Fluorescence Analysis in Pharmaceutical Sciences
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Presented by
P.Sri Jyothi
M.pharmacy
Dpt.Pharmaceutcal
Analysis
PRINCIPLE
FACTORS EFFECTING
FLOURESCENCE
INSTRUMENTATION
APPLICATIONS
CONTENTS
CONCLUSION
REFERENCE
Dept of Analysis
2
FLOURESCENCE:
It is a phenomenon of emission of radiation
when the molecules are excited by radiation
at a certain wavelength.
Dept of Analysis
FLUORIMETRY:
It is a measurement of a fluorescence intensity
at a particular wavelength with the help of filter
flourimetry or spectrofluorimetry.
Dept of Analysis
Dept of Analysis
Concentration
Intensity
of incident
light
Adsorption
Oxygen
Ph
Temperature&
viscosity
Photodecomposition
Factors
effecting
fluorescence:
Fluorescence intensity is
proportional
to
concentration
of
substance only when
the
absorbance
is
less
than
0.02
.
A=log
I
ₒ
\It
or
A=
abc
I
ₒ
=intensity
of
incident light
a= absorptivity of constant
b=
Pathlength
c=
concentration
Dept of Analysis
INTENSITY OF LIGHT:
Increase In The Intensity Of Incident Light On The Sample
Fluorescence Intensity Also
Increases
.
ADSORPTION:
Adsorption Of Sample Solution In The Container May Leads
To
A
Serious
Problem.
PH:
OXYGEN:
In presence of oxygen the non fluorescent compounds shows
decreased fluorescence.
Alteration
of ph of a solution will have
significant
effect
on
fluorescence.
Dept of Analysis
TEMPERATURE :
Temperature
increases can increase
the
collisional
de
activation, and
reduce fluorescent
intensity.
VISCOSITY:
If
viscosity
of
solution is more
the frequency of
collisions are
reduced and increase in fluorescent
intensity.
PHOTOCHEMICAL DECOMPOSITION:
Absorption
of
intense radiation leads
to
photochemical
decomposition
of
a fluorescent substance
to
less fluorescent or non
fluorescent
substance
.
Dept of Analysis
I
Dept of Analysis
SOURCE OF LIGHT
FILTERS AND
MONOCHROMATORS
SAMPLE CELLS
DETECTORS
INSTRUMENTATION
Dept of Analysis
1
)
S
O
U
R
C
E
O
F
L
I
G
H
T
:
-
M
ercury
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.
Dept of Analysis
xenon
arc lamp
:
It
give
more
intense radiation than mercury
vapour lamp.
it
is used
in spectrofluorimeter
.
tungsten lamp
:-
If
excitation has
to
be done in
visible region
this
can be used.
It
is used in low
cost
instruments
.
Dept of Analysis
2)
FILTERS
AND
MONOCHROMATORS
:-
Filter
s
:
I
n fluorimetry 2 things are important i.e.excitation
wavelength and emission
In an inexpensive
instruments
like filter fluorimeter,
primary
filter and secondary
filter are
present.
Primary
filter:
-
absorbs
visible
radiation
and transmit UV
radiation.
Secondary
filter
:-absorbs
UV
radiation and trasmit visible
radiation.
Dept of Analysis
They convert
polychromatic light
into a
monochromatic light
.
They
can isolate a specific range of wavelength or a
particular
wavelength of radiation from a
source
s
In spectrophotometers, excitation monochromators and emission
monochromators are present
Excitation monochromators
: provides a suitable radiation for
excitation of molecule
Emission monochromators
: isolates only the radiation emitted by
the flourescent molecule
.
Dept of Analysis
3)
Sample
cells:
These
are ment for
holding
liquid
samples. These are made up
of
quartz and can have
various shapes
ex
: cylindrical
rectangular
Barrier layer cell/Photovoltaic
cells
Photomultiplier
cells
4
)
D
e
t
e
c
t
o
r
s
:
P
h
o
t
o
m
e
t
r
i
c
d
e
t
e
c
t
o
r
s
a
r
e
u
s
e
d
t
h
e
y
a
r
e
Dept of Analysis
1.
Barrier
layer
/photovoltaic
cell :
It
is employed in inexpensive instruments.
Ex: Filter fluorimetry.
It
consists
of a
copper plate coated with
a
thin layer
of
cuprous
oxide
(Cu
2
o).
A
semi transparent film
of
silver is laid on
this
plate
to
provide good
contact
.
When external light falls on
the
oxide
layer,
the
electrons
emitted from the
oxide
layer
move
into
the
copper
plate
.
Then
oxide
layer becomes positive and copper plate becomes
negative.
Dept of Analysis
Hence an
emf
develops between
the
oxide
layer
And copper
plate and behaves like a voltaic cell. So
it
is called
photovoltaic
cell..
A
galvanometer
is
connected externally between silver film and
copper plate
and
the
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
Dept of Analysis
Dept of Analysis
2.
Photomultiplier
tubes:
These are incorporated in expensive instruments like
spectrofluorimeter.
Its
sensitivity
is
high due
to
measuring weak intensity of
light.
This is achieved by using a photo cathode and a series
of
anodes
(Dyanodes). Up
to
10 dyanodes are used.
Each
dyanode is maintained
at
75- 100Vhigher than the preceding
one.
Dept of Analysis
At
each stage,
the
electron emission is multiplied by a
factor
of
4
to
5 due
to
secondary emission
of
electrons and hence
an
overall
factor
of
10
6
is
achieved.
PMT
can
detect very
weak signals, even 200
times
weaker
than
that
could be done using photovoltaic cell.
Hence
it
is useful in fluorescence measurements.
PMT
should be shielded
from stray
light in order
to
have
accurate
results.
Dept of Analysis
Photomultiplier
tube
Dept of Analysis
1.
Determination
of inorganic
substances.
Al3+,Li+,ZN2+
2.
Determination
of
thiamine Hcl.
3.
Detemination
of
phenytoin.
4.
Determination
of
indoles, phenols,
&
phenothiazines
5.
Determination
of
napthols, proteins, plant pigments and
steroids.
6.
Fluorimetry ,nowadays can be used in
detection of
impurities
in nanogram
level.
Dept of Analysis
Fluorimetric
methods
are
not useful
in
qualitative
analysis ,and much used in quantitative
analysis.
Fluorescence
is
the most
sensitive analytical
techniques.
Detection
studies
will
increase
the
development of
fluorescence
field.
Dept of Analysis
SKOOG ,Principles of Instrumental
Analysis.
Practical pharmaceutical chemistry by
A.H.
BECKETT&
J.B.STENLAKE ,volume
2,
B.K.Sharma Instrumental methods of
chemical
analysis.
A textbook of pharmaceutical
analysis
by
Dr.S.RAVISANKAR.
Dept of Analysis
Fluorescence analysis is a crucial technique in pharmaceutical analysis, involving the emission of radiation by molecules when excited at specific wavelengths. Factors influencing fluorescence, such as concentration, light intensity, adsorption, oxygen presence, pH, temperature, viscosity, and photodecomposition, impact the accuracy of measurements. Fluorimetry aids in quantifying fluorescence intensity, while maintaining optimal conditions for accurate results is essential for reliable analysis in pharmaceutical applications.
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Presentation Transcript
Presented by P.Sri Jyothi M.pharmacy Dpt.Pharmaceutcal Analysis
CONTENTS PRINCIPLE PRINCIPLE FACTORS EFFECTING FACTORS EFFECTING FLOURESCENCE FLOURESCENCE INSTRUMENTATION INSTRUMENTATION APPLICATIONS APPLICATIONS CONCLUSION CONCLUSION REFERENCE REFERENCE 2 Dept of Analysis
FLOURESCENCE: It is a phenomenon of emission of radiation when the molecules are excited by radiation at a certain wavelength. Dept of Analysis
FLUORIMETRY: It is a measurement of a fluorescence intensity at a particular wavelength with the help of filter flourimetry or spectrofluorimetry. Dept of Analysis
Factors effecting fluorescence: Concentration Intensity of incident light Adsorption Oxygen Ph Temperature& viscosity Photodecomposition
CONCENTRATION: Fluorescence intensity is proportional to concentration of substance only when the absorbance is less than 0.02. A=log I \It or A= abc I =intensity of incident light a= absorptivity of constant b= Pathlength c= concentration Dept of Analysis
INTENSITY OF LIGHT: Increase In The Intensity Of Incident Light On The Sample Fluorescence Intensity Also Increases. ADSORPTION: Adsorption Of Sample Solution In The Container May Leads To A Serious Problem. OXYGEN: In presence of oxygen the non fluorescent compounds shows decreased fluorescence. PH: Alteration of ph of a solution will have significant effect on fluorescence. Dept of Analysis
TEMPERATURE : Temperature increases can increase the collisional deactivation, and reduce fluorescent intensity. VISCOSITY: If viscosity of solution is more the frequency of collisions are reduced and increase in fluorescent intensity. PHOTOCHEMICAL DECOMPOSITION: Absorption of intense radiation leads to photochemical decomposition of a fluorescent substance to less fluorescent or non fluorescent substance. Dept of Analysis
I Dept of Analysis
INSTRUMENTATION SOURCE OF LIGHT SOURCE OF LIGHT FILTERS AND FILTERS AND MONOCHROMATORS MONOCHROMATORS SAMPLE CELLS SAMPLE CELLS DETECTORS DETECTORS Dept of Analysis
1)SOURCE OF LIGHT:- 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. Dept of Analysis
xenon arc lamp: It give more intense radiation than mercury vapour lamp. it is used in spectrofluorimeter. tungsten lamp:- If excitation has to be done in visible region this can be used. It is used in low cost instruments. Dept of Analysis
2) FILTERS AND MONOCHROMATORS:- Filters: In fluorimetry 2 things are important i.e.excitation wavelength and emission In an inexpensive instruments like filter fluorimeter, primary filter and secondary filter are present. Primary filter:-absorbs visible radiation and transmit UV radiation. Secondary filter:-absorbs UV radiation and trasmit visible radiation. Dept of Analysis
They convert polychromatic light into a monochromatic light. They can isolate a specific range of wavelength or a particular wavelength of radiation from a sources In spectrophotometers, excitation monochromators and emission monochromators are present Excitation monochromators : provides a suitable radiation for excitation of molecule Emission monochromators : isolates only the radiation emitted by the flourescent molecule. Dept of Analysis
3) Sample cells:These are ment for holding liquid samples. These are made up of quartz and can have various shapes ex: cylindrical rectangular 4) Detectors: Photometric detectors are used they are Barrier layer cell/Photovoltaic cells Photomultiplier cells Dept of Analysis
1. Barrier layer /photovoltaic cell : It is employed in inexpensive instruments. Ex: Filter fluorimetry. It consists of a copper plate coated with a thin layer of cuprous oxide (Cu2o). A semi transparent film of silver provide good contact. is laid on this plate to When external light falls on the oxide layer, the electrons emitted from the oxide layer move into the copper plate. Then oxide layer becomes positive and copper plate becomes negative. Dept of Analysis
Hence an emf develops between the oxide layer And copper plate and behaves like a voltaic cell. So it is called photovoltaic cell.. A galvanometer is connected externally between silver film and copper plate and the 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 Dept of Analysis
BARRIER LAYER CELL Dept of Analysis
2. Photomultipliertubes: These are incorporated in expensive instruments like spectrofluorimeter. Its sensitivity is high due to measuring weak intensity of light. This is achieved by using a photo cathode and a series of anodes (Dyanodes). Up to 10 dyanodes are used. Each dyanode is maintained at 75- 100Vhigher than the preceding one. Dept of Analysis
At each stage, the electron emission is multiplied by a factor of 4 to 5 due to secondary emission of electrons and hence an overall factor of 106 is achieved. PMT can detect very weak signals, even 200 times weaker than that could be done using photovoltaic cell. Hence it is useful in fluorescence measurements. PMT should be shielded from stray light in order to have accurate results. Dept of Analysis
Photomultiplier tube Dept of Analysis
APPLICATIONS 1. Determinationof inorganic substances. Al3+,Li+,ZN2+ 2. Determination of thiamine Hcl. 3. Detemination of phenytoin. 4. Determination of indoles, phenols, & phenothiazines 5. Determination of napthols, proteins, plant pigments and steroids. 6. Fluorimetry ,nowadays can be used in detection of impurities in nanogram level. Dept of Analysis
CONCLUSION : Fluorimetric methods are not useful in qualitative analysis ,and much used in quantitative analysis. Fluorescence isthe most sensitive analytical techniques. Detection studies will increase the development of fluorescence field. Dept of Analysis
REFERENCES : SKOOG ,Principles of InstrumentalAnalysis. Practical pharmaceutical chemistry byA.H. BECKETT& J.B.STENLAKE ,volume 2, B.K.Sharma Instrumental methods of chemical analysis. A textbook of pharmaceutical analysisby Dr.S.RAVISANKAR. Dept of Analysis
