m.pharm analysis, pharmaceutical analysis, food analysis, cosmetic analysis and evaluation, modern bio analytical techniques, advanced pharmaceutical analysis, phamaceutical validation

1. SUBJECT: MPAT
KARNATAKA COLLEGE OF PHARMACY
TOPIC: INSTRUMENTATION & APPLICATION OF
SPECTROFLOURIMETRY
SUBMITTED TO:
MR. T. SREENIVASA RAO
PROFESSOR
DEPT. OF PHARMACEUTICAL CHEMISTRY
KARNATAKA COLLEGE OF PHARMACY,
BANGALURU
SUBMITTED BY:
ABHISHEK
M. PHARM 1ST SEMESTER
DEPT. OF PHARMACEUTICAL CHEMISTRY
KARNATAKA COLLEGE OF PHARMACY,
BANGALURU
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2. CONTENTS
 INTRODUCTION
 INSTRUMENTATION
 APPLICATIONS
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3. SPECTROFLUORIMETRY
 It is a process of phenomenon in which find out the concentration of unknown
sample with the help of fluorescence and phosphorescence.
Fluorescence:-
 When beam of light is incident on certain substances, they emit visible light or
radiations.
Phosphorescence:-
 When beam of light is incident on certain substances, they emit light
continuously even after the incident of light is cut-off.
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4. INSTRUMENTATION OF FLUORIMETRY 4

5. COMPONENTS OF INSTRUMENTATION
 Source of light
 filters and monochromators
 sample cells
 detectors
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6. A. SOURCE OFLIGHT:-
Mercury vapor lamp: mercury vapor at high pressure gives intense lines on
continuous background above 350nm.
 Low pressure mercury vapor gives an additional line at 254nm. It is used in filter
fluorimetry.
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7. Xenon arc lamp:- it give more intense radiation then mercury vapor lamp. It is
used in spectrofluorometer .
Tungsten lamp :- if excitation has to be done in visible region this can be used.
It is used in low-cost instruments.
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8. B. FILTERS AND MONOCHROMATORS
Filters: - these are nothing, but optical filters works on the principle of
absorption of unwanted light & transmitted the required wavelength of light.
In inexpensive instrument fluorimetry primary filter & secondary filter are
present.
Primary filter :- absorbs visible radiation and transmit UV radiation .
Secondary filters:- absorbs UV radiation and transmit visible radiation .
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9. 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.
Excitation monochromators:- provide suitable radiation for excitation of molecule.
Emission monochromators:- isolate the only radiation emitted by the fluorescent
molecule.
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10. C. SAMPLE CELLS:-
 These are a ment of holding liquid samples. These are made up of quartz & can
have various shapes ex: cylinder or rectangular shape.
 The majority of fluorescence assay are carried out in solution.
 The Path length 10mm or 1cm. All the surface of the sample holder are polished in
fluorimetry.
D. DETECTORS :-
photometric detectors are used they are:-
1. Barrier layer cell / photo voltaic cells.
2. Photomultiplier cells
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11. Barrier layer cell:-
 It is an inexpensive instruments. ex: Filter Fluorimeter
 It consist of a copper plate coated with thin layer of cuprous oxide (Cu2O). A
semi-transparent films of silver is laid on this plate to provide good contact’
 When external light fall on the oxide layer, the electrons emitted from the oxide layer
move into the copper plate.
 The oxide layer becomes positive (+) & copper plate become negative (-)
 Hence an emf develops between oxide layer & copper plate and behave like a voltaic
cells. So, it is called photo voltaic calls.
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12.  A galvanometer is connected externally between silver film & copper plate &
deflection in the galvanometer shows the current flow through it. The amount of
current is found to be proportional to the intensity of incident light.
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13. Photomultiplier tubes:-
 These are incorporated in expensive instruments like spectrofluorometer. It is sensitivity is
high due to measuring weak intensity of light.
 The principle employed in this detector id that, multiplication of photoelectrons by
secondary emission of electrons’
 This is achieved by using a photo cathode and a series of anodes. Up to 10 dynodes are
used. Each dynodes is maintained at 75 – 100 V higher than the preceding one.
 At each stage the electron emission is multiplied by a factor of 4 to 5 due to secondary
emission of electron and hence an overall factor of 10 is achieved.
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14.  PMT can detect very weak signals even 200 times weaker than that could
be done using photovoltaic cells. Hence it is used in fluorescence
measurement
 PMT should be shielded from stray light in order to have a accurate
results.
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16. APPLICATIONS:-
 Determination of inorganic substance;-
 Determination of ruthenium ions in presence of other platinum metals.
 Determination of boron in steel, aluminum in alloys & manganese in steel.
 Estimation of cadmium with 2-(2 hydroxy phenyl ) benzoxazole in presence of
tartarate.
 Determination of thiamine HCL.
 Determination of phenytoin.
 Determination of indoles, phenols & phenothiazines
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17. REFERENCE
 Textbook of pharmaceutical analysis 4th edition dr. Ravi Shankar
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18. THANK YOU
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