3. 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 (fluorescence) when
exposed to electromagnetic radiation in the
ultraviolet wavelength.
4. Introduction
• Fluorescence is the phenomenon where
by a molecule, after absorption radiation, emits
radiation of a longer wavelength
• A compound absorbs radiation in the UV –
region 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 k/a the Stokes
shift.
5.
6.
7. Principle
• Fluorescence is an emission
phenomenon where an energy
transition from a higher to a lower state
is accompained by radiation.
• Only molecules in their excited forms
are able to emit fluorescence ; thus they
to be brought into a state of higher
energy prior to the emission
phenomenon.
8. Important Terms :-
•Singlet ground state : state in which electrons in a
molecule are paired.
•Singlet excited state: state in which electrons are
unpaired but of opposite spins .
•Triple state : state in which unpaired electrons of
some 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 .
9. Basic concept :-
•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 Second electronic excited
state S2, both of which are singlet states.
•Relaxation to the ground state from these excited
states occur by emission of energy through heat or
photons.
10.
11.
12. • 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 .
13.
14. 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
(approximately 1,000 times greater than
absorption spectrophotometric methods
) .
15. 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 Io
*molar absorption coefficient ɛ
*path length of the cell b
*concentration of the fluorophor in
solution c
F=φIo(1- e`ɛbc)
16. * 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 increases is progressively
less linear, and the intensity
eventually decreases as concentration
increases further .
17. 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 occurs.
18.
19.
20. A molecule’s fluorescence quantum yield
is influenced by external variables such as
• Temperature
• Viscosity of solvent
• pH .
• Increase temperature generally decrease Φf because
more frequent collisions between the molecule and
the solvent increases external conversion .
• Decrease 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 .
24. Light sources : -xenon arc lamp or mercury
lamp
* A high pressure xenon arc lamp is used
for spectrometer ,which provides intense
,stable and continuous beam in UV and
visible region.
* Filter fluorimeters generally use low –
power mercury vapour lamp which emits
distinct line spectra in the UV and visible
region .
25. Monochromator
• Two sets of monochromator are used :-
• primary monochromator ,
• secondary monochromator .
• Primary monochromator :- it includes slit
and dispersive device to isolate the
wavelength for excitation of sample .
• Secondary monochromator :- isolates the
wavelength of emitted fluorescence .
26. * Cuvettes :- usually quartz cuvettes are
used .
* Detector :- A phototube, photovoltaic
cell or photomultiplier tube is used .
* Read out device (Recorder) :- A
galvanometer or a potentiometer is used
as read out device .
27.
28.
29.
30.
31. Application of spectroflurometer
• For the chemical modification such as oxidation
,reduction ,hydrolysis, couping and self condensation .
• The determination and comparison of both excitation
and fluorescence spectra of a compound may help to
identify it .
• The assay of vitamin in foodstuffs, NADH in
mitochondria , microorganism , hormones' like
cortisol , oestradiol ,drugs (pharmaceuticals )
,cholesterol ,and porphyrins .
• Enzyme assay and kinetic analysis .
• Study of protein structure .
32. Fluorescent indicators
• Mainly used in acid base titration.
• Intensity of colour of the fluorescence of many
substances depend upon the pH of solution.
• These are called as fluorescent indicators and
are generally used in acid base titration .
• Eg. – pH (3.0-4.0) eosin -colourless to green .
• Fluorescien –pH(4.0-6.0) colourless to green .
• Quinine sulphate-: blue – violet.
• Acridine : green – violet .
33. Advantages
• Fluoremetric techniques have a high degree of specificity .
• Nuclear research ( uranium salts).
• 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 attogram rang .
• As both excitation & emission wave length are
characteristic ,it is more specific than absorption methods
.
• Qualitative and quantitative analysis of organic aromatic
compounds present in cigarette smoke, air pollutants,
automobile exhausts etc.
34. Disadvantages
• The susceptibility to environmental
conditions and the virtual impossibility of
predicting whether a compound will
fluorescence .
• The other major problem is quenching,
whereby the energy, transferred to the other
molecules .
• Contamination can quench the fluorescence
and hence give false /no result .
35. References
Principle and techniques of BIOCHEMISTRY
and MOLECULAR BIOLOGY-keith Wilson and
john walker.
TIETZ-fundamentals of clinical chemistry.
Wikipedia .
Google .
