Instrumentation and Applications of fluorimetry – Instrumental Methods of Analysis B. Pharma 7th Semester

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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