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Fluorescence
Spectroscopy
AYA BENDAHMAN
GHASSANE SIRAJ SANI
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.
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).
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.
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.
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
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
01
Luminescence!
SpECTROSCOPY
Mechanism and utilities
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.
Luminescence
spectroscopy
procedures
DAY 1
Preparation of
Samples
DAY 2
Exposure &
Re-emission
3
Spectrum
analysis
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
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.
DisAvdantages of fluorescence
spectroscopy
 Not really useful for identification
 Not all compounds fluorescence
 Contamination can quench the
fluorescence and hence give
false/no results
Applications of luminescence spectroscopy
Analytical Chemistry
Environmental studies
Biochemistry
01
02
03
04 Pharmacy
05 Medecine
Thank you for your
attention

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Fluorescence

Notes de l'éditeur

  1. 10 to the minus 8 power
  2. phosphorescence exhibits a longer lifetime (~10 to -4 power – 10 to -2 power seconds) compared with fluorescence (~10^-9 – 10^-6 seconds)
  3. 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
  4. 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
  5. 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 —------- 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. —-------------- 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.
  6. 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
  7. 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
  8. -Detecting compounds from HPLC flow - Plant pigments, steroids and proteins can be determined at low concentrations. —--- Detecting and quantifying polluants such as polycyclic aromatic hydrocarbons —--- Used generally as a non-destructive method of traking biofluorescent compounds —--- Direct and indirect analysis for multiple drugs -vitamins -catecholamins(dopamin) - Measuring the impurities of a sample —--- Ability to identify different levels skin tumors. lIdentifying blood stains using fluoresdcent reagents.