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 and Applications of Fluorimetry
Fluorimetry, a powerful analytical technique, plays a crucial role in various scientific disciplines. It involves the measurement of fluorescence, where substances emit light upon excitation.
Principles of Fluorimetry
Understanding the basics is essential to grasp fluorimetry’s depth. At its core, fluorescence is the emission of light after absorbing photons. Fluorophores, the molecules responsible for fluorescence, absorb light at specific wavelengths, subsequently re-emitting light at longer wavelengths. This phenomenon forms the foundation of fluorimetry.
Instrumentation of Fluorimetry:
• 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 photoelectrons 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 FLUOROMETER:
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.
| 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
FAQs
- Is fluorimetry only used in scientific research? Fluorimetry is widely used in scientific research, but its applications extend to various industries, including environmental monitoring and pharmaceuticals.
- What is autofluorescence, and how does it impact fluorimetry? Autofluorescence is the natural fluorescence exhibited by certain substances. It can interfere with measurements in fluorimetry, affecting the accuracy of results.
- How has nanotechnology influenced fluorimetry? Nanotechnology integration has enhanced fluorimetry’s precision, allowing for targeted imaging and analysis at the molecular level.
- Can fluorimetry be used in medical diagnostics? Yes, fluorimetry plays a crucial role in medical diagnostics, especially in areas like personalized medicine and disease detection.
- Are there any upcoming technologies that could revolutionize fluorimetry? Advanced fluorophores and improved instrumentation are among the upcoming technologies that hold the potential to revolutionize fluorimetry.
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