Instrumentation and Applications of fluorimetry
Objectives
At the end of the session the student will be
able to
• Identify
the different components in a spectro-fluorometer
• Categorise
the components
• Explain
the construction and working of a
fluorimeter
• Outline
the applications of Fluorimetry
Instrumentation:
• Source of light
• Filters and monochromators
• Sample cells
• Detectors
Light source:
• Mercury arc lamp.
• Xenon arc lamp.
• Tungsten lamp.
• Tunable dye lasers.
Mercury arc lamp:
• Produce intense line spectrum above
350nm.
• High pressure lamps give lines at
366, 405, 436, 546, 577, 691, 734nm.
• Low pressure lamps give additional
radiation at 254nm.
Xenon arc lamp:
• Intense radiation by passage of
current through an atmosphere of xenon.
• Spectrum is continuous over the range between over
250-600nm,peak intensity about 470nm.
Tungsten lamp:
• Intensity of the lamp is low.
• If excitation is done in the visible
region this lamp is used.
• It does not offer UV radiation.
Tunable dye lasers:
• Pulsed nitrogen laser as the primary
source.
• Radiation in the range between 360
and 650 nm is produced.
Filters and monochromators:
FILTERS
• Primary filter- transmits excitation
wavelength of light.
• Secondary filter- transmits
fluorescent light.
MONOCHROMATORS
• Excitation monochromators-isolate
only the radiation which is absorbed by the molecule.
• Emission monochromators-isolate only
the radiation emitted by the molecule.
Sample holders:
• The majority of fluorescence assays
are carried out in solution.
• Cylindrical or rectangular cells
fabricated of silica or glass used.
• Path length is usually 10mm or 1cm.
• All the surfaces of the sample
holder are polished in fluorimetry.
DETECTORS:
• Photovoltaic cell
• Photo emissive cell
• Photomultiplier tubes
• Diodes– Best and accurate.
PHOTOMULTIPLIER TUBE:
• Multiplication of photo electrons by
secondary emission of radiation.
• A photo cathode and series of
dynodes are used.
• Each cathode is maintained at
75-100v higher than the preceding one.
• Over all amplification of 106 is
obtained.
INSTRUMENTS DESIGNS:
• SINGLE BEAM FLUORIMETER
• DOUBLE BEAM FLUORIMETER
• SPECTROFLUORIMETER(DOUBLE BEAM)
SINGLE BEAM FLUORIMETER
• Tungsten lamp as source of light.
• The primary filter transmits a
narrow range of Excitation radiation.
• Emitted radiation measured at 90o
by secondary filter.
• Secondary filter transmits a narrow
range of emitted radiation.
Advantages:
• Simple in construction
• Easy to use.
• Economical
Disadvantages:
• It is not possible to use reference
solution & sample solution at a time.
• Rapid scanning to obtain Exitation
& emission spectrum of the compound is not possible.
Double beam fluorimeter:
• Similar to single beam instrument.
• Two incident beams from light source
pass through primary filters separately and fall on either sample or reference
solution.
• The emitted radiation from sample or
reference pass separately through secondary filter.
Advantages:
• Sample & reference solution can
be analyzed simultaneously.
Disadvantage
• Rapid scanning is not possible due
to use of filters.
SPECTROFLURIMETER:
• The primary filter in double beam
fluorimeter is replaced by excitation monochromators.
• The secondary filter is replaced by
emission monochromators.
• The incident beam is split into
sample and reference beam using a beam splitter.
• The detector is photomultiplier
tube.
Advantages
• Rapid scanning to get Excitation
& emission spectrum.
• More sensitive and accurate when
compared to filter fluorimeter.
SCHEMATIC DIAGRAM OF FLUROMETER:
SCHEMATIC DIAGRAM OF
SPECTROFLUORIMETER
APPLICATIONS
• Determination of inorganic
substances
• Determination of ruthenium ions in
presence of other platinum metals.
• Determination of aluminum (III) in
alloys.
• Determination of boron in steel by
complex formed with benzoin.
• Estimation of cadmium with 2-(2
hydroxyphenyl) benzoxazole in presence
of tartarate.
• Nuclear research
• Field determination of uranium
salts.
• Fluorescent indicators
• Mainly used in acid-base titration.
e.g.: Eosin: colorless-green.
Fluorescein:colourless-green.
Quinine sulphate: blue-violet.
Acridine: green-violet
4]
Fluorimetric reagents
• Aromatic
structure with two or more donor functional groups
|
Reagent |
Ion |
Fluorescence wavelength |
Sensitivity |
|
Alizarin garnet B |
Al3+ |
500 |
0.007 |
|
Flavanol 8-Hydroxy quinoline |
Sn4+ Li2+ |
470 580 |
0.1 0.2 |
• Organic
analysis
• Qualitative
and quantitative analysis of organic aromatic compounds present in cigarette
smoke, air pollutants, automobile exhausts etc.
• Pharmaceutical
Analysis
|
compound |
reagent |
excitation wavelength |
fluorescence |
|
Hydrocortisone |
75%v/v H2SO4 in ethanol |
460 |
520 |
|
Nicotinamide |
cyanogen chloride |
250 |
430 |
·
Liquid
chromatography
• Fluorescence is an imp method of
determining compounds as they appear at the end of chromatogram or capillary electrophoresis
column.
• Determination of vitamin B1 &B2.
Summary
• A
fluorimeter essentially consists of a
radiation source , two mono chromators, sample compartment and a detector
• One
monochromator is located before and the other after the sample compartment at
right angles to each other
• Essentially
the components used in UV spectrophotometers can be used in fluorimeters also
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