Instrumentation and Applications of fluorimetry
At the end of the session the student will be
the different components in a spectro-fluorometer
the construction and working of a
the applications of Fluorimetry
• Source of light
• Filters and monochromators
• Sample cells
• Mercury arc lamp.
• Xenon arc lamp.
• Tungsten lamp.
• Tunable dye lasers.
Mercury arc lamp:
• Produce intense line spectrum above
• 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.
• 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
• Radiation in the range between 360
and 650 nm is produced.
Filters and monochromators:
• Primary filter- transmits excitation
wavelength of light.
• Secondary filter- transmits
• Excitation monochromators-isolate
only the radiation which is absorbed by the molecule.
• Emission monochromators-isolate only
the radiation emitted by the molecule.
• 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.
• Photovoltaic cell
• Photo emissive cell
• Photomultiplier tubes
• Diodes– Best and accurate.
• 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
• 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.
• Simple in construction
• Easy to use.
• 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
• The emitted radiation from sample or
reference pass separately through secondary filter.
• Sample & reference solution can
be analyzed simultaneously.
• Rapid scanning is not possible due
to use of filters.
• The primary filter in double beam
fluorimeter is replaced by excitation monochromators.
• The secondary filter is replaced by
• The incident beam is split into
sample and reference beam using a beam splitter.
• The detector is photomultiplier
• Rapid scanning to get Excitation
& emission spectrum.
• More sensitive and accurate when
compared to filter fluorimeter.
SCHEMATIC DIAGRAM OF FLUROMETER:
SCHEMATIC DIAGRAM OF
• Determination of inorganic
• Determination of ruthenium ions in
presence of other platinum metals.
• Determination of aluminum (III) in
• Determination of boron in steel by
complex formed with benzoin.
• Estimation of cadmium with 2-(2
hydroxyphenyl) benzoxazole in presence
• Nuclear research
• Field determination of uranium
• Fluorescent indicators
• Mainly used in acid-base titration.
e.g.: Eosin: colorless-green.
Quinine sulphate: blue-violet.
structure with two or more donor functional groups
Alizarin garnet B
and quantitative analysis of organic aromatic compounds present in cigarette
smoke, air pollutants, automobile exhausts etc.
75%v/v H2SO4 in ethanol
• Fluorescence is an imp method of
determining compounds as they appear at the end of chromatogram or capillary electrophoresis
• Determination of vitamin B1 &B2.
fluorimeter essentially consists of a
radiation source , two mono chromators, sample compartment and a detector
monochromator is located before and the other after the sample compartment at
right angles to each other
the components used in UV spectrophotometers can be used in fluorimeters also
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