This document discusses instrumentation used in fluorimetry. It describes various light sources like mercury arc lamps, xenon arc lamps, and tungsten lamps. It also discusses filters, monochromators, sample cells, and detectors like photomultiplier tubes that are used. Different types of fluorimeters are described, including single beam, double beam, and spectrofluorimeters. Finally, some applications of fluorimetry are mentioned such as determination of inorganic substances, use in nuclear research, as fluorescent indicators, and in organic analysis.

1. INSTRUMENTATION OF
FLOURIMETRY
PRESENTED BY
C.HARISH
(pharmaceutics)

2. SOURCE OF LIGHT
FILTERS AND MONOCHROMATORS
SAMPLE CELLS
DETECTORS

3. MERCURY ARC LAMP.
XENON ARC LAMP.
TUNGSTEN LAMP.

4. 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.

5.  It gives more intense radiation than mercury lamp
 It is used in spectro flourimetry
Spectrum is continuous over the range between 250-
600nm,peak intensity about 470nm.

6. Intensity of the lamp is low.
If excitation is done in the visible
region this lamp is used.
It does not offer UV radiation.

7. FILTERS
Primary filter-absorbs visible light & transmits uv light.
Secondary filter-absorbs uv radiations & transmits visible light.
MONOCHROMATORS
Exitation monochromaters-isolates only the radiation which is
absorbed by the molecule.
Emission monochromaters-isolates only the radiation emitted by
the molecule.

8. 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.

9. PHOTOVOLTAIC CELL
PHOTO TUBE
PHOTOMULTIPLIER TUBES – Best
and accurate.

10. PHOTOVOLTIC CELL

11. PHOTOVOLTIC CELL
 It consists of a copper plate coated with a thin layer of
cuprous oxide
 when external light fails on oxide layer ,it emmites electrons
and moves to copper plate
 Then oxide layer becomes positive and copper plate
becomes negative
A galvoanmeter connected to measure the intensity of
light

12. PHOTOTUBES

13. PHOTOMULTIPLIER
TUBE

14. SINGLE BEAM FLUORIMETER
DOUBLE BEAM FLUORIMETER
SPECTROFLUORIMETER(DOUBLE
BEAM)

15. Tungsten lamp as source of light.
The primary filter absorbs visible radiation
and transmits uv radiation.
Emitted radiation measured at 90o
by
secondary filter.
Secondary filter absorbs uv radiation and
transmits visible radiation.

16. SINGLE BEAM FLUORIMETRY

17. 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.

19.  The primary filter in double beam fluorimeter is
replaced by excitation monochromaters.
 The secondary filter is replaced by emission
monochromaters.
 The incident beam is split into sample and reference
beam using a beam splitter.
 The detector is photomultiplier tube.

22. 1] 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.

23. 2]Nuclear research
 Field determination of uranium salts.
3]Fluorescent indicators
 Mainly used in acid-base titration.
e.g.:
eosin- colorless-green.
Fluorescein:colourless-green.
Quinine sulphate: blue-violet.
Acridine: green-violet

24. 5] organic analysis
Qualitative and quantitative analysis of organic
aromatic compounds present in cigarette smoke, air
pollutants, automobile exhaust
6]Liquid chromatography
Fluorescence is an imp method of determining
compounds as they appear at the end of
chromatogram or capillary electrophoresis column.
7]determination of vitamin B1 &B2.