluorescence sont souvent appelés "fluorophores". Ils sont fréquemment utilisés comme sondes pour marquer des cibles spécifiques dans la recherche scientifique et médicale.
La fluorescence est un phénomène important dans de nombreuses disciplines
2. Summary:
1. What is the phenomenon of Fluorescence?
2. How does fluorescence spectrometry work ?
3. What are it’s pros and cons?
4. Some of its implication and utilities.
3.
4. Fluorescence
The emission of electromagnetic waves,
(luminescence) caused by the excitation
of atoms or nanostructure, which they
re-emit immediately (with-in 10^-8 s).
5. Phosphorescence
Another type of luminescence that is
caused by the absorption of radiations,
and that continues for noticeable
amounts of time. Even after the
radiation source stops.
6. The commun point between
fluorescence and
phosphorescence is the release
of photons while the electrons
return to the ground state.
However they differ in the
duration in which they do it.
7. The actual difference between them:
Fluorescence
When the atom relaxes
to the ground state (and
emits photons) without
any change in electron
spins.
Phosphorescence
there is a change in
electron spin, which
results in a longer
lifetime of the excited
state (second to
minutes).
8. 8
Absorption
Of the radiation’s
energy
E = h*f.
Excitation
State
Where the electron’s
energy levels change.
Re-emission
Where lies the difference
between the two
phenomenons.
Re-cap
10. Fluo/phosphorescence
spectroscopy
➢ Photoluminescence spectroscopy is a contactless,
nondestructive method of probing the electronic
structure of materials.
It consists of the study of UV, visible, and near-
infrared (NIR) light that is emitted by a chemical species
after having absorbed light.
14. Background
effects due to
light scattering
Solvent effects
Interfering
nonspecific
fluorescence
Concentration
effects
Inner filter
effects,
concentration
quenching
Sample effects
Light scattering,
interfering
fluorescence,
sample
adsorption
Here you could
talk about this
person
Limitations of fluorescence Measurements
15. Avdantages of fluorescence
spectroscopy
SENSITIVITY:
•It is more sensitive as concentration is low as ug/ml or
ng/ml.
PRECISION:
• Upto 1 % can be achieved.
SPECIFICITY:
• More specific than absorption method where absorption
maxima may be same for two compounds.
RANGE OF APPLICATION:
Even non fluorescent compounds can also be converted
to fluorescent compounds by chemical compounds.
16. DisAvdantages of fluorescence
spectroscopy
Not really useful for identification
Not all compounds fluorescence
Contamination can quench the
fluorescence and hence give
false/no results
phosphorescence exhibits a longer lifetime (~10 to -4 power – 10 to -2 power seconds) compared with fluorescence (~10^-9 – 10^-6 seconds)
The Pauli exclusion principle requires that no two electrons in an atom have the same identical set of quantum numbers; hence when two electrons reside in a single
While absorption occurs on the timescale of less than 10-15 seconds, the relaxation process from the excited to the ground state is much slower. Therefore, fluorescence can provide information on a fluorophores’ interactions with surrounding molecules and solvents, unlike absorption.
Fluorescence intensity is directly proportional to the excitation light intensity
F=2.303 * K * I0 * εbc
The fraction of a parallel beam of light absorbed by a sample is independent of the intensity of the incident beam and is related to the concentration of the absorbing species by the familiar Beer-Lambert Law:
I = intensity of transmitted light Io = intensity of incident light E = molecular extinction coefficient c = concentration in gm moles/L-1 l = pathlength of sample
Solid materials do not really need any preparations except for cutting
Powdered materials need to be grinded and pressed.
Liquids are prepared by pouring them into a plastic cup
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We expose the sample to the radiation source through the excitation monochromator then the emitted radiation is treated through the emission monochromator in order to obtain both spectrums.
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Excitation spectra plot the intensity at a fixed emission wavelength while varying the excitation wavelengths. Since most emission spectra are independent of the excitation wavelength, the excitation spectra are frequently duplicates of the fluorophore’s absorption spectrum.
While absorption occurs on the timescale of less than 10-15 seconds, the relaxation process from the excited to the ground state is much slower. Therefore, fluorescence can provide information on a fluorophores’ interactions with surrounding molecules and solvents, unlike absorption.
Fluorescence intensity is directly proportional to the excitation light intensity
F=2.303 * K * I0 * εbc
The fraction of a parallel beam of light absorbed by a sample is independent of the intensity of the incident beam and is related to the concentration of the absorbing species by the familiar Beer-Lambert Law:
I = intensity of transmitted light Io = intensity of incident light E = molecular extinction coefficient c = concentration in gm moles/L-1 l = pathlength of sample
While absorption occurs on the timescale of less than 10-15 seconds, the relaxation process from the excited to the ground state is much slower. Therefore, fluorescence can provide information on a fluorophores’ interactions with surrounding molecules and solvents, unlike absorption.
Fluorescence intensity is directly proportional to the excitation light intensity
F=2.303 * K * I0 * εbc
The fraction of a parallel beam of light absorbed by a sample is independent of the intensity of the incident beam and is related to the concentration of the absorbing species by the familiar Beer-Lambert Law:
I = intensity of transmitted light Io = intensity of incident light E = molecular extinction coefficient c = concentration in gm moles/L-1 l = pathlength of sample
-Detecting compounds from HPLC flow
- Plant pigments, steroids and proteins can be determined at low concentrations.
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Detecting and quantifying polluants such as polycyclic aromatic hydrocarbons
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Used generally as a non-destructive method of traking biofluorescent compounds
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Direct and indirect analysis for multiple drugs
-vitamins
-catecholamins(dopamin)
- Measuring the impurities of a sample
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Ability to identify different levels skin tumors.
lIdentifying blood stains using fluoresdcent reagents.