SPECTROFLUORIMETRY.pptx instrumentation, principle

1. SPECTROFLUORIMETRY
Presented by : Dr.Vijaya .U.
Barge
Pune District Education
Association’s Shankarrao Ursal
College of Pharmaceutical
Sciences & Research Centre,
Kharadi, Pune.

2. LEARNING OBJECTIVES
• Introduction to spectroflurometry.
• To study luminescence and its type.
• To learn electronic transitions and energy level diagram.
• To study fluorescence and phosphorescence and factors affecting it.
• To learn about instrumentation and working of fluorimetry.

3. INTRODUCTION
• Fluorescence is the molecular absorption of light energy at one wavelength and
its nearly instantaneous re-emission at another, usually longer wavelength.
• Fluorometry or fluorimetry is the measurement of fluorescence.
• The instrument used to measure fluorescence is called a fluorometer.
• The emitted light is proportional to the concentration of the analyte being
measured (up to a maximum concentration).
CONTIUNE…..

4. • Fluorescent behavior is observe in simple as well as in complex gaseous, liquid, and solid
systems.
• Light is absorbed by molecules which causes electrons to become excited to a higher
electronic state.
• The electrons remain in the excited state till excitied electron loses excess energy is not
lost by collisions with other molecules,
• After loss of excess of energy ,the electron returns to the ground state.
• Energy is emitted during the electrons return to their ground state.

5. TYPES OF LUMINESCENCE
1) Photoluminescence - As the excitation of the molecule is due to the
absorption of a photon (light), these types of luminescence are called
photoluminescence.
(a) Fluorescence: Fluorescence is the emission of visible light
by a substance that has absorbed light of a different wavelength. The
emitted photon has a longer wavelength.
(b) Phosphorescence: Phosphorescence is related to
fluorescence in emitting a photon however, a phosphorescent material does
not immediately re-emit the radiation it absorbs.

6. IMPORTANT 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.

7. ENERGY LEVEL DIAGRAMS FOR
PHOTOLUMINESCENT MOLECULES
• A Jablonski diagram is basically an energy diagram,
arranged with energy on a vertical axis.
• The diagram is arranged into columns.
• Every column usually represents a specific spin
multiplicity for a particular species.
• As electronic energy states increase, the difference in
energy becomes continually less.
• Additionally, as the electronic energy levels get closer
together, the overlap of vibronic energy levels increases.

8. DEACTIVATION PROCESSES
• An excited molecule can return to its ground state by a combination of several
mechanistic steps. The basic two of these steps are fluorescence and
phosphorescence, involve the release of a photon of radiation.
• The other deactivation methods are-
1. Vibrational relaxation
2. Internal conversion
3. External conversion
4. Intersystem crossing

9. 1. Vibrational relaxation
• Collisions between the molecules of the excited species and those of the solvent lead to
rapid energy transfer and a very small increase in temperature of the solvent.
• Vibrational relaxation process is so efficient that the average lifetime of a vibrationally
excited molecule is very less. As a consequence, fluorescence from solution always
involves a transition from the lowest vibrational level of an excited state.
2. Internal conversion
• The term internal conversion is employed to describe inter molecular processes by when
a molecule passes to a lower energy electronic state without emission of radiation.
• Internal conversion takes when two electronic energy levels are sufficiently close the
existence of an overlap in vibrational levels.

10. 3. External conversion
• Deactivation of an excited electronic state may involve interaction and energy transfer
between the excited molecule and the solvent or other solutes. These processes are called
external conversions.
• Evidence for external conversion includes the marked effect on the fluorescent intensity
of most species.
4. Intersystem crossing
• It is a process in which the spin of an excited electron is reversed and a change à
multiplicity of the molecule results.
• It most common in molecules that contain heavy atoms, such a iodine or bromine. Spin
and orbital interactions increase in the presence of such atoms, and change in spin is thus
more favored.

11. FACTORS AFFECTING FLUORESCENCE AND
PHOSPHORESCENCE INTENSITY
1. Adsorption : Sensitivity requires very dilute solutions that is 10-100 times
weaker than those employed in absorption spectroscopy which leads to
adsorption of the fluorescent substance on the container walls.
2. Intensity of incident light: Increase in intensity of incident light on the
sample increases fluorescence intensity.
Intensity of incident light depends on:
(a) Intensity of light emitted from the lamp.
(b) Excitation monochromator transmission properties.
(c) Excitation slit width.

12. 3. Turbidity : Fluorescence measurements are significantly more immune to the effects of
turbidity compared to absorption techniques like UV/VIS spectrophotometers. If the
interfering substance is reflective, turbidity can create light scatter and reading. If the
interfering substance absorbs light, fluorescence will be reduced.
4. Bubbles in the Sample: Since fluorescence is such a sensitive measurement, and since
accuracy of readings depend on light exciting the molecules of the compound, bubbles
in the sample can result in fluctuating readings
5. Effect of pH:Fluorescence can be affected by pH. The effect of pH depends on the
chemical structureof the molecule.
6. Temperature:Fluorescence is affected by changes in temperature. As temperature
increases, fluorescence decreases. And wise vesa.
7. Viscosity:Increase viscosity leads to decreased collisions of molecules which lead to
enhancement of fluorescence intensity and vice a versa.

13. QUENCHING
• It is a process that decrease the fluorescence intensity of given substance. It may
occur due to various factors ike pH, temperature, viscosity, complex formation.
FLUROSCENCE CAN BE LOST BY
1. Concentration quenching: At low concentration linearity is observed. But as
the concentration of the same substance increased in the sample fluroscence
intensity decreases.

14. 2. Chemical quenching: it may be of various factor like
a) pH: aniline at pH 5-13 gives blue flouresence But at pH 5 and pH >13 it does not
exhibit flouresence
b) Oxygen: presence of oxygen leads to quenching because of its parmegnatic
property.
c) Halides and electron withdrawing group: Halides likechloride bromide, iodide
and electron withdrawing group like nitro and carboxylic group decreases the
flouresence intensity.
d) Heavy metals: Presence of heavy metals also leads toquenching because of
collision and triplet ground state.
3. Static quenching: In these process the quenching agent forms a non-fluorescent
complex .
4. Collisional quenching: Quenching take place when number of collision are increased
due to several factor like presence of halides, heave metals increased temperature and
decrease in viscosity.

15. INSTRUMENTATION

16. 1. LIGHT SOURCE
a) Mercury vapour lamp: Mercury vapour at high
pressure give intense lines on continuous background
above 350nm.low pressure mercury vapour gives an
additional line at 254nm.it is used in filter fluorimeter.
b) xenon arc lamp: It give more intense radiation than
mercury vapour lamp. it is used in spectrofluorimeter.
c) tungsten lamp:- If excitation has to be done in visible
region this can be used. It is used in low cost
instruments.

17. 2. MONOCHROMATOR /FILTER
• Filters: these are nothing but optical filters works on the principle of absorption of unwanted
light and transmitting the required wavelength of light. In inexpensive instruments
fluorimeter primary filter and secondary filter are present.
• Monochromators: they convert polychromatic light into monochromatic light. They can
isolate a specific range of wavelength or a particular wavelength of radiation from a source.
3. SAMPLE CELLS
• These are ment for holding liquid samples. These are made up of quartz which are known as
cuvettes and can have various shapes ex: cylindrical or rectangular etc.

18. 4. LIGHT DETECTOR
• The light detector is most often a photomultiplier tube, though photodiodes are
increasingly being used. The light passing through the emission filter is detected by the
photomultiplier or photodiode. The light intensity, which is directly proportional (linear)
to the compound's concentration, is registered as a digital readout.

19. APPLICATION OF SPECTROFLUOROMETRY
• Determinaton of inorganic substance.
• Determination of uranium salt.
• Used in acid base titration.
• Oraganic anaylsis.
• Pharmaceutical analysis.
• Liquid chromatography.
• Determination of vitamin B1 and B2.

20. REFERENCES
• Dr Sanjay G. Walode , Sr Chandan R.S , Instrumental Methods Of Analysis , First
Edition , September 2020.
• 1 . Willard , Merrit , Dean , Settle , Instrumental Method Of Analysis , 7th edition ,
CBS publishers & Distributors Pvt. Ltd.
• Gurudeep R . Chatwal , Sham K Anand , Instrumental Method Of Chemical
Analysis.
• Douglas A Skoog , Donald M West , F . James Holler , Skoog & West's
Fundamental of Analytical Chemistry , Cengage Technology Edition.