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7/28/2019 Flu Instru
1/21
SPECTROFLUORIMETRY
INSTRUMENTATION
Trainer: Chandramouli R
7/28/2019 Flu Instru
2/21
A generalized Spectrophotometry
consists of:
Source of radiation
A sample cell
PhotodetectorRead out device
7/28/2019 Flu Instru
3/21
A generalized Fluorimeter/
Spectrofluorimeterconsists of: Source of radiation
A primary filter / excitation Monochromator
A sample cell
A secondary filter / emission Monochromator
Photodetector
Read out device
7/28/2019 Flu Instru
4/21
FILTER FLUORIMETER
Secondary filterPrimary filter
AbsorbsVisible
radiation
and
transmits
UV
radiation
AbsorbsUV
radiation
and
transmits
Visible
radiation
7/28/2019 Flu Instru
5/21
SPECTRO FLUORIMETER
Secondary / Emission
Monochromator
Primary / Excitation
Monochromator
Provides a
suitable
radiation for
excitation of
molecule
Isolates only
the radiation
emitted by the
fluorescent
molecule
7/28/2019 Flu Instru
6/21
BLOCK DIAGRAM
Source of
lightPrimary filter /
MonochromatorSAMPLE Secondary filter /
Monochromator
DETECTOREmitted
radiation
from source
Specific
wavelength
of radiation
Resultant
luminescence
Radiation
emitted by the
fluorescent
molecule
7/28/2019 Flu Instru
7/21
Flourimeter over Filter
Flourimeter
Spectroflourimeter can measure the
spectral distribution of Flourescenceemission (emission spectrum)
And
the variation in emission spectral radiancewith excitation wavelength (excitationspectrum)
7/28/2019 Flu Instru
8/21
INSTRUMENTATION
Source of radiation
A primary filter / excitation monochromator
A sample cell A secondary filter / emission monochromator
Detectors
Read out device
7/28/2019 Flu Instru
9/21
INSTRUMENTATION
Source of radiation
A primary filter / excitation monochromator
A sample cell A secondary filter / emission monochromator
Detectors
Read out device
7/28/2019 Flu Instru
10/21
Source of light
Source of light should emit a radiation
over a continuous region and be of
adequate intensity and stability Mercury vapour lamp Xenon arc lamp
Tungsten lamp
7/28/2019 Flu Instru
11/21
INSTRUMENTATION
Source of radiation
A primary filter / excitation monochromator
A sample cell A secondary filter / emission monochromator
Photodetector
Read out device
7/28/2019 Flu Instru
12/21
Filters and Monochromators
Primary filter/Monochromator selects specificbands or wavelengths of radiation from the sourceand directs them through the sample in the samplecell
Secondary filter/Monochromator Resultantluminescence from primary filter/monochromator isisolated by the secondary filter or emissionmonochromator and directed to the photodetector
which measures the power of the emitted radiation.
7/28/2019 Flu Instru
13/21
INSTRUMENTATION
Source of radiation
A primary filter / excitation monochromator
A sample cell A secondary filter / emission monochromator
Detectors
Read out device
7/28/2019 Flu Instru
14/21
Sample cells
Cylindrical /Quadrangular in shape Path length 10mm or 1cm
7/28/2019 Flu Instru
15/21
amp e ce s con
Types :1. Right angle method (90)
2. Frontal method (37)
3. Rotating cell method
sample
Monochromatic excitation light90
sample
Monochromatic excitation light
37
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16/21
7/28/2019 Flu Instru
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Detectors
Photovoltaic cell Phototubes Photomultiplier tubes BEST &
ACCURATE
7/28/2019 Flu Instru
18/21
PHOTOVOLTAIC CELL /
BARRIER LAYER CELL
A Steel support (positive contact)
B SeleniumSemiconductor layer
C Metallic Layer coated
with silver or gold
D Negative contact
E Metallic spray coating to
protect from corrosion
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19/21
PHOTO TUBES / PHOTO
EMISSIVE CELLS
7/28/2019 Flu Instru
20/21
PHOTO MULTIPLIER TUBE
7/28/2019 Flu Instru
21/21
PHOTO MULTIPLIER TUBE