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Definido NS: F Ore Cence

Fluorimetry involves exciting fluorescence substances with UV light and measuring the emitted light. Factors like concentration, pH, and temperature affect fluorescence intensity. A fluorimeter consists of a light source, monochromators, a cuvette, detector, and data analyzer. Quantitative analysis is based on the relationship between fluorescence intensity and concentration. Fluorimetry is used to determine inorganic/organic substances and detect impurities at very low levels.

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59 views19 pages

Definido NS: F Ore Cence

Fluorimetry involves exciting fluorescence substances with UV light and measuring the emitted light. Factors like concentration, pH, and temperature affect fluorescence intensity. A fluorimeter consists of a light source, monochromators, a cuvette, detector, and data analyzer. Quantitative analysis is based on the relationship between fluorescence intensity and concentration. Fluorimetry is used to determine inorganic/organic substances and detect impurities at very low levels.

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Paolo Peps
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© © All Rights Reserved
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DEFINIDO

NS
Fluorescence:
,. When a beam of light passes through a
substances, the would absorbs some form
particles radiation.
of
,. Itenergy and emits in
is phenomenon thewhich the excited singlet
electrons(the electrons are paired) emits
emission
radiation when brought down to
ground level at the certain wavelength.
, Fluorescence occurs at (10-9 -10-6 sec).
FLUORJMETRY:

~ It is a type of electromagnetic spectroscopy that


analyses the fluorescence from the sample.
~ It involves the use of beam of light, usually ultra
violet light that excites the electrons in the
molecules
of certain compounds (fluorescence substances) and
that produces a light of lower energy
PRINCIPL
E,
~ The principle involved is Vibrationalrelaxation.
~ Molecules have bonding (c) electrons and
(1t)electrons and n non bonding electrons.
~ When the excited electrons absorbs the radiant
energy, the bonding electrons gets down to
anti
~ bonding orbital.
~ Has high energy but less stable.
it is a instantenous process
FACTORS
INVOLVED
The factors effecting fluorescence intensity are
·=· Concentration
·=· Quantum yield of
fluorescence
·:· Intensity of incident light
·=· Adsorption
·=· Oxygen
·:· pH
·:· Temperature & Viscosity
·:· Photodecomposition
·=· Quenchers
Quenching
It is the process of reductionof fluorescence
intensity by the presence of substances in the
sample other than fluorescent analyte.
Fonns of quenching
;.... Self quenching
;... Collision quenching
;.... Static quenching
;.... Inner fluorescent effect
Contd.,
Self
quenching:
Fluorescenc in~ensi w
e concentration.
• T
Static quenching:
Due to complex formation between fluorescent
analyt and
e substances.
Collis quenching:
between substance and
ion fluorescent s
Collisi
Inner ions.
fluorescent
on
effect:
Absorption of light by filters
halide
incident
decreases the intensity
fluorescent .
PREPARATION OF
SOLUTION
~ Test solutions prepared for fluorescence
spectrophotometer are usually 10 times to 100
less concentrated than those times
used in absorption
spectre photometer.
~ Concentrations of 10-5-10- mol/ are frequently
used. 7 1
~ Blank should be not less than 0.40 and not more than
2.50.
r (
(
1_
INS'l'RUMENTATI
ON
The basic components of fluorimeter includes
;... An excitation light source
;... An excitation monochromator
:,.. A cuvette
;... An emission monochromaror
;... A detector
:,.. A data analyser
Instrumentatoi
n Monochromator
Xenon white light source
,_ (7SW)
...--:: _......
."" .-- ~ -
J IMI
£,c,:italion fllter ~ Sample
~00
~ ox. 504, nm
Monochromator t ex. 590 nm

--- 1
75

Sample I ~ 25
so

f
0 .i--.-::;::::::::;=~
500 550 600 650 700
EmlS$IOnfil ter Wavelength (nm]
Photo-multiplier
Light
source
~ Mercury lamps are the most commonly
employed
~ light sources.
They have the property that their spectral output
depends upon
~ Mercury the pressure of@254
arc lamp-radiation the filler
nm gas.
~ Xenon arc lamp- spectrum over the range between
250nm-600nm
~ Tungsten lamp- intensity of lamp is low
~ Turn able dye laser- radiation about 360nm-650nm
Monochromato
rs
• Monochromator produces monochromatic light by
removing unwanted wavelengths from the source light
beam.
~ The function of the monochromator is to isolate a
single
atomic resonance line from the spectrum of lines
,, emitted by
. the hollow
Isolates
cathode lamp.
only the radiation absorbed by the molecule
,,.Excitation
Emission monochromators:
monochromators:
Isolates only the radiation emitted by the molecule
Filter
s
The proper selection of filters requires
familiarity with the emission spectrum and
the excitation spectrum.
Primary filter:
It absorbs the visible light and transmits UV light.
Secondary filter:
It the UV light and transmits visible light.
absorbs
Cuvette
~ Cuvettte are cylindrical or rectangular cells fabricated
of silica or glass.
~ They are meant for holding the diluted
~ samples.
~ Path length is about lOmm or lcm
Cells and other glassware used for fluorimetric
analysis should be carefully cleaned, preferably by
boiling in 50 % nitric acid followed by thorough
rinsing in distilled water.
Detector
s
Detectors are device or instrument designed to
detect the presence of a particular object or
substance.
Three forms of detectors are
,. Photovoltaic cell
,. Phototube
,. Photomultiplier tube.
Photomultiplier tube
• A photomultipleir tube is a photo ernissive device in
which
the absorption of a photon results in the emission of an
electron.
• These detectors work by amplifying the electrons generated
by a photocathode exposed to a photon flux.
• Cathode voltage is 75-100 V.
• Amplification of 10"6 is obtained.
• Electron Mulunher Continuous Dvnodc.m -I

El=lro11
Rontl-01,r

1<71,,,., 4
- ru,
....J,t'!'al411V-lll

~1rJrJ..
- • tf,/1,1>4~
r so» l'J
TYPES OF
FLUORIMETER
• In single • In double beam fluorimeter the
fluorimeter,
beam all the Ji ght beam splits i11to two
parts and on] y one part
light waves pass through the passes
sample.
• through the sample.
• Primary filter- absorbs
Primary filter- absorbs the
visible light.
• light and passes to the sample.
• Secondary filter- absorbs Secondary filter - absorbs the
UV light. light from the sample and sent
• Reference and sample cant ~ to detector.
be analyzed Reference and sample can be
simultaneously analyzed simultaneously
SINGLE DOUBLE
BEAM BEAM
FLUORIMETE FLUORIMETE
R R
Contd.,
Single beam fluorimeter Double beam fluorimeter

Double Bmn
F1uorimeter
~ex

goo

\ I I

t
N1••

6
Quantitative analysis
The fluorescence intensity is proportional to the initial
radiation times abc.

log(Fo/Fo-F)= abc
Fo-100 (standard)
F- fluorescence measured
b- cell length
c- concentration
a- proportionality constant
By Simplification,
abc=2-log(100-F)
InShol 2019 ll28 123357970.mp-
'
Application
s
~ Determination of inorganic substances;
~ Determination of thiamine
~ Determination of indoles, phenols,
phenothiazines.
~ Napthols ,proteins ,plant pigment and steroids can
be
~ determined.
~ Detection of impurities at nanogram
~ quantities. Detection of respiratory tract
infection. Determines phenyltoin.

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